Multilevel Systematic Optimization To Achieve Efficient Integrated Expression of Escherichia coli.

Genomic integration of heterologous genes is the preferred approach in industrial fermentation-related strains due to the drawbacks associated with plasmid-mediated microbial fermentation, including additional growth burden, genetic instability, and antibiotic contamination. Synthetic biology and genome editing advancements have made gene integration convenient. Integrated expression is extensively used in the field of biomanufacturing and is anticipated to become the prevailing method for expressing recombinant proteins. Therefore, it is pivotal to strengthen the expression of exogenous genes at the genome level. Here, we systematically optimized the integrated expression system of Escherichia coli from 3 aspects. First, the integration site slmA with the highest expression activity was screened out of 18 sites in the ORI region of the E. coli BL21 (DE3) genome. Second, we characterized 16 endogenous promoters in E. coli and combined them with the T7 promoter. A constitutive promoter, Plpp-T7, exhibited significantly higher expression strength than the T7 promoter, achieving a 3.3-fold increase in expression levels. Finally, to further enhance the T7 expression system, we proceeded with overexpression of T7 RNA polymerase at the chassis cell level. The resulting constitutive efficient integrated expression system (CEIES_Ecoli) showed a 2-fold increase in GFP expression compared to the pET3b recombinant plasmid. Therefore, CEIES_Ecoli was applied to the integrated expression of nitrilase and hyaluronidase, achieving stable and efficient enzyme expression, with enzyme activities of 22.87 and 12,195 U·mL-1, respectively, comparable to plasmid levels. Overall, CEIES_Ecoli provides a stable and efficient method of gene expression without the need for antibiotics or inducers, making it a robust tool for synthetic biology, enzyme engineering, and related applications.

HOCl forms lipid N-chloramines in cell membranes of bacteria and immune cells.

Neutrophils orchestrate a coordinated attack on bacteria, combining phagocytosis with a potent cocktail of oxidants, including the highly toxic hypochlorous acid (HOCl), renowned for its deleterious effects on proteins. Here, we examined the occurrence of lipid N-chloramines in vivo, their biological activity, and their neutralization. Using a chemical probe for N-chloramines, we demonstrate their formation in the membranes of bacteria and monocytic cells exposed to physiologically relevant concentrations of HOCl. N-chlorinated model membranes composed of phosphatidylethanolamine, the major membrane lipid in Escherichia coli and an important component of eukaryotic membranes, exhibited oxidative activity towards the redox-sensitive protein roGFP2, suggesting a role for lipid N-chloramines in protein oxidation. Conversely, glutathione a cellular antioxidant neutralized lipid N-chloramines by removing the chlorine moiety. In line with that, N-chloramine stability was drastically decreased in bacterial cells compared to model membranes. We propose that lipid N-chloramines, like protein N-chloramines, are involved in inflammation and accelerate the host immune response.

Genetic traits and transmission of antimicrobial resistance characteristics of cephalosporin resistant Escherichia coli in tropical aquatic environments.

This study investigates the genetic traits and transmission mechanisms of cephalosporin-resistant Escherichia coli in tropical aquatic environments in Singapore. From 2016 to 2020, monthly samples were collected from wastewater treatment plants, marine niches, community sewage, beaches, reservoirs, aquaculture farms, and hospitals, yielding 557 isolates that were analyzed for antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) using genomic methods. Findings reveal significant genotypic similarities between environmental and hospital-derived strains, particularly the pandemic E. coli ST131. Environmental strains exhibited high levels of intrinsic resistance mechanisms, including mutations in porins and efflux pumps, with key ARGs such as CMY-2 and NDM-9 predominantly carried by MGEs, which facilitate horizontal gene transfer. Notably, pathogenic EPEC and EHEC strains were detected in community sewage and aquaculture farms, posing substantial public health risks. This underscores the critical role of these environments as reservoirs for multidrug-resistant pathogens and emphasizes the interconnectedness of human activities and environmental health.

UPRER-immunity axis acts as physiological food evaluation system that promotes aversion behavior in sensing low-quality food.

To survive in challenging environments, animals must develop a system to assess food quality and adjust their feeding behavior accordingly. However, the mechanisms that regulate this chronic physiological food evaluation system, which monitors specific nutrients from ingested food and influences food-response behavior, are still not fully understood. Here, we established a low-quality food evaluation assay system and found that heat-killed E. coli (HK-E. coli), a low-sugar food, triggers cellular UPRER and immune response. This encourages animals to avoid low-quality food. The physiological system for evaluating low-quality food depends on the UPRER (IRE-1/XBP-1) - Innate immunity (PMK-1/p38 MAPK) axis, particularly its neuronal function, which subsequently regulates feeding behaviors. Moreover, animals can adapt to a low-quality food environment through sugar supplementation, which inhibits the UPRER -PMK-1 regulated stress response by increasing vitamin C biosynthesis. This study reveals the role of the cellular stress response pathway as physiological food evaluation system for assessing nutritional deficiencies in food, thereby enhancing survival in natural environments.

We quickly learn to steer clear of eating the moldy apple, the foul-smelling piece of chicken or the leftovers that taste a little 'off'. This survival instinct is shared across most animal species ­ even those with extremely simple and limited visual or taste systems, like the tiny worm Caenorhabditis elegans. Indeed, assessing the safety and quality of available food items can also rely on cells activating built-in cascades of molecular reactions. However, it remains unclear how these 'cellular stress response programs' actually help guide feeding behaviors. To better understand this process, Liu et al. conducted a series of experiments using C. elegans worms exposed to heat-killed bacteria, which are devoid of many nutrients essential for growth. After initially consuming these bacteria, the worms quickly started to avoid feeding on this type of low-quality food. This suggests that mechanisms occurring after ingestion allowed the worms to adjust their feeding choices. Further work showed that the consumption of heat-killed bacteria triggered two essential stress response pathways, known as the unfolded protein response and the innate immune response. The activation of these pathways was essential for the animals to be able to change their behavior and avoid the heat-killed bacteria. These biochemical pathways were particularly active in the worms' nerve cells, highlighting the importance of these cells in sensing and reacting to food. Finally, Liu et al. also found that adding sugars like lactose and sucrose to the low-quality food could prevent the activation of the stress response pathways. This result suggests that specific nutrients play a central role in how these worms decide what to eat. These findings shed light on the complex systems that ensure organisms consume the nutritious food they need to survive. Understanding these processes in worms can provide insights into the broader biological mechanisms that help animals avoid harmful food.

EspH utilizes phosphoinositide and Rab binding domains to interact with plasma membrane infection sites and Rab GTPases.

Enteropathogenic E. coli (EPEC) is a Gram-negative bacterial pathogen that causes persistent diarrhea. Upon attachment to the apical plasma membrane of the intestinal epithelium, the pathogen translocates virulence proteins called effectors into the infected cells. These effectors hijack numerous host processes for the pathogen's benefit. Therefore, studying the mechanisms underlying their action is crucial for a better understanding of the disease. We show that translocated EspH interacts with multiple host Rab GTPases. AlphaFold predictions and site-directed mutagenesis identified glutamic acid and lysine at positions 37 and 41 as Rab interacting residues in EspH. Mutating these sites abolished the ability of EspH to inhibit Akt and mTORC1 signaling, lysosomal exocytosis, and bacterial invasion. Knocking out the endogenous Rab8a gene expression highlighted the involvement of Rab8a in Akt/mTORC1 signaling and lysosomal exocytosis. A phosphoinositide binding domain with a critical tyrosine was identified in EspH. Mutating the tyrosine abolished the localization of EspH at infection sites and its capacity to interact with the Rabs. Our data suggest novel EspH-dependent mechanisms that elicit immune signaling and membrane trafficking during EPEC infection.

Oxacillinase-484-Producing Enterobacterales, France, 2018-2023.

We examined the emergence and characteristics of oxacillinase-484-producing Enterobacterales in France during 2012-2023. Genomic analysis identified 2 predominant sequence types in Escherichia coli: ST410 and ST1722. Plasmid analysis revealed that blaOXA-484 genes were carried mostly on an IncX3-type plasmid associated with genetic elements including insertion sequences IS3000 and ISKpn19.

Systematic evaluation of the induction of efficient neutralizing antibodies by recombinant multicomponent subunit vaccines against monkeypox virus.

Mpox (formerly known as monkeypox), which has symptoms similar to smallpox, is a zoonotic disease caused by the monkeypox virus (MPXV). From 1 January 2022 to 31 March 2024, 117 countries, territories, or areas reported 95,226 laboratory-confirmed cases of Mpox (including 185 deaths) to the World Health Organization. However, as there is no licensed specific MPXV vaccine available globally, the vaccines currently used for mpox prevention are mostly smallpox vaccines. Thus, the rapid development of safe and effective vaccines is urgently required. In the present study, the key MPXV proteins A35, B6R, E8L, A29, M1R, and H3L were expressed and prepared using a prokaryotic expression system (Escherichia coli) and a eukaryotic expression system (yeast), and the fusion antigens A35-A29 and A35-M1R were constructed based on the dimerization characteristics of the A35 protein. By combining the antigens with aluminum hydroxide and CpG adjuvants in different combinations, we developed nine multicomponent MPXV subunit vaccine candidates. Each antigen (10 µg) and fusion antigen (20 µg) were used to immunize the mice. The first two doses produced a mean titer of 10(Petersen et al., 2016 [5]), and the third dose maintained the same potent antibody-specific response as the previous two immunizations. The protective activity of different antigen combinations was determined using the cell neutralization test of vaccinia virus (VACV), which showed that the subunit vaccine candidates with two to six components (MPXV6/5/4/3a/3b/Fa/2a) had good neutralizing activity, and antigens A35 and M1R could produce neutralizing antibodies against VACV. The neutralizing antibody titer of the fusion antigen MPXVFa (A35-M1R), detected 2 weeks after the second booster dose, was comparable with that of MPXV2a (A35 and M1R). The A35-M1R fusion protein not only provided a high level of protection as a protective antigen but also simplified the preparation of candidate antigens. In summary, we systematically investigated the different protective antigen candidates of MPXV that have been widely studied and provided critical insights into the key protective antigen composition for vaccines, thus establishing a technical and theoretical foundation for the development of MPXV subunit vaccines.

Atmospheric pressure dielectric barrier discharge plasma for in-situ water treatment using a bubbling reactor.

Non-thermal plasma has been an emerging technology for water treatment for decades. In this study, we have designed and fabricated a bubbling plasma batch reactor using an atmospheric pressure dielectric barrier discharge with a hydrophobic porous membrane. The reactor performance is assessed for purifying synthetic contaminated water samples containing chemical contaminant sulfamethoxazole (SMX), a widely used antibiotic, and biological contaminant E. coli K12. The SMX decontamination tests indicate that the degradation process is not first-order and the reaction rate dwindle with increasing initial concentration. The yield at 50% removal achieves its highest value of 8.12 g/kWh for 50 mg/L SMX sample. For inactivation of E. coli K12 tests, the inactivation process is also not first-order, and the pathogen is completely inactivated for 102 CFU/mL and 104 CFU/mL cases after 10 min and 45 min of plasma treatment, respectively. For the 108 CFU/mL sample, a 5-log reduction is achieved after 60 min of treatment. The developed plasma reactor can achieve fast deployment in point of use, low cost for manufacturing, and simple for maintenance. Moreover, it can be used for in-situ water purification in future long duration crewed space missions as well as tackling with water pollution issues on our planet.

Selection for amoxicillin-, doxycycline-, and enrofloxacin-resistant Escherichia coli at concentrations lower than the ECOFF in broiler-derived cecal fermentations.

Antimicrobial resistance (AMR) is an emerging worldwide problem and a health threat for humans and animals. Antimicrobial usage in human and animal medicine or in agriculture results in selection for AMR. The selective concentration of antimicrobial compounds can be lower than the minimum inhibitory concentration and differs between environments, which can be a reason for bacterial resistance. Therefore, knowledge of the minimal selective concentration (MSC), under natural conditions, is essential to understand the selective window of bacteria when exposed to residual antimicrobials. In this study, we estimated the MSCs of three antimicrobials, amoxicillin, doxycycline, and enrofloxacin in a complex microbial community by conducting fermentation assays with cecal material derived from broilers. We examined the phenotypic resistance of Escherichia coli, resistome, and microbiome after 6 and 30 hours of fermenting in the presence of the antimicrobials of interest. The concentrations were estimated to be 10-100 times lower than the epidemiological cut-off values in E. coli for the respective antimicrobials as determined by EUCAST, resulting in an MSC between 0.08 and 0.8 mg/L for amoxicillin, 0.4 and 4 mg/L for doxycycline, and 0.0125 and 0.125 mg/L for enrofloxacin. Additionally, resistome analysis provided an MSC for doxycycline between 0.4 and 4 mg/L, but amoxicillin and enrofloxacin exposure did not induce a significant difference. Our findings indicate at which concentrations there is still selection for antimicrobial-resistant bacteria. This knowledge can be used to manage the risk of the emergence of antimicrobial-resistant bacteria.IMPORTANCEAntimicrobial resistance possibly affects human and animal health, as well as economic prosperity in the future. The rise of antimicrobial-resistant bacteria is a consequence of using antimicrobial compounds in humans and animals selecting for antimicrobial-resistant bacteria. Concentrations reached during treatment are known to be selective for resistant bacteria. However, at which concentrations residues are still selective is important, especially for antimicrobial compounds that remain in the environment at low concentrations. The data in this paper might inform decisions regarding guidelines and regulations for the use of specific antimicrobials. In this study, we are providing these minimal selective concentrations for amoxicillin, doxycycline, and enrofloxacin in complex environments.

The direct inhibitory effects of Lactobacillus acidophilus, a commensal urinary bacterium, on calcium oxalate stone development.

BACKGROUND: Lactobacillus acidophilus is a commensal urinary bacterium found more abundantly in healthy individuals than in stone patients. Hence, it has been proposed to play an inhibitory role in kidney stone disease (KSD) but with unclear mechanisms. We therefore investigated the direct effects of L. acidophilus on calcium oxalate (CaOx) stone development compared with Escherichia coli, which is known to promote CaOx stone formation. RESULTS: L. acidophilus at 1 × 103 CFU/ml  significantly reduced the abundance of newly formed crystals, enlargement and aggregation of seeded crystals, and crystal adhesion on renal cell membranes. By contrast, E. coli at 1 × 103 CFU/ml significantly enhanced crystal growth and aggregation but did not affect crystallization and crystal-cell adhesion. Oxalate consumption assay showed that neither L. acidophilus nor E. coli significantly reduced the remaining oxalate level after 1 - 3 h incubation. However, both of them adhered to CaOx crystals. Surface component detection revealed that only L. acidophilus expressed S-layer protein, whereas only E. coli exhibited flagella on their surfaces. Removal of L. acidophilus S-layer protein and E. coli flagella completely abolished the inhibitory and promoting effects of L. acidophilus and E. coli, respectively. CONCLUSIONS: L. acidophilus inhibits CaOx stone development by hampering crystallization, growth, aggregation and cell-adhesive ability of CaOx. By contrast, E. coli enhances CaOx stone development by promoting CaOx growth and aggregation. Their contradictory effects are most likely from differential surface components (i.e., S-layer protein on L. acidophilus and flagella on E. coli) not from oxalate-degrading ability. Video Abstract.

BracketMaker: Visualization and optimization of chemical protein synthesis.

Chemical protein synthesis (CPS), in which custom peptide segments of ~20-60 aa are produced by solid-phase peptide synthesis and then stitched together through sequential ligation reactions, is an increasingly popular technique. The workflow of CPS is often depicted with a "bracket" style diagram detailing the starting segments and the order of all ligation, desulfurization, and/or deprotection steps to obtain the product protein. Brackets are invaluable tools for comparing multiple possible synthetic approaches and serve as blueprints throughout a synthesis. Drawing CPS brackets by hand or in standard graphics software, however, is a painstaking and error-prone process. Furthermore, the CPS field lacks a standard bracket format, making side-by-side comparisons difficult. To address these problems, we developed BracketMaker, an open-source Python program with built-in graphic user interface (GUI) for the rapid creation and analysis of CPS brackets. BracketMaker contains a custom graphics engine which converts a text string (a protein sequence annotated with reaction steps, introduced herein as a standardized format for brackets) into a high-quality vector or PNG image. To aid with new syntheses, BracketMaker's "AutoBracket" tool automatically performs retrosynthetic analysis on a set of segments to draft and rank all possible ligation orders using standard native chemical ligation, protection, and desulfurization techniques. AutoBracket, in conjunction with an improved version of our previously reported Automated Ligator (Aligator) program, provides a pipeline to rapidly develop synthesis plans for a given protein sequence. We demonstrate the application of both programs to develop a blueprint for 65 proteins of the minimal Escherichia coli ribosome.

Towards Understanding Tumour Colonisation by Probiotic Bacterium E. coli Nissle 1917.

The last decade has seen a rapid increase in studies utilising a genetically modified probiotic, Escherichia coli Nissle 1917 (EcN), as a chassis for cancer treatment and detection. This approach relies on the ability of EcN to home to and selectively colonise tumours over normal tissue, a characteristic common to some bacteria that is thought to result from the low-oxygen, nutrient-rich and immune-privileged niche the tumour provides. Pre-clinical studies have used genetically modified EcN to deliver therapeutic payloads that show efficacy in reducing tumour burden as a result of high-tumour and low off-target colonisation. Most recently, the EcN chassis has been expanded into an effective tumour-detection tool. These advances provide strong justification for the movement of genetically modified EcN into clinical oncology trials. What is currently unknown in the field is a deep mechanistic understanding of how EcN distributes to and localises within tumours. This review summarises the existing EcN literature, with the inclusion of research undertaken with other tumour-homing and pathogenic bacteria, to provide insights into possible mechanisms of EcN tumour homing for future validation. Understanding exactly how and why EcN colonises neoplastic tissue will inform the design and testing of the next generation of EcN chassis strains to address biosafety and containment concerns and optimise the detection and treatment of cancer.

Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans.

Loss-of-function Parkin mutations lead to early-onset of Parkinson's disease. Parkin is an auto-inhibited ubiquitin E3 ligase activated by dual phosphorylation of its ubiquitin-like (Ubl) domain and ubiquitin by the PINK1 kinase. Herein, we demonstrate a competitive binding of the phospho-Ubl and RING2 domains towards the RING0 domain, which regulates Parkin activity. We show that phosphorylated Parkin can complex with native Parkin, leading to the activation of autoinhibited native Parkin in trans. Furthermore, we show that the activator element (ACT) of Parkin is required to maintain the enzyme kinetics, and the removal of ACT slows the enzyme catalysis. We also demonstrate that ACT can activate Parkin in trans but less efficiently than when present in the cis molecule. Furthermore, the crystal structure reveals a donor ubiquitin binding pocket in the linker connecting REP and RING2, which plays a crucial role in Parkin activity.

Sonodynamic inactivation of gram-negative and gram-positive bacteria in the presence of phenothiazine compounds toluidine blue and azurin A.

BACKGROUND: Sonodynamic antimicrobial chemotherapy (SACT) is an effective antimicrobial treatment that can avoid the production of drug-resistant bacteria. Design and development of new high-efficiency sonosensitizers play a key role in the practical application of SACT. METHODS: The bacteriostatic effects of two phenothiazine compounds, toluidine blue (TB) and azure A (AA) combined with ultrasonic (US) on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were studied, and the sonodynamic antibacterial activities of TB and AA were compared. The reactive oxygen species (ROS) and the types of ROS produced in the sonodynamic system were detected and the sonodynamic mechanisms of TB and AA were proposed. RESULTS: The sonodynamic bacteriostasis mediated by TB and AA increased with the increasing concentration of sonosensitizer, the extension of sonication time and the increase of reaction temperature. The production of ROS was the main reason that TB and AA had excellent sonodynamic antibacterial performance. Singlet oxygen (1O2) and hydroxyl radical (•OH) were the main ROS types in the sonodynamic antibacterial system. The ROS produced by the combined action of AA and US was higher than that of TB. CONCLUSION: Both TB and AA displayed excellent sonodynamic antibacterial activities. Moreover, AA had a higher sonodynamic activity than TB. The electron donation effect and steric hindrance effect of the methyl group of phenothiazine parent nucleus of TB might be the cause of the decrease of its sonodynamic activity. These results would provide a valuable reference for the further study of phenothiazines sonosensitizers and their clinical application in SACT.

Antibiotic Resistance and Virulence Gene Patterns Associated with Multi Drug Resistant Avian Pathogenic Escherichia coli (APEC) Isolated from Broiler Chickens in India.

In the present study, a total of 102 samples were collected from chickens of different flocks, died due to suspected colibacillosis. Bacteriological and PCR methods were applied to detect avian pathogenic Escherichia coli (APEC). Phenotypic antimicrobial resistance (AMR) was determined by disk diffusion method. Extended spectrum beta lactamases (ESBL) detection was carried out via PCR by targeting blaTEM, blaSHV, blaOXA, and blaCTX-M groups 1, 2, and 9. Genes of eight virulence factors and class I integrons were also detected by PCR using gene specific primers. Culture, microscopic, biochemical tests and PCR recognised 69/102 (67.64%) samples as E. coli. Phenotypic AST revealed higher resistance against fluoroquinolone antibiotics, i.e., enrofloxacin (72.46%), levofloxacin (69.56%) & ciprofloxacin (66.66%), followed by amoxyclav (63.77%) and tetracycline (59.42%). Six isolates were found as pan-drug-resistant E. coli. A total of 48 (69.56%) and 7 (10.14%) isolates were positive for the presence of blaTEM and blaCTX-M-G9 genes, respectively, whereas 2 (2.90%) isolates each were found positive for blaSHV, blaOXA, and blaCTX-M-G1 genes. Among APEC associated virulence genes, iss (79.71%) was the most predominant, followed by tsh (50.72%), ast (30.43%), cvaf (26.08%), pap (23.18%), vat (8.69%) and stx-1 (1.44%). Thirty-two isolates harboured class I integrons, either with or without ESBL genes. Conclusively, the isolates under study showed pan and multiple-drug resistance, specifically against fluoroquinolone drugs. ESBL production was mediated principally through bla TEM and blaCTX-M-G9. Multiple virulence factors, toxins, and carriage & spread factor render these as zoonotically potential pathogens for humans. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01132-2.

An Additional L451G452N453 in the RpoB Protein Suppressed the Synthetic Lethality in Escherichia coli at 37 Degrees Caused by Depletion of DnaK/J and Trigger Factor.

Escherichia coli depletion of chaperone trigger factor and DnaK/J were not viable at 37°C, but viable below 30°C. Among the engineered E. coli depleted of trigger factor and DnaK/J, one strain Z625, exhibited survival at 37°C, while another strain Z629 only survived below 30°C. Comparative analysis of fatty acid profiles of Z625 and Z629 revealed absence of numerous saturated fatty acids in Z625 as compared to the wild-type E. coli BW25113. In addition, increased unsaturated fatty acids were present in Z625, whereas the fatty acids profile of Z629 closely resembled that of BW25113. Whole genome sequencing revealed a 9-bp insertion in rpoB of Z625. Combined structural analysis of simulated RpoB protein bearing the amino acid sequence L451G452N453 insertion and susceptibility analysis to rifampicin suggested that the insertion did not disturb the individual RpoB structure as beta subunit of RNA polymerase. Comparative transcriptomic analysis of Z625 and Z629 suggested that this insertion impacted transcription of the overall RNA polymerase in Z625, leading to potential repression of some genes whose overexpression was toxic to E. coli. Additionally, Z625 exhibited distinctive metabolic adaptations, likely contributing to its survival at 37°C. In summary, our study elucidated one LGN insertion in rpoB that impacts transcriptional regulation in E. coli, thereby explaining the survival of E. coli depletion of trigger factor and DnaK/J at 37°C, and these founding suggested that some simple mutations in critical genes like rpoB might play an important role in driving adaptive evolution.

Genetic landscape of ESBL producing international clone ST410 of Escherichia coli from pediatric infections in Shenzhen, China.

Background: The emergence of ESBLs producing cephalosporin-resistant Escherichia coli isolates poses a threat to public health. This study aims to decipher the genetic landscape and gain insights into ESBL-producing E. coli strains belonging to the high-risk clone ST410 from pediatric patients. Methods: 29 E. coli ST410 isolates were collected from young children and subjected to antimicrobial susceptibility testing, Whole-genome sequencing (WGS), serotype analysis, MLST, ESBL genes, virulence genes, and plasmid profiling. Results: Antimicrobial susceptibility testing demonstrated a high level of resistance to cephalosporins followed by aminoglycoside, sulfonamide, carbapenem and penicillin group of antibiotics. However, n=20/29 shows MDR phenotype. Phylogenetic group B2 (n=15) dominated, followed by group D (n=7), group A (n=4), and group B1 (n=3). Serotyping analysis identified O1:H7 (n=8), O2:H1 (n=6), O8:H4 (n=5), O16:H5 (n=4), and O25:H4 (n=3). Other serotypes identified included O6:H1, O15:H5, and O18:H7 (n=1 each). The most commonly detected ESBL genes were bla CTX-M, (n=26), followed by bla TEM (n=23), and bla SHV (n=18). Additionally, bla OXA-1 (n=10), bla OXA-48 (n=5), bla KPC-2 (n=3), bla KPC-3 (n=2), bla NDM-1 (n=4), bla NDM-5 (n=1), bla GES-1 (n=2), bla GES-5 (n=1), and bla CYM-1 (n=3). Notable virulence genes identified within the ST410 isolates included fimH (n=29), papC (n=24), hlyA (n=22), and cnf1 (n=18), among others. Diverse plasmids were observed including IncFIS, IncX4, IncFIA, IncCol, IncI2 and IncFIC with transmission frequency ranges from 1.3X10-2 to 2.7X10-3. Conclusion: The ST410 clone exhibited a complex resistance profile, diverse serotypes, the presence of specific resistance genes (ESBL genes), virulence gene repertoire, and diverse plasmids. The bla CTX-M was the most prevalent ESBL gene detected.

Using a dual immunoinformatics and bioinformatics approach to design a novel and effective multi-epitope vaccine against human torovirus disease.

Human Torovirus (HToV), a member of the Coronaviridae family, causes severe enteric diseases with no specific medication available. To develop novel preventative measures, we employed immunoinformatics techniques to design a multi-epitope-based subunit vaccine (HToV-MEV) triggering diverse immune responses. We selected non-allergenic, non-toxic, and antigenic epitopes from structural polyproteins, joined them with suitable linkers, and added an adjuvant 50S ribosomal L7/L12 peptide. The vaccine's solubility score of 0.903678 and physiochemical properties were found effective. Molecular dynamics simulations and free energy calculations revealed strong binding affinity for Toll-like receptor 3 (TLR-3), with a lowest energy score of -303.88, indicating high affinity. In-silico cloning and codon optimization showed significant production potential in E. coli. Immune simulations predicted a human immunological response. Our results are promising, but subsequent in vivo research is recommended. The HToV-MEV vaccine design demonstrates potential for preventing HToV-related diseases, and further investigation is warranted to explore its therapeutic applications.

A method for producing protease pS273R of the African swine fever virus.

The pS273R protease of the African swine fever virus (ASFV) is responsible for the processing of the viral polyproteins pp220 and pp62, precursors of the internal capsid of the virus. The protease is essential for a productive viral infection and is an attractive target for antiviral therapy. This work presents a method for the production of pS273R in E. coli cells by fusing the protease with the SlyD chaperone. The chimeric protein pS273R protease, during expression, is formed in a soluble form possessing enzymatic activity. Subsequently, pS273R separates from SlyD through autocatalytic cleavage at the TEV protease site in vivo. This work devised a straightforward protocol for chromatographic purification, resulting in the production of a highly purified viral protease. Additionally, we suggest using a fluorescence method to assess the activity of pS273R. This method is predicated on a shift in the chimeric protein thioredoxin-EGFP's electrophoretic mobility following its protease cleavage. It was shown that thioredoxin-EGFP substrate is effectively and selectively cleaved by the pS273R protease, even in complex protein mixtures such as mammalian cell lysates.

Microbiological quality of vegan alternatives to dairy and meat products in England during 2022-2023.

AIMS: Plant-based alternatives to meat and dairy products have become increasingly popular in the UK. Despite a public perception that they have a relatively low microbiological risk, outbreaks of illness have been linked with these foods. This study aimed to assess the microbiological safety and quality of vegan alternatives to dairy and meat products available in England. METHODS AND RESULTS: Samples were collected between September 2022 and March 2023 from retail, production and catering premises, and tested for a range of bacterial pathogens and hygiene indicators using standard procedures. A total of 937 samples were tested, of which 92% were of a satisfactory microbiological quality, 3% were borderline and 5% were unsatisfactory. Those interpreted as unsatisfactory were due to elevated counts of Enterobacteriaceae and E. coli (indicators of poor hygiene) rather than pathogenic micro-organisms. Listeria monocytogenes was present in five samples of tofu, all from the same producer (all at counts of < 100 cfu g-1), while other Listeria species were detected at counts of < 20 cfu g-1 in two burgers and two 'vegan chicken' products. The majority of samples did not have pH and water activity values that would significantly contribute to preventing microbial growth: 62.4% had pH > 5.0 and 82.4% had Aw > 0.94. CONCLUSIONS: The majority of vegan products examined were of a satisfactory quality, but results demonstrate that microbiological control must be maintained using appropriate processing and storage temperatures and application of a safe length of shelf-life.