Search Results (308)

Pathogenic Vibrio species are a major cause of disease outbreaks in aquaculture, leading to substantial economic losses and posing risks to food safety. The increasing prevalence of antibiotic resistance among these pathogens has created an urgent need for alternative antimicrobial strategies. Phage-derived endolysins, which specifically degrade bacterial peptidoglycan, have emerged as promising candidates to replace or supplement conventional antibiotics. In this study, we systematically characterized the endolysin LysV039C from the Vibrio alginolyticus phage phiV039C. Whole-genome analysis identified a 462 bp endolysin gene, which was cloned into the pET-28a vector, expressed in E. coli BL21, and purified. The lytic activity of the recombinant enzyme was evaluated using a turbidity reduction assay. LysV039C showed the strongest activity (58.9%) against host bacteria in the logarithmic phase, with peak activity (60.7–64.1%) achieved at 53 μg/mL, 35 °C, and neutral pH. The addition of 2.5 mM EDTA enhanced activity to 63.8%, whereas 10 mM divalent metal ions strongly inhibited the enzyme (<6.5%). Lytic spectrum analysis demonstrated that LysV039C exhibited a broader lytic spectrum against six Vibrio species than its parent phage phiV039C. Overall, LysV039C combines high environmental adaptability (alkaline tolerance, suitability for aquaculture temperature ranges) with efficient and broadened lytic activity against multiple Vibrio species. These findings provide a foundation for developing eco-friendly agents for the prevention and control of pathogenic Vibrio and offer a valuable reference for the mining of other phage-derived antibacterials against aquatic pathogens. Full article

Rapidly increasing manufacturing waste intensifies the need for highly efficient and economically feasible waste-to-resource technologies. This research presented an upcycling strategy that transforms silver (Ag)-bearing waste into functional silver oxide nanoparticles (Ag₂O NPs) for potential use in biomedical applications. A two-step process of silver purification and the bio–sonochemical synthesis of Ag₂O NPs was employed. First, the sequential purification of Ag via melt-refining and electrorefining was utilized to efficiently achieve a silver purity of ≥99.90%. This purification encouraged the recycling of various forms of Ag-bearing waste. Subsequently, the purified Ag was prepared as the precursor for the bio–sonochemical synthesis of Ag₂O NPs, using Cannabis sativa SUT CBD12 flower extract as a natural reducing agent. The optimal conditions were adapted from a preliminary test using AgNO₃ as the precursor. At initial concentrations of 0.01 M Ag solution, 1 mM PVP solution, and 10 g dried weight of Cannabis sativa SUT CBD12 added to 100 mL DI water, the optimal condition was obtained at the capping agent:reducing agent:precursor volume ratio of 0.6:0.006:1, pH 10–12, under a short sonication time of 2 min. The purified Ag (waste-derived) and AgNO₃-derived Ag₂O NPs shared similar spherical shapes and sizes of ~100–130 nm. The Ag₂O NPs showed antibacterial effectiveness against S. aureus (ZOI of 26–29 mm) and E. coli (ZOI of 16–20 mm). Preliminary observations into the incorporation of Ag₂O NPs into polycaprolactone (PCL) to produce electrospun PCL/Ag₂O NPs nano fabrics shows a bead-free morphology, raising the possibility of a potential use in wound dressing. Following these preliminary explorations into potential uses and emission controls, a circular design maximizing the use of recycled resources is emphasized. Full article

Repeated exposure to subinhibitory concentrations of antiseptics may lead to reduced susceptibility or resistance and potentially promote cross-resistance to antibiotics. However, the underlying molecular mechanisms remain incompletely understood. This study investigated whether antiseptic-adapted Escherichia coli strains exhibit altered protein expression profiles compared with wild-type (WT) E. coli. Protein expression was analysed in E. coli strains previously adapted over ten passages to subinhibitory concentrations of chlorhexidine (CHX), cetylpyridinium chloride (CPC), and benzalkonium chloride (BAC). WT bacteria were exposed to sub-minimum inhibitory concentrations (sub-MICs) of antiseptics for 3 h to induce stress. Untreated WT and heat-shocked WT E. coli (42 °C, 2 h) served as controls. Protein expression profiles were assessed using SDS-PAGE and Western blotting targeting stress-associated proteins. SDS-PAGE demonstrated altered protein expression patterns in antiseptic-adapted strains, including differences in band intensities and additional protein bands. Western blot analysis showed increased DnaK and GroEL expression with reduced LexA levels in heat-shocked WT bacteria, whereas RecA remained largely unchanged. Antiseptic-adapted strains exhibited increased DnaK, GroEL, and OmpF expression together with reduced LexA and RecA expression. These findings indicate that adaptation to antiseptics involves complex mechanisms among multiple proteins rather than a single adaptive mechanism. Full article

Background/Objective: Antimicrobial resistance poses a critical public health crisis, highlighting the urgent requirement to investigate bacterial evolutionary adaptations and pioneer alternative therapeutics. Consequently, bacteriophages and metal-based compounds are emerging as viable options to combat drug-resistant infections. Building on our finding that T4 phage resistance in E. coli B also confers adaptation to high iron(III), we used RNA-sequencing (RNA-seq) to explore bacterial gene expression in resistant and control populations. We analyzed samples from our five experimental groups—Ancestor (ANC), control (CON), phage-selected (Phage), iron(III)-selected (FE), and phage/iron(III)-selected (PF), to understand how these regimes drive transcriptional changes. Method: Total RNA was extracted using the TRIzol protocol, and sequencing libraries were prepared with the Illumina RNA Total Library Prep Kit. Sequencing was performed on the Illumina NextSeq 1000/2000 platform. Reads were aligned to the E. coli B ATCC 11303 reference genome, and pairwise comparisons between the five experimental groups were conducted to determine differential gene expression profiles. Results: Principal component analysis (PCA) showed that iron-adapted populations (FE and PF) separated distinctly from the Ancestor and control populations along PC1 (capturing 40% of the variance), while the phage-selected replicates were split, with one (Phage5) clustering with CON3 and two (Phage2, Phage4) falling closer to, but clearly separated from, the Ancestor. Differential expression analysis (P_adj < 0.05 and |log₂FC| ≥ 1) revealed extensive transcriptional rewiring, with 482 and 381 differentially expressed genes (DEGs) in the FE and PF populations, respectively, compared to the Ancestor, and 177 DEGs in the phage-selected population compared to the Ancestor. The direct pairwise comparison between the iron-selected and phage/iron-selected populations yielded zero DEGs, demonstrating that both iron-adapted populations converged on a near-identical gene expression profile regardless of their distinct genetic and evolutionary backgrounds. Conclusions: This study suggests that pmrB and arn pathway genes may serve as primary markers for resistance to iron stress. These results are significant because they demonstrate a coordinated, multi-gene defense mechanism in E. coli B against high iron(III) stress, in which the arn operon and eptA remodel lipid A and the outer membrane, while glycerol-3-phosphate metabolism and phage-shock/chaperone pathways are repressed. Full article

Water scarcity is an increasingly urgent global challenge, prompting the development of new water purification technologies that surpass conventional solutions. Decentralized greywater treatment is emerging as a viable option for enhancing water reuse in multifunctional systems that contribute to microclimate regulation, cooling, and urban climate adaptation. In this context, water-storage mats have been identified as a form of decentralized, roof-based biofilter for greywater treatment. The aim of this study was to assess the performance of newly developed, innovative, bio-based textile mats and assess their effectiveness in treating pre-treated greywater from a canteen (CGW) with a high organic content, in both laboratory- and pilot-scale experiments. The findings from the lab-scale testing revealed that the mats made from polyethylene terephthalate (PET) nonwoven fabric materials had the highest water storage capacity and dried out more slowly in outdoor conditions than mats made from polylactide (PLA) spunbonded fabric and polyhydroxyalkanoate (PHA) spunbonded nonwoven fabric. The PET hydroentangled nonwoven fabric mat (PET-WS) performed better than the other sample mats in the lab-scale experiment, and also outperformed the PHA mat consistently in the pilot-scale experiment when treating CGW. Apparent reductions in the concentration of the macro-pollutant parameters were observed at the outflow of the PET-WS mat compared to the inflow (p < 0.05) at the pilot-scale. Mean concentration reductions were comparatively higher for the five-day biochemical oxygen demand (BOD₅), chemical oxygen demand (COD), total nitrogen (TN), and total suspended solids (TSS), with mean reductions of 64%, 54%, 39% and 60%, respectively. This indicated the superior treatment performance of the PET-WS mat compared to the PHA mat, with mean reductions of only 36%, 25%, 6%, and 32%, respectively. However, the lower E. coli counts of 1.1 and 0.5 log reduction for the PET-WS and PHA mats, respectively, indicated that an additional disinfection unit was necessary. The findings of this study may help to determine the performance, stability and reliability of using lightweight, nonwoven fabric mats to treat high-strength GW, which is currently considered as an intermediate treatment step. The study also provides recommendations for process optimization. Additional post-treatment steps are required to produce high-quality treated effluent for non-potable reuse, particularly in urban areas facing high water scarcity, provided that the relevant reuse regulations or discharge criteria are met. Full article

Escherichia coli is increasingly recognised as an important cause of ventilator-associated pneumonia (VAP), particularly in intensive care units (ICUs) with high antimicrobial selective pressure. Unlike classical respiratory pathogens, ICU-associated E. coli often originates from the patient’s intestinal microbiota and harbours a complex mobilome enriched with antimicrobial resistance determinants. In this study, a total of 200 nosocomial endotracheal aspirate samples were aseptically collected from patients admitted to the Respiratory ICU at Assiut University hospital. Antimicrobial susceptibility testing, serotyping and screening for various virulence and antimicrobial resistance genes (e.g., extended-spectrum β-lactamase (ESBL) and carbapenems genes) were carried out. In total, E. coli isolates were recovered from 54/200 (27%) endotracheal aspirates, with a high prevalence of multidrug resistance (MDR) observed (74.1%). Resistance to β-lactams was common with phenotypic evidence suggestive of ESBL production detected in 64.8% of isolates. Genome sequencing of three MDR E. coli isolates confirmed that they carried multiple antimicrobial resistance genes, which included ESBL genes (e.g., bla_CTX-M-15 and bla_TEM-1B). Each strain was also found to be high-risk extraintestinal pathogenic E. coli (ExPEC) clones, belonging to either sequence type ST131 or ST405. These findings support an endogenous infection model for VAP, whereby ICU selective pressure favours highly mobile, multidrug-resistant E. coli lineages adapted for extraintestinal survival. The high production of ESBLs and the prevalence of carbapenemase genes highlight the urgent need for molecular surveillance and antimicrobial stewardship strategies for the control of such high-priority pathogens in this part of the world. Full article

Tropical coastal waters are increasingly recognized as critical reservoirs for virulent, antibiotic-resistant enteric pathogens, yet seasonal dynamics governing their spatial distribution remain poorly characterized. We hypothesized that hydrological shifts and anthropogenic nutrient enrichment drive the seasonal distribution, virulence profiles, and antimicrobial resistance (AMR) of Escherichia coli, Salmonella spp., and Shigella spp. in the Jaffna Peninsula, Sri Lanka. Across 25 coastal sites during dry and transitional seasons, we integrated physicochemical water quality assessment, culture-based enumeration, PCR-based virulence gene profiling, Minimum Inhibitory Concentration (MIC) assays, GIS mapping, and statistical analyses. Key water quality parameters, including ammonium, nitrite, and total phosphorus, showed significant seasonal variation (p < 0.05), reflecting distinct hydrological regimes across seasons. A total of 220 E. coli, 200 Salmonella spp., and 100 Shigella spp. isolates were examined for virulence gene profiles and antibiotic tolerance. E. coli was detected at 80–88% of sites, Salmonella spp. at 72–88%, and Shigella spp. at 32–48%. Among E. coli isolates, stx1 was detected at 20–28% of sites and eae at 16% across both seasons. The stn gene was detected in Salmonella spp. at 12–28% of sites seasonally. Virulence profiling confirmed STEC harbouring stx1, stx2, and eae; Salmonella spp. carried stn; and Shigella spp. possessed invasion-associated genes. Trimethoprim–sulfamethoxazole resistance was recorded in 63.2% of E. coli, 33.0% of Salmonella spp., and 31.0% of Shigella spp. isolates at the lowest tested concentration of 4 µg/mL., while ciprofloxacin and piperacillin–tazobactam retained greater efficacy. Correlation analyses revealed significant associations among faecal contamination, nutrient enrichment, and virulence gene prevalence, implicating untreated sewage discharge and eutrophication as likely ecological factors associated with pathogen occurrence. These findings designate the Jaffna coastal zone as a significant reservoir of virulent AMR enteric pathogens, underscoring the urgent need for integrated One Health surveillance and seasonally adaptive coastal water quality management. Full article

The misuse of antibiotics is causing widespread antibiotic resistance, creating an urgent need for new treatment options such as nanoparticle-based therapies. This study aimed to compare silver nanoparticles (AgNPs) produced via green synthesis methods with those made through traditional chemical processes. Furthermore, the study investigated and contrasted the bacterial responses to these two types of AgNPs over a 21-day period of selection pressure using experimental evolution techniques. Analysis using scanning electron microscopy and transmission electron microscopy revealed a consistent, uniform morphology among the AgNPs produced via chemical methods. In contrast, AgNPs synthesized through green methods displayed an irregular morphology. Despite these morphological differences, all nanoparticles from both synthesis approaches were under 100 nm in diameter. These findings were further supported by the absorption spectrum data, which showed a maximum absorption peak between the 400 and 500 nm wavelength range. E. coli exposed to green synthesized AgNPs for 21 days adapted to their presence, exhibiting both enhanced resistance to the green synthesized AgNPs themselves and the development of cross-resistance to ionic silver, a pattern not observed in chemically synthesized AgNP-selected populations. Populations selected using chemical synthesized AgNPs did not develop increased resistance to either chemically or green synthesized AgNPs; however, they showed a slight increase in resistance to ionic silver. Genomics analysis identified polymorphism in genes in a green synthesized AgNP-resistant line including but not limited to the multidrug efflux transporter system (EmrAB), DUF4756 family protein (D1792_RS05680), putative zinc-binding protein YnfU/cold shock-like protein (ynfU/cspB) and imcF-related family protein (D1792_RS10035). Bacterial resistance to chemical AgNPs involves specific polymorphisms in key bacterial components like the RNA polymerase sigma factor (RpoE) and the EmrAB efflux pump. Collectively, the method used to synthesize the AgNPs influences their antibacterial efficacy and the likelihood of bacteria developing resistance. Understanding this interaction is vital for developing effective and resistance-controlled applications of AgNPs across medicine, environmental science, and industry. Full article

The antimicrobial resistance crisis has led to the use of metals and bacteriophages as possible alternatives to antibiotics. Experimental studies have examined interactions between ionic/nano-silver and bacteriophages against multidrug-resistant bacteria. However, these approaches have often failed to examine whether silver affects the stability and infectivity of bacteriophages. Here, we utilized experimental evolution to evolve resistance to ionic silver in bacteriophage T7. High ionic silver concentrations that do not represent physiological exposure conditions were used to impose strong selective pressure. Evolution of ionic silver resistance in phage T7 was rapid, as evidenced by recovery of bacteriophage growth in E. coli following repeated exposures to ionic silver, enhanced infectivity of silver-selected populations relative to parallel control and ancestral populations under increasing ionic silver concentrations, and greater suppression of E. coli growth in standard medium. Furthermore, silver resistance evolved without loss of thermal or pH stability under the conditions tested. The genomic foundation of silver resistance was relatively simple, with positive and negative natural selection differentiating the silver-selected populations from the controls and ancestral populations across serial passages in silver. Support for replication-associated adaptation under ionic silver selection may be reflected in recurrent mutations identified in genes involved in transcription, DNA replication, and genome maintenance, including T7p07 (RNA polymerase), T7p10 (DNA ligase), and T7p29 (DNA polymerase I). These findings highlight the importance of evaluating phage –silver combination strategies within an evolutionary framework that accounts for the adaptive capacity of bacteriophages under silver selection. Full article

Background/Objectives: The increased prevalence of multidrug-resistant Escherichia coli and carbapenemase-producing Enterobacteriaceae poses a critical threat to global health and food safety. Antimicrobial Blue Light (aBL) in the 400–470 nm spectrum has emerged as a promising, chemical-free disinfection strategy that targets intracellular porphyrins and flavins to induce oxidative stress. However, the influence of wavelength, dosimetry, and environmental stressors on endogenous photoinactivation remains poorly standardized regarding optical parameters and biological exposure protocols. This systematic review aimed to evaluate the antimicrobial efficacy of pure blue light (400–470 nm) against E. coli across various phenotypes and environmental conditions, excluding the use of exogenous photosensitizers. Methods: PubMed, Scopus, and Web of Science were searched for studies that utilized 400–470 nm light as an antimicrobial agent against E. coli. Data extraction focused on spectral efficiency, total fluence (J/cm²), and log₁₀ reduction. The Risk of Bias was assessed using an adapted Office of Health Assessment and Translation tool for in vitro studies. Results: Synthesis of 11 high-quality studies indicated that wavelengths near 405 nm have the highest germicidal efficiency due to the Soret band absorption of endogenous porphyrins. Efficacy is highly dose-dependent: significant log₁₀ reductions were achieved in planktonic cells, although biofilms required substantially higher fluences. Sub-lethal environmental stressors such as acidic pH, high salinity, and thermal fluctuations demonstrated a synergistic effect, which significantly enhanced the rate of photoinactivation. Multidrug-resistant and carbapenemase-producing Enterobacteriaceae strains showed similar susceptibility to aBL relative to antibiotic-sensitive strains, suggesting no cross-resistance between light and traditional drugs. Conclusions: Endogenous blue light is a highly effective, non-thermal technology for E. coli decontamination. Its efficacy is modulated by the interplay between optical parameters and environmental conditions. These findings provide a framework for the development of standardized protocols for applying aBL to clinical wound care and food industry use cases. They also highlight the potential of aBL as a critical tool in the post-antibiotic era. This systematic review was registered in the International prospective register of systematic reviews (PROSPERO) under protocol CRD420261331871. Full article

Background/Objectives: Escherichia coli (E. coli) spans commensal, intestinal pathogenic, and extraintestinal pathogenic lineages distributed across human, animal, and environmental reservoirs, yet the extent to which virulence architectures are shared across these compartments remains incompletely understood. Using a One Health framework, we profiled putative virulence determinants in pooled multidrug-resistant (MDR) E. coli source groups representing human, animal, and environmental sectors. Methods: Virulence genes were predicted with VirulenceFinder, and presence–absence profiles were integrated to define functional composition, sector overlap, source-group distribution breadth, and pathotype-associated signatures. Predicted pathogenic potential was assessed with PathogenFinder and compared with pathogenic family richness. Results: Overall, 114 putative virulence genes were detected, with adhesion/colonization functions dominating the virulome (33/114), followed by toxin-associated genes (12/114). A conserved core of 50 virulence genes was shared across all three sectors, including determinants linked to serum resistance (iss, ompT, traT), adhesion (csgA, fimH), stress adaptation (terC), and iron acquisition (sitA, iutA, fyuA). ExPEC-associated determinants were most numerous in environmental source groups (n = 52), whereas diarrheagenic E. coli markers were most frequent in animal-associated groups (n = 42). LEE-associated effectors were infrequent and largely absent from human source groups. Despite ecological differences in virulence composition, pathogenicity scores remained consistently high across sectors (0.83–0.92) and showed no significant association with pathogenic family richness (Spearman’s ρ = 0.197, p = 0.392). Conclusions: Within the limits of pooled source-group analysis, these findings suggest that MDR E. coli across One Health compartments shares a broadly distributed, ExPEC-associated virulence repertoire overlaid with sector-specific pathotype signals, underscoring the value of integrated genomic surveillance while highlighting the need for isolate-resolved analysis. Full article

Background. The highly mutable influenza virus causes severe annual infections worldwide and results in substantial socioeconomic losses. The spread of infection could be effectively controlled by cross-protective vaccines and universal diagnostic test systems based on the nucleoprotein (NP) as one of the most conserved viral antigens. However, NP also undergoes slow evolutionary changes, and little is known about the influence of these mutations on its antigenicity and immunogenicity. Methods. We expressed the full-length recombinant 6xHis-tagged NPs of ten evolutionary distant influenza A strains of different subtypes in E. coli BL21(DE3) cells and purified these proteins by immobilized metal affinity chromatography. The obtained antigens were identified by mass spectrometry and serological methods. NPs served as antigens for three immunizations of BALB/c mice (15 µg/animal at 14-day interval) and as capturing proteins in ELISA at 2 µg/mL, in order to study the effect of adaptive mutations on the antigenic and immunogenic properties of NPs. Results. A pronounced cross-reactivity of anti-NP antibodies induced in mice by immunization with different NPs was revealed. At the same time, we observed the differences in the humoral immunogenicity of NP, which are in line with the accumulation of evolutionarily driven NP mutations. In general, antibody affinity to heterologous NPs was reduced, indicating the differences in the specificity of anti-NP immunoglobulins, which may be caused by evolutionarily determined variability of immunogenic epitopes leading to the emergence of escape mutations. Conclusions. Overall, our results reflect the slightly evolving nature of the NP antigen, which influences the specificity spectrum of anti-NP antibodies and should be considered as a limitation for the development of NP-based cross-protective vaccines and test systems. Full article

Jet-cutting—the most powerful immobilization technique—was utilized for the entrapment of recombinant E. coli cells expressing a cascade of enzymes, including alcohol dehydrogenase, enoate reductase, and cyclohexanone monooxygenase, within mechanically reinforced barium–calcium alginate beads. Cost-effective alginate beads with entrapped cells were applied in a model process for the production of the industrially relevant ε-caprolactone under bioreactor-controlled conditions, enabling parallel repeated biotransformations. Immobilization resulted in a reduced rate of cell deactivation over four biotransformation cycles, leading to overall ε-caprolactone yield increases of 36% using 0.55 mm beads and 22% using 0.9 mm beads compared to the use of free cells. Additionally, the model bioprocess was employed to investigate the metabolic adaptation of cells to immobilization and repeated biotransformations using viability assays and spectrally resolved confocal microscopy. These measurements, conducted for the first time throughout the entire cellular life cycle, clearly demonstrated that the cells retained high viability during cultivation, immobilization, and repeated use in biotransformations. Moreover, based on characteristic spectral shifts, advanced analysis via spectrally resolved confocal microscopy revealed distinct mechanisms of metabolic adaptation in entrapped cells versus free cells during repeated cascade reactions in parallel bioreactors. Full article

The Escherichia genus includes both commensal and pathogenic species and is characterized by its diversity and adaptability to the mammalian gut and other environments. Among these species, E. coli has facilitated many scientific advances as a model organism. Recently, a new member of the Escherichia genus, Escherichia marmotae, has been described as a phylogenetically distinct clade that shows the greatest genetic divergence from E. coli. This review explores E. marmotae, its cryptic evolution, distinct characteristics, and ecological niches. E. marmotae has recently gained scientific prominence due to its association with animal feces, environmental occurrence, human clinical samples, and emerging as a potential pathogen. While its pathogenicity remains understudied, growing evidence from clinical, environmental, and animal sources suggests the need for heightened surveillance. This review highlights current knowledge gaps, underscores the need for improved diagnostic tools, and proposes future research directions to elucidate the clinical and ecological implications of this emerging pathogen. Full article

Long-term adapted cell wall-deficient (L-forms) bacteria show unique cell shapes and growth patterns compared to wild-type bacteria. However, quantitative analysis to assess the morphological heterogeneity of existing L-forms is limited. In this study, we validated that two stable L-form strains of Escherichia coli (NC-7 and LWF+) hold spherical or pleomorphic morphology in confocal and electron microscopy. Using imaging flow cytometry, we further reported that the variations in cell size distribution and cell viability between L-forms and walled cells are statistically significant. Moreover, freeze-fracture electron microscopy observations revealed a clear presence of an outer membrane in NC-7 but not in LWF+, suggesting that E. coli L-form strains could survive in both spheroplastic and protoplastic forms after adaptive evolution. Accordingly, the mutations in genes associated with cell envelope and outer membrane components are more prevalent in the LWF+ genome, potentially leading to outer membrane depletion. Notably, experimental evidence derived from E. coli LWF+ cells exhibiting a monoderm phenotype may support the diderm-to-monoderm transition hypothesis, implying the monoderm phenotype arose from the evolutionary loss of the outer membrane in diderm ancestors. Taken together, our findings offer insights into quantification analysis and the cell envelope status of two E. coli L-forms, facilitating future investigations into genotype-phenotype associations in these two L-form bacteria models. Full article