Efficient and tunable promoters are essential tools for metabolic engineering and synthetic biology in Streptomyces. In this study, the lytic bacteriophage phiSASD1 was exploited as a source of novel regulatory elements. Fifteen candidate promoter fragments were identified through bioinformatic prediction and systematically evaluated using a catechol 2,3-dioxygenase (xylE) reporter system in Streptomyces lividans TK54 and Escherichia coli JM109. Among these candidates, seven fragments exhibited measurable promoter activity, with PSD13 showing the highest transcriptional strength. In S. lividans, PSD13 displayed up to a 9.79-fold higher activity than the widely used strong promoter PermE*, while retaining detectable activity in E. coli, indicating excellent cross-host compatibility. Sequence analysis combined with 5' RACE revealed that PSD13 possesses a typical σ[70]-dependent promoter architecture, with its core functional region located between - 70 to + 9 bp relative to the transcription start site. Furthermore, PSD13 efficiently drove the soluble expression of phage endolysin in E. coli, potentially reducing inclusion body formation compared with conventional T7-based expression systems. Collectively, these results identify PSD13 as a phage-derived promoter exhibiting strong activity in both Streptomyces and E. coli, suggesting its potential as a useful genetic element for Streptomyces engineering and heterologous gene expression. KEY POINTS : • Strong transcriptional promoters were systematically identified from Streptomyces phages. • The novel SD13 promoter shows higher activity than PermE* and works across hosts. • SD13 promoter efficiently expresses soluble heterologous proteins, advancing Streptomyces synthetic biology.

Precision fermentation (PF) uses engineered microbial cell factories to manufacture defined products under controlled bioprocess conditions. In this review, PF is treated operationally as the integration of engineered hosts with metabolic engineering and process control, while traditional food fermentations without engineered production strains are excluded. The field now spans pharmaceuticals, food ingredients, industrial enzymes, specialty chemicals, and biopolymers, but commercial translation remains constrained by biological non-linearity, substrate and feedstock variability, mass-transfer limitations at scale, downstream recovery, and the cost gap with incumbent processes. This mini-review examines five connected layers: engineered microbial chassis; design build test learn (DBTL) and multi-omics-guided metabolic engineering; bioreactor intensification; Process Analytical Technology (PAT), soft sensors and AI-enabled control; and techno-economic and life cycle assessment. Quantitative case studies illustrate both the progress and the remaining scale-up gaps, including semi-synthetic artemisinin, recombinant insulin, lactic acid, succinic acid, QS-21, and polyhydroxyalkanoates. The central argument is that the next phase of PF will depend less on isolated improvements in strain performance than on coordinated integration of strain design, intensified operation, real-time process understanding, and rigorous economic and environmental assessment. This integration is essential for translating laboratory performance into reproducible, scalable, and commercially credible biomanufacturing. KEY POINTS: PF requires integration of cell-factory engineering with scalable bioprocessing rather than optimisation of any single layer. DBTL and multi-omics improve strain performance, but laboratory titres do not guarantee industrial scalability. Bioprocess intensification and real-time monitoring are required to convert strain performance into robust production. PAT, soft sensors, and AI strengthen process control when supported by reliable online data and qualified models. TEA and LCA should be integrated early to filter commercial and environmental feasibility before scale-up.

This study demonstrates the considerable antagonistic activity of Aspergillus terreus MAK153 against a pathogenic Fusarium oxysporum AUMC3191 isolate. In dual culture assay, soil-isolated Aspergillus terreus Thom effectively hindered F. oxysporum AUMC3191 growth and impressively blocked zearalenone (ZEN) synthesis. Scanning electron microscopy demonstrated significant damage to F. oxysporum hyphae, such as abnormalities, shrinkage, bending, and deformity. Molecular identification through rDNA ITS sequence analysis confirmed the isolate as Aspergillus terreus, registered in GeneBank (Accession No. OR480103). GC/MS profiling of A. terreus MAK153 identified several bioactive secondary metabolites, notably estragole, caryophyllene, 2,4-di-tert-butylphenol, 1-docosene, 1-docosanol, olean-12-ene-3,28-diol, and simvastatin. Statistical analysis revealed a very strong positive correlation (r = 0.99) between chromatographic peak area% and peak area values. The influence of nutrient sources on simvastatin production was evaluated using four culture media. FTIR and HPLC analyses confirmed simvastatin biosynthesis, while HPLC revealed significant medium-dependent variation in yield (p < 0.001). Camellia sinensis Tea broth (CTB) elicited the highest yield, dominating extracellular filtrate accumulation (145.93 ± 1.13 mg/mL). Molecular docking simulations assessed simvastatin's binding affinity to HMG-CoA reductase and 3-ketosteroid reductase, yielding binding energies of -8.0 and -7.8 kcal/mol, respectively. The interactions involved multiple hydrophobic bonds and hydrogen bonds, suggesting simvastatin functions as a multi-target inhibitor capable of disrupting ergosterol biosynthesis at two distinct sites. Notably, this study represents the first computational exploration of simvastatin's interaction with 3-ketosteroid reductase, with implications for estrogen signalling modulation and potential therapeutic applications. KEY POINTS: • Aspergillus terreus MAK153 inhibits F. oxysporum growth and zearalenone production • Aspergillus terreus induces significant hyphal damage in F. oxysporum using SEM • Simvastatin-HMG-CoA and 3-ketosteroid interaction could modulate estrogen-related pathways potentially.

This study evaluates how non-conventional anaerobic microbial consortia from sanitary landfill leachate and mining wastewater respond to selected winery wastewater-associated inhibitors, including alcohols, carboxylic acids, and phenolic compounds. Batch assays were conducted in nutrient broth acetate-fumarate medium under single and combined inhibitory conditions. The enriched consortia were characterized by full-length 16S rRNA gene sequencing. Both consortia exhibited comparable growth rates under control conditions, but showed distinct growth dynamics, fumarate consumption, succinate and acetate profiles, and inhibitor consumption patterns. The landfill-derived consortium was dominated by Escherichia and displayed faster fumarate consumption under control and alcohol-supplemented conditions, indicating higher baseline substrate turnover. In contrast, the mining-derived consortium showed a more distributed taxonomic profile, dominated by Proteus, Wolinella, and Escherichia, and maintained fumarate consumption more consistently under carboxylic acid and phenolic stress. Alcohols had limited effects on growth and fumarate consumption, whereas carboxylic acids significantly reduced growth and altered metabolite profiles. Phenolic compounds exerted a strong inhibitory effect, particularly at high concentrations. Catechol and tyrosol showed only minor concentration changes, consistent with low apparent consumption under the tested anaerobic conditions. Under combined inhibitory conditions, both consortia maintained fumarate consumption and metabolite formation despite reduced growth, with the mining-derived consortium showing increased fumarate consumption and enhanced succinate production. Microbial source, enrichment history, community composition, and inhibitory conditions jointly shaped the resilience and metabolic performance of the anaerobic consortia. This work supports screening non-conventional microbial consortia for future anaerobic and bioelectrochemical treatment of chemically complex agro-industrial wastewater. KEY POINTS: • Non-conventional anaerobic consortia maintained activity under inhibition • Taxonomic composition was associated with distinct metabolic profiles • Phenolics caused strong inhibition and low apparent anaerobic consumption.

Blowflies of the genus Lucilia (Insecta: Diptera: Calliphoridae) are important in forensic science, medical research, and agriculture, particularly due to their role as facultative ectoparasites of domesticated sheep. The Australian sheep blowfly, L. cuprina dorsalis, is a primary causative agent of cutaneous myiasis, leading to substantial economic losses to the wool industry. In contrast, the closely related subspecies L. cuprina cuprina does not induce myiasis in sheep, but functions as a necrophagous subspecies in urban regions of Australia. Differences in the physiology, behavior, and host interactions of these subspecies might be attributed to their bacterial community structures as demonstrated in other blowfly species. However, our understanding of the bacterial communities associated with these flies is limited. This study aimed to characterize the microbiomes of two geographically distinct subspecies of L. cuprina-L. c. dorsalis (rural) and L. c. cuprina (urban) using 16S rRNA gene sequencing. The findings indicate that, while both subspecies harbor broadly similar microbial compositions, their respective urban and rural environments influence the prevalence of specific bacterial genera. The bacterial communities of these flies were predominantly composed of members of the phyla Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. The genus Ignatzschineria was highly abundant in L. c. dorsalis, whereas Pseudomonas was more prevalent in L. c. cuprina, highlighting distinct microbial compositions between the two subspecies. These findings provide fundamental insights into the microbial composition of Lucilia species, contributing to a broader understanding of their ecological and biological roles, with potential applications in forensic entomology, medical research, and agricultural pest management. KEY POINTS: • The microbial communities of Lucilia cuprina dorsalis and Lucilia cuprina cuprina have a broadly similar composition, but the abundance of particular bacterial genera is notably different. • Proteobacteria is the most abundant phylum in both subspecies. • The genus Ignatzschineria is notably abundant in Lucilia cuprina dorsalis, while Pseudomonas is predominant in Lucilia cuprina cuprina, emphasizing differences between the microbial community members between these subspecies.

Tyrosine kinase inhibitors (TKIs) remain a major focus of targeted therapeutic discovery, yet current discovery pipelines are constrained by resistance development, limited scaffold diversity, and high attrition rates. Entomopathogenic fungi (EPF) represent an underexplored microbial resource with significant biosynthetic potential for generating kinase-relevant secondary metabolites. Recent genomic studies reveal that EPF encode diverse and largely cryptic biosynthetic gene clusters (BGCs), including polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), and hybrid pathways capable of producing structurally complex bioactive compounds. This mini-review examines the biotechnological exploitation of EPF for TKI discovery, focusing on fungal metabolites with direct or indirect kinase-related activity, genome mining strategies, biosynthetic logic, pathway regulation, and engineering approaches for metabolite production. Although only a limited number of EPF-derived metabolites have been experimentally validated as direct tyrosine kinase inhibitors, accumulating evidence indicates that many modulate kinase-associated signalling pathways and represent promising starting points for kinase-oriented natural product discovery. Particular emphasis is placed on pathway activation, heterologous expression, metabolic engineering, and scalable bioprocess development. Key translational challenges, including selectivity, toxicity, yield instability, and regulatory considerations, are also discussed. Collectively, this review highlights EPF as promising microbial platforms for kinase-oriented natural product discovery and provides a biotechnology-focused framework for advancing fungal secondary metabolites toward translational development. KEY POINTS: • Entomopathogenic fungi encode diverse kinase-active secondary metabolites. • Genome mining and synthetic biology unlock cryptic TKI pathways. • Biotechnological optimization is critical for scalable fungal TKI discovery.

Among the emerging cancer therapies, immunotherapy using T cell engineering stands out as one of the most promising approaches. While the clustered regularly interspaced palindromic repeats (CRISPR/Cas) system is widely recognized as one of the most common gene-editing system tools, the main challenge for in vivo delivery applications remains the lack of an efficient and safe intracellular carrier. Small extracellular vesicles (s-EVs), as natural nanoparticles with low immunogenicity, emerge as a promising candidate for delivering the CRISPR/Cas system. Two guide RNAs (gRNAs) targeting the programmed cell death (PD-1) gene were cloned into the PX-459 vector, and these CRISPR/Cas9 plasmids were loaded into targeted s-EVs by electroporation. Subsequently, T cells were exposed to these s-EVs, and the disruption to the PD-1 gene was examined using flow cytometry and sequencing. Flow cytometry results indicated that newly produced targeted s-EVs exhibited a higher uptake by T cells compared to non-targeted s-EVs. The successful loading of PX-459 plasmids into targeted s-EVs and their functional delivery to T cells were confirmed. Subsequent carboxyfluorescein succinimidyl ester (CFSE) staining and enzyme-linked immunosorbent assay (ELISA) analyses demonstrated that the knockout (KO) of the PD-1 gene in T cells significantly enhanced both their cytotoxicity and cytokine release compared to untreated T cells. Targeted s-EVs were used in the current study as a means to deliver the CRISPR/Cas9 system into T cells, enabling genetic modification of target cells. This approach holds the potential to improve and simplify immunotherapy. This study demonstrates a novel approach to genetic modification in T cells using targeted s-EVs containing the CRISPR/Cas9 gene-editing system. KEY POINTS: •CRISPR/Cas9 is a kind of powerful gene editing system in cancer therapeutics. •s-EVs can be applied as a biological carrier for CRISPR/Cas9 system. • T cell engineering-based immunotherapy is a promising form of anticancer therapy.

Extremozymes, with their exceptional catalytic activity and stability under extreme environmental conditions, have great potential for pesticide remediation. This study involves the isolation and screening of a new extremozyme-producing microbe, Proteus mirabilis JB1, for the production of alkaline-pH-stable laccase. A primary OFAT screening tested a broad range of carbon sources (glucose, lactose, xylose, sucrose, starch, glycerol, cellulose, trisodium citrate, sodium acetate), nitrogen sources (ammonium sulfate, sodium nitrate, urea, beef extract, peptone, casein, gelatin), salts (0.1 mM and 0.01 mM) such as CuSO4, KCl, ZnCl2, MgCl2, MnCl2, NiCl2, NaCl, and CaCl2, and pH levels (7-10). The highest enzyme activity of 0.999 U/ml was achieved using 0.25% (w/v) cellulose, 0.8% (w/v) urea, 0.1 mM MnCl2, MgSO4, CaCl2, NaCl, and KCl, and 0.01 mM CuSO4, FeSO4, and NiCl2 at pH 9 with good stability to surfactants (Tween 80, SDS), organic solvents (acetone, ethanol, methanol), and inhibitors (EDTA). PBD and CCD were then implemented to screen and optimize critical parameters-cellulose (1.5% w/v), urea (0.7% w/v), and MgSO4 (0.05 mM)-to achieve an efficient increase in enzyme activity to 2.3 U/mL, demonstrating the strength and efficacy of the optimization process. Biogenic selenium nanoparticles (Bio-SeNPs) were fabricated and exhibited very high efficiency in pesticide degradation, achieving 100% degradation of cypermethrin without forming any toxic intermediates within 72 h. This research has provided a comprehensive and replicable technique covering microbial isolation, enzyme optimization, fabrication of a nanobiocatalyst, and its application. KEY POINTS: • A novel isolate, Proteus mirabilis, was employed for the production of the extremozyme laccase. • Optimization increased the yield by 9.58-fold. • Bio-SeNPs were employed to degrade cypermethrin, achieving 100% detoxification within 72 h.

Cold atmospheric plasma (CAP) is emerging as an environmentally friendly disinfection technology, but most plasma-liquid systems remain confined to millilitre‑scale volumes, limiting their applicability as bioprocesses for water treatment. Here, we designed and evaluated a bubble‑enhanced dielectric barrier discharge reactor, the Spiral Plasma Aeration Reactor Column (SPARC), as an environmental technology approach for litre‑scale inactivation of model bacteria in water. Direct CAP exposure achieved rapid, near‑complete inactivation of Escherichia coli and Bacillus subtilis in 200 µL, but performance decreased sharply at 100 mL, confirming strong volume dependence under surface‑limited plasma-liquid contact. Coupling CAP with gas bubbling (2 L·min-1) in SPARC markedly enhanced inactivation at 250-500 mL, achieving approximately 2-3 log reductions (≥ 99-99.9%) for E. coli and B. subtilis within 60 min, whereas non-aerated CAP remained below ≈0.8-log (≤ 60%) reduction under identical conditions. At 1 L under high initial loads (~ 106-107 CFU·100 mL-1), bubble-assisted CAP yielded ~ 4-log inactivation of E. coli and 3-3.5-log reduction of B. subtilis, including continued post-treatment inactivation consistent with plasma-activated water effects. In a protein‑rich organic matrix, early‑stage inactivation was attenuated, but SPARC still achieved ≈2-3‑log reductions, indicating robust yet matrix‑sensitive performance under wastewater‑like conditions. At fixed electrical input (34.5 W), specific energy consumption ranged from 60 kWh·m-3 (250 mL, 30 min, > 99% E. coli inactivation) to 30 kWh·m-3 (1 L, 60 min, ≈4‑log E. coli inactivation), corresponding to ≈7.5 kWh·log₁₀-1·m-3. These results position bubble‑assisted CAP as a promising environmental biotechnology and bioprocess platform for water disinfection and emphasise the central role of reactor and contactor design in determining microbial efficacy and energy demand. KEY POINTS: • Direct CAP exposure in microlitre-scale water (200 µL) produces rapid ≥ 3-log₁₀ (> 99.9%) inactivation of E. coli and B. subtilis, but performance deteriorates at 100 mL, confirming strong volume dependence under surface-limited plasma-liquid contact. • The bubble-enhanced DBD reactor SPARC, operated at 250-500 mL, achieves ≈2-3-log₁₀ (≥ 99-99.9%) bacterial reductions within 60 min, whereas non-aerated CAP under identical electrical conditions generally remains below ≈0.8-log₁₀ (≤ 60%) reduction. • At 1 L, bubble-assisted SPARC attains ~ 4-log₁₀ (E. coli) and 3-3.5-log₁₀ (B. subtilis) inactivation under high initial loads, with specific energy consumption between 30 and 60 kWh·m-3 (≈7.5 kWh·log₁₀-1·m-3) for the laboratory prototype.

Foot-and-mouth disease virus (FMDV) exhibits greater temperature sensitivity compared to many other picornaviruses, which poses a substantial challenge for the stability of inactivated vaccines. Therefore, enhancing the thermal stability of the FMDV capsid is crucial. FMDV exists as a quasispecies with high mutation rates, providing a basis for selection under pressure. Our group previously discovered that heat shock protein HSP60 was recruited to support FMDV replication by promoting the assembly of the viral replication complex (RC). By applying the HSP60 inhibitor mizoribine, we selected FMDV variants with enhanced fitness under conditions of impaired HSP60 function. Subsequent sequencing of the selected populations and reverse genetics approaches identified a key amino acid substitution, T171P in the viral VP1 protein that confers resistance to the HSP60 inhibitor. Structural simulation predicted that the T171P substitution enhances the stability of the VP1 protein, which was further supported by experimental evidence demonstrating increased thermal stability of the whole viral capsid. A series of experiments, including virion stability assessment using the PaSTRy assay, quantification of intact particles following heat challenge, and evaluation of viral infectivity at elevated temperatures, demonstrated that the T171P-VP1 variant possesses enhanced thermostability compared to the wild-type (WT) virus. Furthermore, immunization experiments showed that the inactivated T171P-VP1 variant, after heat challenge, elicited a stronger neutralizing antibody response than the similarly treated WT virus. This study identifies a novel amino acid substitution that significantly enhances FMDV thermostability. This finding not only provides a viable strategy for improving the thermal stability of FMD vaccines but also opens new avenues for exploring the intricate mechanisms of host-virus interaction. KEY POINTS: • A novel HSP60 inhibitor-based selection strategy identified the T171P-VP1 substitution conferring enhanced viral fitness. • The T171P substitution significantly improves FMDV capsid thermostability compared to the wild-type virus. • The inactivated T171P-VP1 variant induces stronger neutralizing antibody responses after heat challenge, offering a promising approach for thermostable FMD vaccine development.

A wide range of microbial prevention and control strategies are applied along the agri-food chain to combat the transmission of foodborne pathogens to the final consumer. These strategies range from traditional thermal treatments to emerging technologies as well as the use of food chain biocides to reduce microbial loads on raw materials and food contact surfaces, and preservatives for the preservation of feed and food products. However, bacteria have demonstrated a remarkable ability to adapt to and overcome these food-related stresses, often resulting in reduced susceptibility to other seemingly unrelated agents through cross-protection mechanisms. In this study, we aimed to evaluate the outcomes of ciprofloxacin (CIP) antimicrobial resistance (AMR) in Salmonella Typhimurium and its potential relationship with cross-protection to food-chain biocides and preservatives, as well as the ability of the resistant variants (RVs) to retain their virulence. The CIP-RVs showed heterogeneous and compound-dependent responses to the tested food-chain antimicrobial compounds. All five RVs displayed increased tolerance to at least one compound, but cross-protection was not uniform across strains or treatments. Notably, SeTRV1 displayed reduced susceptibility to four of the six agents tested, and derivative strains carrying SeTRV1-derived alleles supported an important contribution of ramR, cyaA, and especially rpoD to this broad phenotype. Moreover, several RVs displayed Galleria mellonella pathogenicity comparable to that of the parental strain, despite occasional reductions or dose-dependent effects, suggesting that reduced susceptibility to food-chain antimicrobial compounds may occur without a major loss of virulence-associated traits in this model. KEY POINTS: • CIP-resistant S. Typhimurium variants can show reduced chemical susceptibility • Cross-protection is associated with global regulation and stress response mutations • CIP-RVs showed divergent pathogenicity patterns in the Galleria model.

Salmonella enterica bacteriophages are increasingly studied for bacterial biocontrol and applied microbiology. However, reliable downstream characterization depends strongly on how the phage material is purified and concentrated. This study compared five preparation strategies for a Salmonella bacteriophage: sucrose density gradient centrifugation, cesium chloride gradient centrifugation, ultrafiltration followed by lyophilization, ultrafiltration followed by ammonium sulfate precipitation, and ultrafiltration followed by trichloroacetic acid precipitation. The preparations were assessed for infectious phage recovery, total protein concentration, SDS-PAGE protein profiles, an exploratory molecular weight-based comparison with a reference phage dataset, and targeted real-time PCR screening for selected virulence-associated determinants. Ultrafiltration followed by ammonium sulfate precipitation gave the highest infectious phage recovery among the infectivity-preserving workflows and produced a consistent eight-band SDS-PAGE profile. Trichloroacetic acid precipitation produced a similar banding pattern but was considered mainly a protein precipitation method because of its denaturing effect. Cesium chloride gradient centrifugation gave the most complex SDS-PAGE profile, while ultrafiltration followed by lyophilization gave the least resolved one. Several bands fell within the molecular-mass ranges reported for proteins of related Salmonella phages, but matching by molecular mass alone cannot identify the proteins, which would require LC-MS/MS. Targeted real-time PCR did not detect sopE, sspH1, sseI, or sopE2. Under the conditions tested, ultrafiltration followed by ammonium sulfate precipitation gave the highest infectious recovery and a consistent SDS-PAGE pattern; these results describe how the workflows behaved here rather than showing that one method is generally superior. KEY POINTS: • Five workflows were compared for Salmonella phage purification. • Ammonium sulfate precipitation gave the highest infectious recovery. • Selected virulence-associated genes were not detected by qPCR.

Pulmonary hypertension (PH) is a multifactorial, progressive disease characterized by pulmonary vascular remodeling and right heart failure, representing a substantial global health burden. Dysregulated interactions among multiple cell types within the pulmonary vascular microenvironment, particularly macrophage polarization imbalance with a predominant transition toward the M2 phenotype, play a critical role in driving pro-fibrotic and pro-remodeling processes in PH. Lactylation, a novel post-translational modification that covalently links lactate to lysine residues, translates glycolytic metabolic signals into persistent epigenetic changes. Recent advances have identified lactylation on histones (e.g., H3K18la), non-histone proteins, and mitochondrial proteins, revealing new layers of regulatory complexity. Elucidating the role of lactylation, particularly its impact on macrophage polarization and vascular cell function, may offer novel insights into the pathogenesis of PH and identify potential therapeutic targets. KEY POINTS: • Macrophage polarization imbalance (predominant M2 phenotype) is a critical driver of pulmonary vascular remodeling in pulmonary hypertension (PH), and lactylation serves as a novel metabolic-epigenetic bridge linking macrophage metabolic reprogramming and phenotypic switching. • Lactylation modifications occur extensively on histones, non-histone proteins, and mitochondrial proteins in PH, regulating fibrosis-related pathways, arginine metabolism signaling, and the dysfunction of pulmonary vascular cells. • Targeting lactylation and lactate metabolism represents a promising therapeutic strategy for PH, supported by our previous studies on M2 macrophage polarization and ongoing lactylomic research.

Cefiderocol is a newly developed siderophore-conjugated cephalosporin specifically designed to overcome multidrug-resistant (MDR) Gram-negative bacilli. Nevertheless, reduced susceptibility and resistance to cefiderocol has increasingly been reported, especially derived from alterations in iron transport systems and plasmid-mediated determinants and the expression of structural variants of β-lactamases. Moreover, cefiderocol resistance derived from target alteration remains insufficiently defined. This work aimed to characterize the genomic and structural basis of cefiderocol resistance emerging in vivo during therapy in Klebsiella pneumoniae. Comparative genomics revealed conserved plasmid architecture and no acquisition of novel β-lactamases or iron-uptake determinants. Eighty-two chromosomal variants were identified, among which a missense substitution in ftsI (FtsI G306V), located adjacent to the catalytic serine (S307) of PBP3, emerged as a plausible resistance determinant. Structural modelling suggests that the G306V substitution introduces a significant side-chain volumetric increase, resulting in locally increased steric hindrance within the active-site, possibly impairing cefiderocol interaction with its target. The absence of canonical iron-transport alterations further distinguishes this case from the predominant mechanism described in global molecular epidemiology studies. Large-scale genomic screening revealed a very low prevalence of substitutions affecting ftsI, including in resistance isolates, although possible clonal expansion cannot be ruled out. Our findings align with the broader epidemiological landscape in which cefiderocol resistance is heterogeneous and flexible. Therefore, incorporating analysis of ftsI variants within or proximal to the active-site into genomic surveillance frameworks could improve resistance detection to this last-resort antibiotic. KEY POINTS: • Resistance occurred without alterations in iron-uptake systems or siderophore pathways. • Mutations in FtsI (G306V) and AcrR (E91K) were identified as key candidates. • FtsI G306V is rare across cefiderocol resistant and global K. pneumoniae genomes.

The escalating threat of multidrug-resistant (MDR) bacterial pathogens underscores the urgent need for novel antimicrobial agents. Here, we combined a one-strain many compounds (OSMAC) and untargeted metabolomics approaches to investigate the bioactivities and the chemical space of the endophytic fungus Alternaria alternata P02PL2, isolated from a medicinal plant, Sclerocarya birrea. The antibacterial activities were assessed against contemporary clinical isolates, MDR Pseudomonas aeruginosa 5625574, methicillin-resistant Staphylococcus aureus (MRSA) 5627679, and a collection culture of S. aureus ATCC 25923 for reference. We first tested for bioactivities across eight growth media to optimize the production of bioactive secondary metabolites. The bioactive fungal extract fractions exhibited minimum inhibitory concentrations (MICs) ranging from 0.25 to 0.5 mg/mL. This was followed by an untargeted metabolomics analysis, which identified 41 secondary metabolites. Virtual screening, molecular docking, and 200-ns molecular dynamics simulations targeting S. aureus and P. aeruginosa DNA gyrase revealed lead compounds with superior binding affinities (e.g., fluorescein -9.2 kcal/mol for P. aeruginosa; pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro -8.8 kcal/mol for S. aureus) compared to reference ligands. Binding free energy calculations highlighted strong affinities, with 5,7-dihydroxy-2-(4-hydroxyphenyl)-6,8-bis(3,4,5-trihydroxyoxan-2-yl)chromen-4-one (-42.16 kcal/mol) and fluorescein (-41.42 kcal/mol) outperforming the P. aeruginosa reference ligand evo (-19.80 kcal/mol) and 80s (-48.74 kcal/mol) leading for S. aureus. Per-residue energy decomposition and quantum chemical analyses further elucidated key interactions and reactivity profiles. These results position A. alternata P02PL2 as a promising source of DNA gyrase inhibitors to combat antimicrobial resistance. KEY POINTS: • A. alternata extracts inhibits S. aureus and Pseudomonas with MIC of 0.25-0.5 mg/mL • Inhibitors included Diketopiperazine (S. aureus), C-glycosyl flavone (Pseudomonas) • In silico analyses suggest the metabolites putatively inhibit bacterial DNA gyrase.