Pseudomonas aeruginosa is an important opportunistic pathogen in urinary tract infections (UTIs) and microbial keratitis (MK), but differences in resistance and virulence between exoU and exoS P. aeruginosa isolates across regions and infection sites remain poorly understood. This study compared antibiotic resistance and virulence profiles of exoU and exoS P. aeruginosa isolates from Bangladeshi and Indian UTIs, and from Indian UTIs and MK collections. A total of 23 Bangladeshi UTI isolates were analyzed by broth microdilution for susceptibility to ciprofloxacin, levofloxacin, gentamicin, and tobramycin, and by whole-genome sequencing (WGS) to identify resistance-associated mutations, acquired resistance genes, and virulence factors. These WGS data were compared with 15 publicly available Indian UTI genomes, while the Indian UTI genomes were compared with 19 publicly available Indian MK genomes. In the Bangladeshi UTI cohort, exoU isolates showed greater resistance to fluoroquinolones and aminoglycosides than exoS isolates. Comparative analysis of Bangladeshi versus Indian UTI isolates and Indian UTI versus MK isolates showed that exoU isolates carried a burden of resistance mutations, including enrichment of GyrA Thr83Ile, ParE Asp533Glu, MexR Val126Glu, MexB Ser1041Glu, and MexC Ala262Glu. Aminoglycoside-associated substitutions FusA2 Gln161Leu and ParR Leu153Arg and Ser170Asn were more common in exoU UTI isolates. Acquired resistance genes showed aac(6')-II and aac(3)-Id were linked to Bangladeshi UTI, rmtF associated with Indian UTI, and crpP confined to MK exoU isolates. Virulence profiling showed exoU enrichment in pldA, pvdY, and csrA, whereas exoS was associated with pilC, pilV, pilW, pilY2, and pilA. Collectively, these results indicate that exoU and exoS isolates differ in both their resistance and virulence profiles, while also showing evidence of geographic and infection-site-specific adaptation.

Pseudomonas aeruginosa is a prominent biofilm-forming pathogen associated with chronic infections that are difficult to eradicate due to the protective biofilm matrix. Inducing biofilm dispersion has emerged as a potential therapeutic strategy. Although researchers have compared selected biofilm dispersion mechanisms, the characteristics of cells generated by mechanistically distinct release strategies, particularly enzymatic matrix degradation, remain uncharacterized. In this study, we compared P. aeruginosa cells released from biofilms using four distinct dispersion mechanisms: An external signaling molecule (cis-2-decenoic acid; cis-DA), a nutrient cue (glutamate), an environmental toxin (sodium nitroprusside; SNP), and a matrix-degrading enzyme (amylase). Released cells exhibited distinct antibiotic susceptibility, attachment, and gene expression depending on the release mechanism. Glutamate-released cells exhibited greater tobramycin tolerance and attachment, whereas glutamate and amylase-released populations exhibited differences in the expression of selected genes. Additionally, the release mechanism influenced subsequent biofilm recovery and reformation, and cis-DA significantly enhanced tobramycin-mediated killing of mature biofilms. Collectively, these findings demonstrate that the mechanism used to induce release may influence subsequent bacterial behavior and antibiotic susceptibility. These findings highlight the importance of considering release mechanism when developing dispersion-based strategies for treating biofilm-associated infections.

Acinetobacter baumannii has traditionally been considered a nosocomial pathogen, but cases of community-acquired A. baumannii pneumonia have increasingly been reported in tropical Asia, including Vietnam. Data on its clinical characteristics, antimicrobial resistance, and patient outcomes remain limited. This study aimed to describe the clinical profile, resistance patterns, and outcomes of community-acquired A. baumannii pneumonia in southern Vietnam. We performed a single-center retrospective cohort study at Nhan Dan Gia Dinh Hospital, a 1500-bed tertiary center in Ho Chi Minh City (January 2021-December 2023). Adults (≥18 years) with community-acquired pneumonia and a positive sputum, endotracheal aspirate, or blood culture for A. baumannii obtained within 48 hours of admission were included. Patients with cultures >48 hours after admission, hospitalization in the prior 90 days, residence in/transfer from another facility ≥48 hours preadmission, polymicrobial cultures, or incomplete records were excluded. Thirty-one patients were identified (median age 69.0 years (interquartile range 64.0-84.0); 83.9% male; 83.9% current/former smokers). No clear seasonal predominance was observed within the study window after month-level normalization. On admission, 87.1% had hypoxemia (peripheral oxygen saturation<90%) and nearly half (45.2%) were severe community-acquired pneumonia. Antibiotic escalation was required in 61.3% after a median of 4 days (interquartile range 3-5). Resistance was approximately 69%-83% across the routinely tested β-lactams and fluoroquinolones, whereas aminoglycosides showed comparatively higher in vitro activity than the other tested antibiotic classes but susceptibility remained suboptimal (amikacin 50%, tobramycin 46%, gentamicin 36%). Initial empiric therapy was active against the index isolate in 4/25 patients (16.0%) with available susceptibility results. In-hospital mortality was 13/31 (41.9%). Community-acquired A. baumannii pneumonia is associated with severe disease, extensive antimicrobial resistance, frequent empiric treatment mismatch, and high mortality. In resource-constrained settings, limited therapeutic options highlight the importance of early microbiological diagnosis, comprehensive susceptibility testing, and locally informed treatment strategies.

CO2-responsive wormlike micelles are smart viscoelastic fluids with broad potential in oilfield stimulation and related fields. However, CO2-switchable systems constructed via the pseudo-surfactant strategy have so far been based mainly on flexible aliphatic polyamines, and the use of aminoglycoside antibiotics as multi-amine building blocks remains unexplored. Herein, we report the first construction of an aminoglycoside-based CO2-responsive pseudo-pentameric surfactant by simply mixing tobramycin-bearing five primary amine groups, a cyclohexose skeleton, and abundant hydroxyl groups-with stearic acid at a 1 : 5 molar ratio in aqueous solution. The resulting system forms highly viscoelastic wormlike micelles with a maximum zero-shear viscosity of ∼3200 mPa s. The system exhibits a lower overlap concentration (c* ≈ 75-85 mM) than the previously reported SA/cyclen pseudo-tetrameric system (c* ≈ 80-100 mM), which we attribute to the combined structural features of tobramycin-including its cyclohexose skeleton, abundant hydroxyl groups, and five primary amines. Moreover, antibacterial assays demonstrate that the wormlike architecture effectively shields tobramycin's activity, while CO2-triggered disassembly restores its full efficacy against Staphylococcus aureus. This work expands CO2-responsive wormlike micelles to aminoglycoside-based pseudo-surfactants and provides a dual-function smart fluid platform that integrates CO2-switchable viscoelasticity with on-demand antimicrobial release, opening avenues for applications ranging from wound dressings and antimicrobial coatings to oilfield biocides.

Despite the onset of highly effective modulator therapies people with Cystic Fibrosis continue to experience recurrent microbial lung infections. Many of these individuals will have at least one positive culture per year for Pseudomonas aeruginosa , a bacterium that readily adapts to chronic CF airway disease. Of these adaptations, the formation of a protective biofilm and changes in motility are hallmarks of established infections. We have shown previous evidence that glycerol metabolism and the P. aeruginosa glycerol regulon repressor, GlpR, is linked to enhanced biofilm production and reduced susceptibility to tobramycin. In the current study, we report that loss of GlpR contributes to higher viscosity and elasticity in synthetic cystic fibrosis sputum media. Further, we show that the loss of the glycerol repressor GlpR, or growth on glycerol, both resulting in derepression of the GlpR/glycerol regulon, cause decreased motility in both acute and chronic CF-adapted lab strains. RNA sequencing analysis indicated that loss of GlpR altered the expression of genes involved in motility, iron scavenging, transport, metabolism, and virulence. An in silico search of P. aeruginosa's genome using GlpR's previously determined binding consensus site identified potential bindings sites in genes related to biofilm development, motility, antibiotic resistance, and metabolism, and these binding sites were confirmed using chromatin immunoprecipitation sequencing. Collectively, our results indicate that GlpR regulates P. aeruginosa phenotypes that facilitate persistence in the CF airway and we provide evidence that GlpR regulates genes outside of the canonical glp regulon.

Therapeutic antibodies represent clinically validated binding interfaces that remain largely unexplored as sources of targeting peptides. Here, we employed a structure-guided approach to extract and optimize an EGFR-binding motif from the anti-EGFR monoclonal antibody necitumumab and transformed it into a peptide-drug conjugate for targeted treatment of EGFR-overexpressed non-small-cell lung cancer (NSCLC). Although necitumumab is an FDA-approved therapeutic for EGFR-positive NSCLC, its clinical effectiveness is constrained by limited antitumor efficacy, inadequate penetration into solid tumors, the development of resistance, and clinically significant on-target toxicities. Like other monoclonal antibodies, necitumumab is a large protein biomolecule whose receptor-binding activity is mediated by a relatively small structural interface, providing an opportunity to re-engineer this functional region into a minimal peptide scaffold. We hypothesized that structure-guided extraction of antibody paratopes could provide a rational route for peptide ligand discovery, retaining receptor specificity while improving tumor penetration and eliminating Fc-driven toxicity. Using the resolved necitumumab Fab-EGFR co-crystal structure, we extracted the primary receptor-contact segment from the antibody paratope by implementing protein-protein interaction analysis and re-engineered it through systematic in silico mutation into an optimized peptide scaffold using MD and MM/GBSA combination protocol. A novel peptide scaffold PE3 (YIYYSFSQDYWP) was identified as the most advantageous carrier, showing favorable affinity toward EGFR (∆G = -66.12 kcal/mol) and subsequently validated to exhibit potent cellular uptake. To evaluate its therapeutic potential, PE3 was converted into a peptide-drug conjugate (PDC) through conjugation with Ahx-DA1, a metabolically stable analogue of dolastatin-10, yielding the conjugate PE3-DA1with IC50 0.05 ± 0.01 μM against H1975 NSCLC cell line. PE3-DA1 retained potent and selective antiproliferative activity against EGFR-positive cells. In the NSCLC xenograft model, PE3-DA1 induced complete tumor regression without evidence of off-target toxicity. This study demonstrates for the first time that a defined EGFR-binding interface within the necitumumab antibody can be reduced to an optimized peptide scaffold while preserving its targeting function and subsequently exploited as a PDC carrier to achieve selective antitumor activity against EGFR-overexpressing NSCLC.

The global escalation of multidrug-resistant tuberculosis (MDR-TB) demands identification of novel therapeutic targets. Here, we establish Vitamin B12-independent methionine synthase E 'MetE' (Rv1133c) as a crucial drug target and validate tobramycin as its inhibitor. Through a multi-faceted approach, including virtual screening and MD simulations, we identified interactions between the tobramycin-MetE complex, which were experimentally validated. Through Isothermal calorimetry we found tobramycin binds to MetE with high affinity (Ka⁓10[-8]M). Subsequent biophysical characterization revealed an interesting mechanism of action: tobramycin binding induces conformational perturbations of the MetE enzyme, as confirmed by changes in intrinsic fluorescence, denaturation patterns and circular dichroism. Under In-vitro conditions tobramycin demonstrated potent anti-mycobacterial activity at concentrations nontoxic to human macrophage cells. We hypothesize that this inhibition involves classical 30S ribosomal binding and MetE disruption. Most significantly, we discovered a powerful synergistic effect between tobramycin and the first-line anti-TB drug, rifampicin. Together, these findings establish a robust preclinical foundation for repurposing tobramycin against management of tuberculosis.

Simultaneously recognizing multiple targets and independently decoding their signals in food is critically demanded for safeguarding public health against antimicrobial resistance. Photoelectrochemical (PEC) sensors, promising for on-site detection, face a fundamental constraint: all targets generate unidimensional current signals at the electrode, rendering multiple target signals prone to overlap and difficult to distinguish. To address this issue, we developed a magnetic-bead-assisted dual-target distinguishable Y-shaped probe (MDDYP) based on orthogonal stimuli-responsive triggers, enabling independent detection of tobramycin (TOB) and tetracycline (TET) on a PEC sensor. Three DNA strands are assembled into a Y-shaped nanostructure bearing hydroquinone (HQ)-encapsulated liposomes, in which the PC-linker and C-rich sequence function as orthogonal stimulus-responsive modules, which are selectively activated after TOB or TET recognition events, respectively. Either UV-induced photocleavage at the PC-linker or acidification-induced i-motif formation at the C-rich sequence drives target-concentration-dependent liposome release into the supernatant, achieving solution-phase signal separation without electrode surface modification. HQ liberated from the lysed liposomes is oxidized at the ITO/ZIF-67/Fe3O4/Fe-COFBTC photoanode, altering the electron transfer pathway and reversing the photocurrent polarity as the quantitative readout. The platform demonstrates a wide linear range of 1 fM-100 nM for TOB and 10 fM-100 nM for TET, with detection limits of 0.62 and 2.27 fM, respectively. Featuring simple operation, rapid response, and dual-target detection, this work offers a promising strategy with potential for future integration into on-site platforms for multiplexed detection of antibiotic residues.

Pulmonary infections caused by P. aeruginosa remain challenging to treat because physiological lung barriers limit antibiotic distribution and local availability at the site of infection. Here, we investigated how PEGylation influences the behaviour and antibacterial performance of tobramycin-loaded liposomes across distinct pulmonary microenvironments. Non-PEGylated (LP-TOB) and PEGylated (LP-PEG-TOB) liposomes were prepared, physicochemically characterised and compared with free tobramycin (TOB) using complementary in vitro assays and advanced alveolar and bronchial air-liquid interface co-culture models. Both liposomal formulations displayed suitable physicochemical properties, high colloidal stability and preserved antibacterial activity against P. aeruginosa. PEGylation significantly altered formulation behaviour by reducing epithelial uptake, increasing mucus-associated localisation and promoting faster tobramycin release. These differences resulted in microenvironment-dependent antibacterial responses following bacterial challenge. LP-PEG-TOB achieved greater control of extracellular bacterial burden than LP-TOB in both pulmonary models, with its distribution within the mucus compartment potentially contributing to its performance in the bronchial model. In contrast, LP-TOB demonstrated superior control of the intracellular bacterial signal in macrophages, consistent with its greater cellular interaction. All formulations were non-cytotoxic and attenuated infection-associated inflammatory responses. High-content imaging further revealed that antibiotic exposure mitigated infection-induced cellular and nuclear morphological alterations in macrophages. Overall, these findings demonstrate that PEGylation is a key determinant of liposome distribution and antibacterial performance across pulmonary microenvironments. This study provides a mechanistic basis for tailoring liposomal surface properties to the intended site of action in inhaled antibiotic therapy.