Phage therapy represents a promising alternative for combating bacterial infections. This study employed an A. baumannii isolate harboring the I-F CRISPR-Cas system as a host to isolate phage and evaluate its biological characteristics. Phage-resistant mutants were screened using a double-layer agar plate assay, and the underlying molecular mechanisms were identified through whole-genome sequencing, followed by validation via gene knockout. Transcriptome sequencing was subsequently applied to alterations in the global regulatory networks of these mutants. Our results demonstrate the successful isolation of a novel myovirus, stable at 40-50 ℃, which was successfully isolated and found to utilize the capsule as its adsorption receptor. Whole-genomic analysis confirmed its taxonomic distinction from currently published phages. Investigation into the primary resistance mechanism revealed that the capsule loss, due to an insertional mutation in the UDP-glucose 4-epimerase encoding gene galE. This conclusion was further validated through targeted gene knockout of galE. This defect concurrently attenuated bacterial virulence, as demonstrated by significantly reduced lethality in the Galleria mellonella infection model and enhanced susceptibility to serum killing, while concurrently enhancing the capacity for biofilm formation. Transcriptomic profiling indicated that the ΔgalE significantly upregulated multiple biofilm-associated genes and remodeled the transcriptomic-wide regulatory. Furthermore, the combination of carbenicillin or ceftazidime with the phage exhibited a synergistic effect in vitro, effectively inhibiting biofilm formation and suppressing the emergence of phage resistance. Overall, this work characterizes a novel phage and delineates the host's biological network changes triggered by phage resistance, offering valuable insights for developing phage-based antimicrobial strategies.

Combination therapies of β-lactam antibiotics with β-lactamase inhibitors are widely used, yet resistance to these regimens continues to emerge, highlighting the need for alternative strategies to control β-lactam resistance. Our previous work showed that bacteria can sense β-lactam antibiotics prior to overt cell wall damage. The activation of histidine kinase VbrK by β-lactams initiating transcription of β-lactamase genes, thereby promoting β-lactam resistance. Using an optimized fluorescence polarization-based screening assay followed by hit-to-lead optimization, we identified LPZ-51 as an effective inhibitor of VbrK. LPZ-51 repressed blaA transcription without affecting bacterial growth but markedly potentiated the activity of β-lactam antibiotics. In a bacteria-challenged zebrafish model, LPZ-51 synergized with carbenicillin to reduce bacterial burden, alleviate intestinal inflammation, and improve host survival. Together, these findings demonstrate that targeting VbrK attenuates β-lactam resistance by blocking β-lactamase induction at the transcriptional level, providing a mechanistically distinct antiresistance strategy complementary to existing β-lactam therapies.

The rapid emergence of antimicrobial resistance is a critical global health challenge that renders conventional antibiotics ineffective. Developing innovative strategies to resensitize drug-resistant pathogens to existing antibiotics represents a promising therapeutic approach. Here, we present a biomimetic nanoplatform (Ce-Car@EV NPs) through the rational integration of ginger-derived extracellular vesicles (EVs) and a pH-responsive cerium-carbenicillin coordination nanoparticles (Ce-Car NCPs). This design enables prolonged circulation and targeted degradation in acidic infection sites, releasing Ce4+ ions and carbenicillin. The released Ce4+ ions penetrate the bacterial cells, where they disrupt ATP synthesis, impede oxidative phosphorylation, and inhibit the activity of efflux pump. By depleting ATP, blocking efflux pumps, and thereby reversing bacterial resistance, Ce4+ ions act as a potent adjuvant to carbenicillin. Here we show that this strategy effectively restores carbenicillin efficacy against drug-resistant Pseudomonas aeruginosa both in vitro and in vivo, establishing a therapeutic strategy leveraging metallic adjuvants to counteract antimicrobial resistance.

Bacterial persistence contributes to antibiotic failure and recurrent infectious disease, yet the metabolic cues that promote persister formation remain poorly understood. Here we investigated Escherichia coli persistence after nutrient downshifts from glucose to various carbons. Compared to shifts to gluconeogenic carbons (pyruvate, malate, succinate, and fumarate), the glucose-to-fatty acid shift induced exceptionally high persister levels, with cells tolerating ampicillin (56%), carbenicillin (22%), and gentamicin (1%) after 24-h treatment. With an RNA-based biosensor and HPLC quantification, we detected up to 4-fold higher guanosine tetra- and penta-phosphate [(p)ppGpp] during the prolonged carbon starvation period post glucose-to-fatty acid shift, whereas (p)ppGpp levels remained low after glucose-to-gluconeogenic carbon shifts due to the shorter lag phase. Shortening the lag phase by pre-exposing cells to fatty acid substantially reduced persistence after the glucose-to-fatty acid shift. Overexpression of acyl-ACP synthase, which acylates free acyl carrier protein and thereby suppresses SpoT-dependent (p)ppGpp synthesis, lowered (p)ppGpp levels and reduced persistence. Furthermore, overexpression of PlsB, a growth-essential enzyme in phospholipid biosynthesis that is inhibited by (p)ppGpp, also reduced persistence. In addition, 13C isotope tracing and metabolomic analysis revealed that persisters remain metabolically adaptive, rerouting measurable carbon flux into gluconeogenesis and the pentose phosphate pathway for biomass synthesis. The metabolic remodeling could assist cells to balance redox homeostasis and mitigate oxidative stress. These findings establish the role of (p)ppGpp in nutrient shift persister formation and highlights critical pathways that may be targeted to reduce persistence and improve treatment outcomes against recurrent infections.

V. furnissii is a marine bacterium capable of infecting both invertebrates and humans. However, the mechanisms underlying its drug resistance and virulence remain largely elusive. In this study, we isolated a multidrug-resistant V. furnissii strain, MT14, from bivalve mollusks. Genomic analysis revealed that MT14 carries a novel pAQU-type plasmid, designated pMT14, which harbors six antibiotic resistance genes: qnrS2, qnrVC6, dfrA31, tetA; sul2, and blaGMA-1. Notably, blaGMA-1 and its variants confer resistance to penicillin-class antibiotics, including ampicillin, amoxicillin, and carbenicillin. Comparative genomic analysis further revealed that V. furnissii strains commonly encode T6SS1, T6SS2, and the hemolysin gene vfh, while only five sequenced strains carry zot toxin gene. To our knowledge, this is the first report of zot in V. furnissii. In addition, the absence of drug resistance genes in the majority of genomes (73.3%, 22/30) suggests that such traits are not intrinsic to the species but have been acquired by specific strains, likely through plasmid transfer or other mobile genetic elements. The emergence of blaGMA-1 and zot in V. furnissii represents a potential public health concern, underscoring the need for enhanced surveillance of antimicrobial resistance in marine pathogens.

In this study, a systematic review and meta-analysis were conducted to consider antimicrobial resistance (AMR) rates of Campylobacter in Iran. A systematic search was conducted in the databases of PubMed, Scopus and Web of Science and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Data analysis was done with R software. A number of 58 articles in the current study evaluated AMR in Campylobacter species in human, animal and food isolates. Our findings demonstrated that most of the Campylobacter spp. isolates in Iran have high resistance proportions to beta-lactams (including cefixime (0.97), cephalothin (0.84), ceftriaxone (0.76), cephalexin (0.63), ceftazidime (0.53) and carbenicillin (0.38)), fluoroquinolones (including ofloxacin (0.79), nalidixic acid (0.51) and ciprofloxacin (0.52)), sulfonamides like trimethoprim-sulfamethoxazole (0.68) (potentiated sulfonamides), florfenicol (0.62), tetracycline (0.57) and macrolides (including erythromycin (0.12) and azithromycin (0.17)). Conversely, Campylobacter spp. showed low resistance rates to aminoglycosides (including neomycin (0.08), amikacin (0.08), meropenem (0.06), spectinomycin (0.04), gentamicin (0.04) and imipenem (0.02)) and chloramphenicol (0.07). Regarding Campylobacter coli and Campylobacter jejuni isolates, resistance to erythromycin (0.18-0.09), and gentamycin (0.08-0.04) was higher in C. coli than C. jejuni isolates, respectively, whereas resistance to ciprofloxacin (0.56) and tetracycline (0.56) was higher in C. jejuni. The most prevalent antimicrobial resistance genes (ARGs) in Campylobacter spp. were tetO (0.73) and cmeB (0.48) and blaOXA61 (0.42). Therefore, the use of strict control systems and a restriction on the use of antibiotics in human, agricultural and animal farming are urgently required to reduce the development and spread of AMR.

(1) This study explored Lacticaseibacillus rhamnosus MS27, a newly isolated strain, as a potential probiotic candidate for alleviating the onset and severity of ulcerative colitis (UC). (2) L. rhamnosus MS27 was isolated and subjected to biochemical identification, antibiotic sensitivity testing, and antibacterial activity assessment. Dextran sulfate sodium (DSS) colitis model mice were used to evaluate its alleviating effects. In this study, 16S rRNA microbiome and eukaryotes reference transcriptome analyses were conducted to investigate its impact on intestinal microbial ecology and potential molecular mechanisms. (3) L. rhamnosus MS27 exhibits high acid tolerance at pH 3.23 and maintains a high viable bacterial count for 24 h. It can utilize sucrose, lactose, maltose, inulin, esculin, salicin, and mannitol but not raffinose, and it is sensitive to carbenicillin, erythromycin, tetracycline, chloramphenicol, clindamycin, and penicillin. It effectively increases the abundance of beneficial microbes, particularly Akkermansia, Muribaculaceae, and Limosilactobacillus reuteri (p < 0.05), while significantly reducing microorganisms linked to human pathogens causing diarrhea and gastroenteritis (p < 0.05). Transcriptomic analysis demonstrated that the expression levels of Igkv16-104 and C1qtnf3 were significantly downregulated in the presence of L. rhamnosus MS27 treatment compared to DSS treatment alone (p < 0.05). Further analysis revealed significant differences in genes related to immune functions, antigen presentation, and immune cell markers, indicating potential protein-protein interaction networks, particularly among genes of the major histocompatibility complex (MHC). (4) L. rhamnosus MS27, as a novel strain, demonstrates a significant capacity to alleviate inflammatory phenotypes. L. rhamnosus MS27 exhibits distinctive metabolic characteristics in lactic acid utilization, acetic acid and oleic acid production. Furthermore, it contributes to systemic homeostasis regulation by modulating Turicibacter to link intestinal microbiota composition with host immune function.

Acanthamoeba keratitis (AK) is a painful corneal infection caused by pathogenic free-living amoebae of the genus Acanthamoeba, primarily affecting contact lens users with inadequate hygiene practices. Clinical manifestations include corneal infiltration, epithelial and stromal damage, and severe ocular pain, which may lead to vision loss or enucleation in advanced cases. Current treatments, such as polyhexamethylene biguanide (PHMB), often fail due to the parasite's ability to form drug-resistant cysts. In this study, metabolomic profiling revealed a significant accumulation of γ-aminobutyric acid (GABA) during encystation. Expression analysis of key genes associated with the GABA shunt pathway showed marked upregulation of glutamate dehydrogenase (GDH) and glutamate decarboxylase (GAD). Supplementation with exogenous GABA enhanced encystation rates and upregulated encystation-associated genes, including cellulose synthase I, autophagy-related protein 8, and encystation-mediating serine proteinase, suggesting that GABA may function as a signaling molecule regulating encystation. To explore potential anti-encystation strategies, β-lactam antibiotics, well-known GABAA receptor antagonists, were tested. Cefotaxime, carbenicillin, and penicillin G significantly inhibited cyst formation without inducing cytotoxicity, whereas non-β-lactam antibiotics showed no such effect. These findings suggest that the inhibitory effect is associated with the β-lactam ring structure and may involve interference with GABA-mediated signaling pathways. Collectively, our study reveals a critical role of GABA metabolism in Acanthamoeba encystation and highlights β-lactam antibiotics as potential adjunctive agents for overcoming cyst-associated drug resistance in AK treatment.

Salmonella Typhimurium, a major foodborne pathogen, forms biofilms that enhance its environmental persistence and resistance to antibiotics, presenting significant public health challenges. The CsgD protein, a key transcriptional regulator, orchestrates biofilm formation by regulating curli fimbriae and cellulose production. This study aimed to identify and evaluate potential CsgD inhibitors to disrupt S. Typhimurium biofilms using a combination of computational and experimental methodologies. Molecular docking was performed to screen 145 FDA-approved antibiotics from DrugBank against the CsgD protein. Carbenicillin, identified as a top candidate, was further analyzed through 100 ns molecular dynamics simulations to assess the stability of the carbenicillin-CsgD complex. Experimental evaluations determined the minimum biofilm inhibitory concentration (MBIC), and minimum biofilm eradication concentration (MBEC) of carbenicillin against S. Typhimurium isolates. Biofilm structure and curli production were examined using scanning electron microscopy (SEM) and Congo red agar assays, respectively. Molecular docking revealed carbenicillin's high binding affinity to CsgD. Molecular dynamics simulations confirmed the structural stability of the carbenicillin-CsgD complex. Experimental assays established MBIC and MBEC at 1 and 4 μg/mL, respectively. SEM analysis showed morphological changes and disrupted biofilm architecture at 0.5-1 μg/mL carbenicillin, while Congo red agar assays demonstrated dose-dependent suppression of curli production. Carbenicillin exhibits significant potential as a CsgD-targeted anti-biofilm agent, providing a foundation for novel therapeutic strategies to combat S. Typhimurium infections and address their public health burden.

The efficient treatment of prevalent drug-resistant (DR) bacteria-infected wounds caused by the clinical use of conventional antibiotics remains a significant challenge. Here, a well-integrated intelligent drug-releasing, photodynamic therapy, and visual therapeutic diagnosis strategy combining burgeoning aggregation-induced emission luminogen (AIE-gen) and microneedling technology is innovatively proposed for advanced infected-wound management. This strategy is based on an intelligently responsive two-stage AIE-gen-containing microneedle-based platform (AIE-film/TS-MN) with a double-layer structure. The uppermost layer comprises an on-demand pH-responsive AIE-photosensitizer (AFPP)-based film (AIE-film). This layer is designed to achieve the precise release of reactive oxygen species (ROS) or nitrocefin, which responds to β-lactamase in DR bacteria. The basal layer is a two-stage microneedle (TS-MN) patch with a "core-shell" structure bio-inspired by snake fangs, providing outstanding anti-migratory origins and acid-responsive drug-releasing properties through crustaceous gelatin loaded with Fe-carbenicillin frameworks (Fe-MOF). AFPP and nitrocefin in AIE-film accurately respond to acidic wound-microenvironment and β-lactamase, colorfully changing from yellow and green to orange and red, respectively. This indicates that AIE-film/TS-MN exhibits significant potential as a novel biosensor to record the color-change semiquantitatively at wound-sites through smartphone analysis, visually achieving significantly infected wound-diagnosis/-assessment to facilitate the therapeutic efficacy. Additionally, the smart multiple drug-releasing synergistically eliminates DR-bacteria, including carbenicillin from Fe-MOF and AFPP-activating ROS. Comprehensive in vitro and in vivo experiments confirm that the telemedicine AIE-film/TS-MN offers advanced integrated therapeutic effects for active infected wound management. Additionally, it reduces antibiotic abuse and provides an innovative and promising strategy for visual medical diagnostics and therapeutic interventions.

The objectives of this study were to investigate the etiology of morbidity and mortality in Tibetan sheep from a township in Shannan, Tibet, and to characterize the biological characteristics of the causative pathogen, thereby providing a scientific basis for effective disease prevention and control. Pathological specimens were aseptically collected from deceased animals, and the pathogen was isolated and cultured based on clinical presentations. Identification of the isolate involved morphological observation, 16S rRNA PCR amplification and sequencing, biochemical assays, toxin gene typing, Kirby-Bauer disk diffusion antimicrobial susceptibility testing, and antibiotic resistance genes screening. On tryptose sulfite cycloserine (TSC) agar, the organism formed black, circular colonies. Gram staining revealed short, straight, Gram-positive bacilli arranged singly or in pairs, consistent with the Clostridium perfringens . 16S rRNA gene sequence alignment showed >99 % identity to reference C. perfringens sequences in the NCBI database. Confirming the strain asC. perfringens. Biochemical tests indicated positive reactions for glucose, Biochemical assays were positive for glucose, lactose, and maltose fermentation; gelatin hydrolysis; oxidase activity; nitrate reduction;hydrogen sulfide production; and ornithine decarboxylase, but negative results for indole production, mannitol and sorbitol fermentation, and urea hydrolysis, matching known traits of C. perfringens. . Toxin gene PCR detected cpa,cpb, and etx, but not iap, cpe, or netB, identifying the isolate as C. perfringens type B. Antimicrobial susceptibility testing revealed high sensitivity to sulfadiazine, norfloxacin, nitrofurantoin, and cefazolin, but resistance to amoxicillin, oxacillin, carbenicillin, and tetracycline.Among the resistance genes screened, only aac(6')-Ib-cr was detected.This study reports the first isolation of C. perfringens type B in Shannan, Tibet; elucidates its biological characteristics and toxin profile; identifies effective antibiotics for targeted intervention; and provides foundational data for controlling lamb dysentery caused by this pathogen and for understanding its pathogenicity in sheep.

Antibiotic resistance mediated by β-lactamases, encoded by bla genes, is a significant global health threat, necessitating systematic studies of their diversity and evolution, particularly among pathogenic bacteria lineages. Leveraging over 6,000 quality-filtered Vibrio genomes alongside six newly sequenced marine symbiotic strains representing 128 nominal and 57 unclassified Vibrio species, our study extends taxonomic breadth and resolution for investigating β-lactamase diversity. We identified 4,431 β-lactamases across 41 species, encompassing all four Ambler classes (A-D). Among these, carbenicillin-hydrolyzing Class A β-lactamases encoded by blaCARB family were the most prevalent (60.7%) and exhibited a clade-centric distribution particularly in Harveyi clade and V. cholerae, underscoring the influence of specific ecological and evolutionary pressures. We refined carbenicillin-hydrolyzing Class A β-lactamase classification into two subfamilies: CARB-17-like (blaCARB-17-like) confined to Harveyi clade and CARB-1-like (blaCARB-1-like) found exclusively outside Harveyi clade based on phylogenetic placement, sequence similarity, and inheritance patterns, providing a clearer framework for delineating their functional and phylogenetic nuances. Notably, blaCARB-17-like genes in nonpathogenic Harveyi Subclade II showed significantly relaxed selection, accompanied by unusual mutations within key conserved motifs especially catalytic serine residues, suggesting evolutionary drift that may compromise canonical enzymatic activity. Furthermore, blaCARB-17-like genes, present as a single copy, emerged as a core gene in Harveyi clade, showing promise as a diagnostic marker for clinically significant Harveyi clade species, despite limited yet significant interspecies genetic exchanges mediated by recombination or mobile genetic elements. Our study advances the understanding of β-lactamase evolution and genomic distribution in Vibrio, with broad implications for diagnostic applications and resistance management strategies.