BACKGROUND:The rising prevalence of polymyxin resistance in multidrug-resistant Klebsiella pneumoniae presents a critical situation with limited therapeutic options. METHODS:Methods Genomic sequencing of 15 clinical polymyxin-resistant K. pneumoniae strains with multidrug resistance revealed that MgrB inactivation, predominantly disrupted by insertion sequences (ISs) in the IS1, IS4, and IS5 families, was the leading cause of polymyxin resistance. Comparative transcriptomics of wild-type, ΔmgrB, and ΔmgrBΔphoP were performed to elucidate the MgrB-PhoPQ regulatory network. RESULTS:This study conducted a system-wide analysis of the regulatory network and identified a species-specific PhoPQ regulon in K. pneumoniae.Beyond the classical MgrB-PhoPQ-ArnBCADTEF pathway, we identified a previously unannotated PhoPQ-regulated gene, 144 bp LN739_RS09850, encoding an Ecr homologue from Enterobacter cloacae. This protein has been reported to confer colistin heteroresistance, with the underlying mechanism not yet functionally validated. This study revealed that overexpression of Ecr homologues decreased colistin susceptibility in both K. pneumoniae and E. cloacae, but this phenotype was abolished upon phoP deletion, confirming PhoP's essential role. Consistent with this dependency, comparative transcriptomics of Ecr-overexpressing K. pneumoniae vs. control revealed significant upregulation of mgrB, phoPQ, arnBCADTE, and pmrD. Two-hybrid bacterial assays further demonstrated direct Ecr-PhoQ interaction. Electrophoretic mobility shift assay confirmed that PhoP directly binds to the ecr promoter in vitro, and a β-galactosidase reporter assay demonstrated that PhoP enhanced ecr promoter activity, indicating that PhoP regulates ecr expression by directly controlling its transcription. CONCLUSION:Collectively, these findings suggest that PhoP may directly activate the transcription of Ecr, with Ecr feedback activating the PhoPQ system via interaction with PhoQ, leading to induction of the arn operon and consequent polymyxin resistance.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease with limited therapeutic options. To investigate the roles of the pulmonary microbiota and metabolism in fibrosis, we established a bleomycin (BLM)-induced mouse model at 14- and 28-day timepoints and performed integrated 16S rRNA gene amplicon sequencing and untargeted metabolomic analyses. Histological and Western blot analyses confirmed significant fibrotic changes and the upregulation of fibrotic markers. Microbiome profiling revealed marked dysbiosis after BLM exposure, characterized by reduced microbial diversity and enrichment of Klebsiella. LC-MS-based metabolomic analysis identified substantial perturbations in the lung tissue metabolome, particularly in lipid metabolism, amino acid metabolism, and energy pathways. Correlation analysis indicated a strong positive association between the abundance of Klebsiella and the levels of specific dipeptides, including Ala-Hyp-Gly, Asp-His, and Asp-Asn. The accumulation of these dipeptides may reflect increased collagen degradation and turnover in fibrotic lungs. Collectively, our findings demonstrate that BLM-induced pulmonary fibrosis is accompanied by coordinated alterations in the lung microbiome and metabolome. Notably, microbial dysbiosis, particularly the expansion of Klebsiella, may be associated with alterations in amino acid metabolism and fibrotic progression.

The genetic connection between virulence and antibiotic resistance remains poorly understood. Our previous RNA-Seq analysis of a polymyxin-resistant Klebsiella pneumoniae ATCC BAA2146 mutant identified a highly expressed VirK/YbjX family gene (kpn2146_RS17285), encoding a conserved membrane protein, designated virK (virulence required for Klebsiella pneumoniae). While PhoP-dependent antibiotic resistance is mediated through established pathways such as arn/pmr, we identify VirK as a PhoP-regulated factor specifically contributing to virulence. VirK localizes to the outer membrane and, although not involved in lipopolysaccharide modification, its deletion modestly reduced bacterial virulence in a mouse systemic infection model. Transcriptional and electrophoretic mobility shift assays demonstrated that virK is directly activated by the PhoP protein. A strong positive correlation between virK and phoP expression (r = 0.98) was also observed in multidrug-resistant clinical isolates. Since the PhoP/PhoQ two-component system mediates polymyxin resistance, its direct regulation of VirK uncovers an adaptive mechanism coupling enhanced virulence with antibiotic resistance. These findings reveal a previously unrecognized PhoP/VirK regulatory pathway that contributes to pathogenicity in polymyxin-resistant Klebsiella pneumoniae, offering new insights into bacterial evolution and suggesting that targeting PhoP/PhoQ could provide an effective strategy to combat multidrug-resistant K. pneumoniae infections.

Background Staphylococcus epidermidis, typically regarded as a harmless commensal, has become one of the major causes of nosocomial infections, including ocular, skin, medical device-associated and bloodstream infections. Therefore, we analyze its population structure through genomic analysis integrated with metadata.Results We performed whole-genome sequencing-based population genomic analyses by integrating 1742 publicly available S. epidermidis genomes (accessed by August 2025) with 94 newly sequenced isolates. Our analyses revealed that S. epidermidis represents a species complex composed of four phylogenetic lineages (phylogroups 1-4) with diverse clonal backgrounds and a broad global distribution. The species harbors an open pan-genome and demonstrates a strong capacity to acquire novel genetic traits through mobile genetic elements. Extensive antimicrobial resistance and substantial virulence potential were observed across lineages. Notably, phylogroup 1, dominated by ST 2, exhibited a 97.8% detection rate of the methicillin resistance gene mecA, likely driven by clonal expansion and horizontal gene transfer, identifying it as a high-risk lineage. The analysis of enriched genes in blood-derived strains showed that the adaptability of S. epidermidis in bloodstream-associated environments is controlled by multiple genes, involving antimicrobial resistance, cell wall remodeling, environmental adaptation, and core metabolism.Conclusions This study provides a comprehensive population genomic framework for S. epidermidis, elucidating its population structure, genomic diversity, antimicrobial resistance, and virulence-associated genetic features. These findings offer valuable insights into the evolutionary dynamics and pathogenic potential of S. epidermidis and provide an important genomic resource to inform infection control strategies and clinical management.

Idiopathic pulmonary fibrosis (IPF) denotes a chronic, advancing, and life-threatening lung disorder. Dysregulated cytokines, particularly those in the transforming growth factor-β (TGF-β)-associated signaling pathway, drive the pathological development of IPF. Natural products derived from traditional Chinese medicine hold great potential as promising therapeutic candidates for IPF. This study integrated machine learning (ML) with experimental validation to identify TGF-β/small mother against decapentaplegic (SMAD) pathway inhibitors from natural compounds. An in-house library was screened by means of a dual-luciferase reporter assay, revealing the flavonoid dihydromyricetin (DHM) as the most potent inhibitor. In vitro, DHM suppressed TGF-β1-triggered epithelial–mesenchymal transition (EMT) in A549 cells and fibroblast transdifferentiation in medical research council cell strain 5 (MRC-5) cells. In vivo, DHM attenuated fibrosis and inflammatory responses in a bleomycin (BLM)-triggered pulmonary fibrosis mouse model. Mechanistic studies revealed that DHM targets the type I TGF-β receptor (known as ALK5), reduces its membrane expression, binds directly to the receptor and represses its kinase activity, ultimately downregulating the TGF-β/ALK5 pathway. The present research is the first to report DHM as a TGF-β/SMAD inhibitor identified through ML with therapeutic efficacy against IPF. DHM’s anti-fibrotic effects are mediated through ALK5 blockade, suppressing downstream signaling, EMT, and fibroblast activation. These findings not only highlight DHM’s latent ability to act as a novel remedy for IPF but also underscore the utility of computational approaches in natural product drug discovery.

Pseudomonas aeruginosa is a widespread Gram-negative opportunistic pathogen in environmental and hospital settings, frequently causing respiratory diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disorder (COPD), and ventilator-associated pneumonia. In our previous study, a galU-deleted clinical P. aeruginosa was found to exhibit increased susceptibility to polymyxins. The galU gene plays an important role in the biosynthesis of lipopolysaccharide (LPS) O-antigen. Here, we systematically evaluated the effects of galU deletion on the phenotype and virulence of P. aeruginosa PAO1. A galU deletion mutant was successfully constructed in P. aeruginosa PAO1 by CRISPR/Cas9, and the complementation was accomplished by pUCP18 plasmid carrying wild-type galU. The changes in phenotype, virulence, and pathogenicity were systemically studied. The results revealed that knockout of galU led to the loss of O-antigen, which affected growth, virulence, and pathogenicity through various ways in P. aeruginosa, and significantly affected the susceptibility of P. aeruginosa to polymyxins. Mechanism study suggested the involvements of quorum sensing, Entner-Doudoroff pathway, and tyrosine metabolism on bacterial virulence and antibiotic susceptibility changes after galU deletion. galU and the related pathways may serve as effective targets for the treatment of P. aeruginosa infection, providing a theoretical basis for the development of novel antibacterial drugs.

OBJECTIVES:To elucidate the characteristics of a multidrug-resistant hypervirulent Klebsiella quasipneumoniae subsp. similipneumoniae strain Kpn1587 isolated from a 77-year-old male patient's sputum with lung infection using whole-genome sequencing and various phenotypic assays. METHODS:MIC of Kpn1587 was measured using the VITEK 2 system and broth microdilution method. String test, mucoviscosity assay, siderophore production assay, capsular polysaccharide extraction and quantification, cell assays and mouse systemic infection model were used to analyse the hypervirulent phenotype of the strain. The fitness of Kpn1587 was evaluated by growth curve and in vitro competition. A conjugation experiment was conducted to verify the transferability of the resistant plasmid. Elucidation of genomic features was carried out using whole-genome sequencing and comparative genomics. RESULTS:Kpn1587 showed multiple antibiotic resistance and a high virulence phenotype with the resistant plasmid being able to transfer to an Escherichia coli recipient strain at a conjugative efficiency of 6.8 × 10-2 per donor. Genomic analysis revealed that the Kpn1587 strain belongs to the ST367-KL1 type, harbouring a repB-type virulence plasmid, pKpn1587-Vir, which carries typical virulence genes, and an IncFII resistance plasmid, pKpn1587-CTXM, which carries blaCTX-M-14. CONCLUSIONS:Kpn1587 demonstrates, for the first time, the coexistence of an IncFII-type transferable plasmid carrying blaCTX-M-14 and a repB virulence plasmid in an ST367-KL1 type K. quasipneumoniae subsp. similipneumoniae strain. The co-occurrence of these plasmids underscores the potential for horizontal dissemination of this dual-threat phenotype, with important implications for clinical management and public health.

Staphylococcus aureus remains a leading cause of morbidity and mortality worldwide, with persistent and relapsing infections posing a major global health threat. Here, we report that baloxavir, an FDA-approved influenza antiviral, exhibits antibacterial activity against S. aureus. Baloxavir demonstrated potent activity against both MSSA and MRSA clinical isolates with MICs of 2-4 μg/mL and exhibited concentration-dependent antibacterial activity in time-kill assays. Notably, baloxavir effectively eliminated intracellular S. aureus in both A549 alveolar epithelial cells and RAW264.7 macrophages at 10 μg/mL and achieved complete eradication in A549 cells at 50 μg/mL. In vivo, baloxavir (20-40 mg/kg) significantly improved survival in MRSA-infected mice from 12.5% to 75-87.5%. Transcriptomic analysis revealed significant downregulation of purine de novo biosynthesis genes, including purF and purK, which was validated by RT-qPCR (r = 0.862, p = 0.027). This study demonstrates for the first time that baloxavir possesses significant antibacterial activity against S. aureus including MRSA, positioning it as a promising repurposed candidate for treating persistent intracellular infections and post-viral superinfections.

This study investigated how Staphylococcus aureus adapts under pentoxifylline (PTX) therapy through analyzing three phylogenetically related isolates (L1, L2, L3) from a bacteremia patient. Whole genome sequencing revealed L1 contained a 2400-bp insertion disrupting nupC , while L3 harbored mutations in the anti-Shine-Dalgarno sequences of 16S rRNA. Each isolate evolved distinct mechanisms to reduce c-di-AMP levels, accompanied by changes in survival ability and virulence. Molecular analysis demonstrated PTX noncompetitively inhibits GdpP phosphodiesterase, directly modulating cyclic diadenosine monophosphate (c-di-AMP) signaling. Our findings provide novel insights into how non-antibiotic medications shape bacterial adaptation through second messenger modulation, highlighting the complex trade-offs between stress resistance and fitness in clinical settings. Importance This study reveals how pentoxifylline, a non-antibiotic drug, drives Staphylococcus aureus adaptation by modulating c-di-AMP signaling. Our findings highlight the unintended consequences of non-antibiotic medications on bacterial evolution, offering new insights for clinical treatment strategies to mitigate resistance development. Highlights

Ilaprazole, a proton pump inhibitor, has been approved and marketed in Korea and China for the treatment of gastric ulcer, duodenal ulcer, gastroesophageal reflux disease, and erosive esophagitis. This study evaluated the in vitro antibacterial activity of ilaprazole against Helicobacter pylori (H. pylori), both as a single agent and in combination with other components used in the standard quadruple therapy. The antibacterial activity of ilaprazole was tested on 25 H. pylori strains, including the clinical isolates resistant to clarithromycin (CLA), amoxicillin (AMX), levofloxacin, and/or metronidazole. Antibacterial activities and killing kinetics were evaluated by the minimal inhibitory concentration (MIC) and time-kill curve determination, respectively. Synergistic effects were assessed in checkerboard and time-kill assays. Resistance development was assessed through serial passage over 12 cycles. Ilaprazole exhibited potent in vitro antibacterial activity against H. pylori, with MIC50 and MIC90 values of 8 µg/mL, demonstrating activity against drug-resistant strains. When combined with CLA, ilaprazole showed synergistic effects against 36% of the tested strains. Notably, the quadruple combination of ilaprazole + AMX + CLA + bismuth potassium citrate exhibited an obvious synergistic effect. Importantly, repeated exposure to ilaprazole over 12 passages did not induce resistance. These findings highlight the promising in vitro antibacterial activity of ilaprazole against H. pylori, including drug-resistant strains, and its potential to enhance the efficacy of quadruple therapy. The study supports the inclusion of ilaprazole in treatment regimens for H. pylori infections, offering a compelling rationale for its clinical use. IMPORTANCE H. pylori infection remains a major global health issue, contributing to a wide range of gastric diseases. Despite current treatment regimens, rising antibiotic resistance limits their effectiveness, emphasizing the need for novel therapeutic approaches. This study highlights the promising in vitro antibacterial activity of ilaprazole against H. pylori, including drug-resistant strains. Ilaprazole not only exhibits direct antimicrobial effects but also enhances the efficacy of combination therapy, particularly in quadruple therapy regimens, which are recommended as first-line treatment. The findings demonstrate that ilaprazole, in combination with clarithromycin, shows synergistic effects, offering a potential solution to overcome antibiotic resistance challenges. Importantly, repeated exposure to ilaprazole did not induce resistance, a critical factor for its long-term use. These results provide compelling evidence for ilaprazole's inclusion in clinical treatment strategies, contributing to improved eradication rates and better patient outcomes in H. pylori management.

TEM and SHV are among the most prevalent β-lactamases contributing to β-lactam antibiotic resistance in clinical settings,leading to treatment challenges and increased mortality rates.Except for penicillin and early cephalosporins,TEM and SHV variants have evolved with the ability to hydrolyze the second-and third-generation cephalosporins,monobactams,and even β-lactamase inhibitors.Accurate detection of β-lactamases is of paramount importance for optimizing antibiotic use and combating antimicrobial resistance(AMR).While genetic detec-tion methods,such as polymerase chain reaction(PCR),are widely employed,their positive results may lack phenotypic correlation due to the low or absent expression of blasHV and blaTEM in many strains[1].Therefore,a direct protein-level detection method such as targeted proteomics is more precise and clinically relevant.This study highlights the development of a rapid detection method using targeted proteomics with high-resolution accurate mass(HRAM)Orbitrap MS for the direct detection of TEM and SHV in Enterobacteriaceae strains,which offers greater clinical relevance compared to conventional ge-netic approaches.

Methicillin-resistant Staphylococcus aureus (MRSA) infection is a serious clinical threat, and D-Serine (D-Ser) showed significant sensitization effects on β-lactams against MRSA in our previous study. Quantitative PCR analysis found the elevated expression of the dlt operon with D-Ser combination, which is responsible for wall teichoic acid (WTA) modification involving D-Alanine (D-Ala). This study aims to verify the effect of D-Ser on WTA modification through the dlt pathway and explore the related effects on bacteria. The DltA and DltC were recombined, and enzyme kinetic evaluations with different D-amino acids were then conducted; it was found that D-Ser is the second-best substrate for DltA (just after D-Ala), no matter whether DltC is present or not. D-Ser treatment also lowered WTA generation as demonstrated by WTA phosphate quantification and native-PAGE electrophoresis, increased the susceptibility of S. aureus to polymyxins, and elevated the mouse survival rate in the MRSA intraperitoneal infection model without affecting the bacterial loads in the main organs, indicating possible effects of D-Ser on MRSA virulence through WTA modification. In conclusion, the current study provided evidence for D-Ser modification of WTA via the dlt pathway, and its possible involvement in D-Ser sensitization deserves further investigation.

Idiopathic pulmonary fibrosis (IPF) is a progressive and chronic interstitial lung disease with unclear underlying pathogenic mechanisms. Dysbiosis of the lung microbiota is believed to be associated with the development of fibrosis; however, the roles of the microbiome in the respiratory functions of hosts with IPF remain poorly understood. To investigate the relationship between the lung microbiome and the pathological processes of idiopathic pulmonary fibrosis under laboratory conditions, C57BL/6 J mice were exposed to bleomycin and observed at 7, 14, 21, and 28 days post-exposure. 16S rDNA analysis revealed that the lung microbial community exhibited dysbiosis in the bleomycin-induced pulmonary fibrosis model, characterized by an abnormally high proportion of Klebsiella quasipneumoniae (K. quasipneumoniae), as confirmed by RNA fluorescence in situ hybridization. Throughout the progression of experimental pulmonary fibrosis, Tax4Fun analysis indicated that the abundance of K. quasipneumoniae differed significantly between model mice and control mice, correlating with the sustained activation of reactive oxygen species (ROS) pathways. Importantly, the dysbiosis of K. quasipneumoniae may serve as a critical factor triggering increased ROS levels, accompanied by macrophage mitophagy, ultimately leading to the overexpression of TGF-β1, a key player in the pathogenesis of pulmonary fibrosis. These findings suggest that lung microbiota dysbiosis exacerbates the progression of bleomycin-induced pulmonary fibrosis related to macrophage mitophagy.

In previous studies, polymyxin MRX-8 demonstrated potent in vitro antibacterial activity with a reduced nephrotoxicity risk and an improved PK/PD profile compared with polymyxin B. In this study, the in vivo antibacterial efficacy of intravenously administered MRX-8 was evaluated in murine models of systemic infection (induced by intraperitoneal injection), lung infection (induced by intratracheal route inoculation), and ascending urinary tract infection (induced by intraurethral inoculation) with P. aeruginosa, K. pneumoniae, E. coli, and A. baumannii as the challenging pathogens. MRX-8 demonstrated superiority to polymyxin B against carbapenem-resistant K. pneumoniae, P. aeruginosa and E. coli infections; however, the efficacy of MRX-8 was less than or comparable with that of polymyxin B against carbapenem-resistant A. baumannii infections. The results suggest MRX-8 could be an efficacious treatment alternative versus Gram-negative, treatment-resistant pathogens that can confound current antimicrobial agents.

The treatment of sepsis caused by multidrug-resistant (MDR) Gram-negative bacterial infections remains challenging. With these pathogens exhibiting resistance to carbapenems and new generation cephalosporins, the traditional antibiotic polymyxin B (PMB) has reemerged as a critical treatment option. However, its severe neurotoxicity and nephrotoxicity greatly limit the clinical application. Therefore, we designed negatively charged high-density lipoprotein (HDL) mimicking nanodiscs as a PMB delivery system, which can simultaneously reduce toxicity and enhance drug efficacy. The negative charge prevented the PMB release in physiological conditions and binding to cell membranes, significantly reducing toxicity in mammalian cells and mice. Notably, nanodisc-PMB exhibits superior efficacy than free PMB in sepsis induced by carbapenem-resistant Acinetobacter baumannii (CRAB) strains. Nanodisc-PMB shows promise as a treatment for carbapenem-resistant Gram-negative bacterial sepsis, especially caused by Acinetobacter baumannii, and the nanodiscs could be repurposed for other toxic antibiotics as an innovative delivery system. STATEMENT OF SIGNIFICANCE: Multidrug-resistant Gram-negative bacteria, notably carbapenem-resistant Acinetobacter baumannii, currently pose a substantial challenge due to the scarcity of effective treatments, rendering Polymyxins a last-resort antibiotic option. However, their therapeutic application is significantly limited by severe neurotoxic and nephrotoxic side effects. Prevailing polymyxin delivery systems focus on either reducing toxicity or enhancing bioavailability yet fail to simultaneously achieve both. In this scenario, we have developed a distinctive HDL-mimicking nanodisc for polymyxin B, which not only significantly reduces toxicity but also improves efficacy against Gram-negative bacteria, especially in sepsis caused by CRAB. This research offers an innovative drug delivery system for polymyxin B. Such advancement could notably improve the therapeutic landscape and make a significant contribution to the arsenal against these notorious pathogens.

Antibacterial resistance is a global health threat that requires further concrete action on the part of all countries. In this context, one of the biggest concerns is whether enough new antibacterial drugs are being discovered and developed. Although several high-quality reviews on clinical antibacterial drug pipelines from a global perspective were published recently, none provides comprehensive information on original antibacterial drugs at clinical stages in China. In this review, we summarize the latest progress of novel antibacterial drugs approved for marketing and under clinical evaluation in China since 2019. Information was obtained by consulting official websites, searching commercial databases, retrieving literature, asking personnel from institutions or companies, and other means, and a considerable part of the data covered here has not been included in other reviews. As of June 30, 2023, a total of 20 antibacterial projects from 17 Chinese pharmaceutical companies or developers were identified and updated. Among them, two new antibacterial drugs that belong to traditional antibiotic classes were approved by the National Medical Products Administration (NMPA) in China in 2019 and 2021, respectively, and 18 antibacterial agents are in clinical development, with one under regulatory evaluation, five in phase 3, six in phase 2, and six in phase 1. Most of the clinical candidates are new analogs or mono-components of traditional antibacterial pharmacophore types, including two dual-acting hybrid antibiotics and a recombinant antibacterial protein. Overall, despite there being 17 antibacterial clinical candidates, our analysis indicates that there are still relatively few clinically differentiated antibacterial agents in stages of clinical development in China. Hopefully, Chinese pharmaceutical companies and institutions will develop more innovative and clinically differentiated candidates with good market potential in the future research and development (R&D) of original antibacterial drugs.

IntroductionMycobacterium tuberculosis (Mtb), the main cause of tuberculosis (TB), has brought a great burden to the world's public health. With the widespread use of Mtb drug-resistant strains, the pressure on anti-TB treatment is increasing. Anti-TB drugs with novel structures and targets are urgently needed. Previous studies have revealed a series of CYPs with important roles in the survival and metabolism of Mtb. However, there is little research on the structure and function of CYP138.MethodsIn our study, to discover the function and targetability of CYP138, a cyp138-knockout strain was built, and the function of CYP138 was speculated by the comparison between cyp138-knockout and wild-type strains through growth curves, growth status under different carbon sources, infection curves, SEM, MIC tests, quantitative proteomics, and lipidomics.Results and discussionThe knockout of cyp138 was proven to affect the Mtb's macrophage infection, antibiotics susceptibility, and the levels of fatty acid metabolism, membrane-related proteins, and lipids such as triacylglycerol. We proposed that CYP138 plays an important role in the synthesis and decomposition of lipids related to the cell membrane structure as a new potential anti-tuberculosis drug target.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a global threat, but the mechanism of non-carbapenemase carbapenem resistance is still unclear. In the current study, we investigated the contributions of point mutations in mexR, oprD, and ftsI to carbapenem resistance in P. aeruginosa during in vivo evolution studies with consecutive clinical isolates. Real-time qPCR and Electrophoretic Mobility Shift Assay demonstrated that MexR (Gln55Pro) mutation increased MexAB efflux pump genes expression by altering MexR's binding capacity, leading to a four- to eight-fold increase in meropenem MIC in the Pae d1 Green ∆mexR and PAO1∆mexR mutants. The OprD (Trp415*) truncation affected porin structure, and the constructed mutant Pae d1 Green oprD Trp415* increased meropenem MIC by 16-fold (from 0.25 to 4 µg/mL). The contribution of ftsI mutation to meropenem resistance was confirmed by clinical linkage analysis and was estimated to cause a two-fold increase in meropenem MIC by comparing the resistant clinical isolate with the Pae d1 Green oprD Trp415*∆mexR double mutant. The study found that the oprD Trp415* allele alone accounts for the imipenem MIC in clinical isolates, while the ∆mexR and ftsI Arg504Cys alleles do not contribute to imipenem resistance. In conclusion, we identified and explored the contributions of mexR, oprD, and ftsI mutations to high level non-carbapenemase carbapenem resistance in P. aeruginosa. These findings highlight the interplay of different mutations in causing non-carbapenemase carbapenem-resistance in P. aeruginosa. IMPORTANCE:The emergence of carbapenem-resistant Pseudomonas aeruginosa (CRPA) poses a significant global health threat, complicating treatment options for infections caused by this pathogen. Understanding the mechanisms behind non-carbapenemase carbapenem resistance is critical for developing effective therapeutic strategies. This study provides crucial insights into how specific point mutations in key genes-mexR, oprD, and ftsI-contribute to carbapenem resistance, particularly the MexR (Gln55Pro) mutation's effect on efflux pump expression and the OprD (Trp415*) truncation's impact on porin structure. The findings elucidate the complex interplay of these mutations, highlighting their roles in conferring high-level resistance, and underscore the imperative for continued research to inform therapeutic strategies against CRPA infections.

Polymyxin B and polymyxin E (colistin) are presently considered the last line of defense against human infections caused by multidrug-resistant Gram-negative organisms such as carbapenemase-producer Enterobacterales, Acinetobacter baumannii, and Klebsiella pneumoniae. Yet resistance to this last-line drugs is a major public health threat and is rapidly increasing. Polymyxin S2 (S2) is a polymyxin B analogue previously synthesized in our institute with obviously high antibacterial activity and lower toxicity than polymyxin B and colistin. To predict the possible resistant mechanism of S2 for wide clinical application, we experimentally induced bacterial resistant mutants and studied the preliminary resistance mechanisms. Mut-S, a resistant mutant of K. pneumoniae ATCC BAA-2146 (Kpn2146) induced by S2, was analyzed by whole genome sequencing, transcriptomics, mass spectrometry and complementation experiment. Surprisingly, large-scale genomic inversion (LSGI) of approximately 1.1 Mbp in the chromosome caused by IS26 mediated intramolecular transposition was found in Mut-S, which led to mgrB truncation, lipid A modification and hence S2 resistance. The resistance can be complemented by plasmid carrying intact mgrB. The same mechanism was also found in polymyxin B and colistin induced drug-resistant mutants of Kpn2146 (Mut-B and Mut-E, respectively). This is the first report of polymyxin resistance caused by IS26 intramolecular transposition mediated mgrB truncation in chromosome in K. pneumoniae. The findings broaden our scope of knowledge for polymyxin resistance and enriched our understanding of how bacteria can manage to survive in the presence of antibiotics.

Background: Sepsis has become a global health concern owing to its increasing incidence and high mortality rate. In the present study, we investigated a novel drug candidate ASK0912 on its protective effects in mice with Acinetobacter baumannii 20-1-induced sepsis, and studied the related mechanisms.Material and methods: To analyze the protective effect of ASK0912 on septic mice, survival rates, body temper-ature, organ and blood bacterial loads, white blood cell and platelet counts, organ damage, and cytokine levels were determined.Results: ASK0912 remarkably increased the survival rate of mice with sepsis induced by A. baumannii 20-1 at a low dose of 0.6 mg/kg. Rectal temperature measurements showed that ASK0912 treatment prevented the body temperature decrease of septic mice to some extent. Treatment with ASK0912 can notably reduce the organ and blood bacterial loads and alleviate platelet count reduction due to sepsis. ASK0912 attenuated organ damage, including reduced levels of total bile acids, urea, and creatinine, aggregation of inflammatory cells, and miti-gation of structural changes in septic mice, as demonstrated by biochemical analysis and hematoxylin & eosin staining. Additionally, multiplex assay showed that abnormally increased cytokine levels (IL-1 & beta;, IL-3, IL-5, IL-6, IL-10, IL-13, MCP-1, RANTES, KC, MIP-1 & alpha;, MIP-1 & beta;, and G-CSF) in septic mice decreased after ASK0912 treatment.Conclusions: ASK0912 can not only improve the survival rate, hypothermia, lower the bacterial loads in the organs and blood, but also alleviate the pathophysiological manifestations such as intravascular coagulation abnormalities, organ damages, and immune system disorder of sepsis mice induced by A. baumannii 20-1.