Gram-negative bacterial (GNB) bloodstream infections (BSIs), particularly those caused by carbapenem-resistant GNB (CR-GNB) and Acinetobacter baumannii (AB), are associated with substantial morbidity and mortality. This study aimed to provide, to our knowledge, the first comprehensive meta-analysis integrating overall GNB-BSI risk factors with both pathogen-specific and resistance-defined subgroup analyses. A systematic literature search was conducted across PubMed, Web of Science, Embase, the Cochrane Library, and four major Chinese databases from inception to August 2025. We included observational studies evaluating risk factors for GNB-related BSIs in patients with GNB infections. Two reviewers independently performed study screening, data extraction, and quality assessment. Methodological quality was evaluated using the Newcastle–Ottawa Scale and the Agency for Healthcare Research and Quality tool. Meta-analyses were performed using R software. Pooled odds ratios (ORs) or standardized mean differences (SMDs) with 95

Pathogenic bacteria continually evolve under antimicrobial pressure through acquired resistance genes, making it crucial to understand their evolutionary strategies. We identify a clinical Klebsiella pneumoniae isolate resistant to ceftazidime/avibactam (CZA), harboring heterogeneous multicopy bla CTX-M , among which a bla CTX-M-249 variant mediates CZA resistance. Both bla CTX-M-249 and its closely related allele bla CTX-M-65 are dominant within the clonal population and are located at two loci on the same plasmid, with their proportions shifting under antibiotic pressure. Using experimental and mathematical models, we demonstrate that the heterogeneous arrangement of bla CTX-M variants on the same plasmid confers greater stability and competitive advantage than that across separate plasmids, particularly during drug switching. Re-analysis of large genomic datasets supports the universality of this phenomenon. Our findings reveal an evolutionary strategy in which β-lactamase genes, through multicopy heterogeneity on a single plasmid, ensure stable inheritance of resistance and enhance bacterial adaptability under fluctuating clinical antibiotic pressures.

OBJECTIVES:To investigate the genomic and resistance features of a blaNDM-1-positive Acinetobacter soli isolate and assess the global distribution and genomic traits of A. soli. METHODS:We isolated a blaNDM-1-positive A. soli strain from a patient in 2023. Antimicrobial susceptibility testing was performed and whole-genome sequencing was used to investigate the genomic characteristics and resistance features of A. soli. Conjugation was performed to assess the transferability of the blaNDM-1-carrying plasmid, and plasmid stability was evaluated using serial-passaging experiments. Additionally, genomic analysis of 74 publicly available A. soli genomes was performed to explore their global distribution, resistance genes, plasmid replicons and phylogeographic relationships. RESULTS:SZL7 exhibited resistance to meropenem (8 mg/L) and ceftazidime (≥64 mg/L), but remained susceptible to tigecycline, polymyxin B, fluoroquinolones, cefiderocol and cefoperazone-sulbactam. Among the 74 global genomes, blaNDM-1 was detected in 9.46% (7/74) of the isolates. The blaNDM-1 gene was located on a plasmid and was stably inherited after 10 days of serial passage. A total of 83.78% of the isolates originated from Asia, particularly China and Japan. Notably, all Chinese isolates were collected from coastal cities, with environmental samples predominating, indicating the importance of environmental monitoring in these high-risk areas. CONCLUSIONS:This study presents the first global genomic analysis of A. soli, highlighting its potential as a reservoir and vector for clinically significant resistance genes, such as blaNDM-1. These findings emphasize the importance of continuous genomic surveillance and environmental monitoring within the One Health framework to mitigate emerging resistance threats.

OBJECTIVES:Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major cause of hospital- and ventilator-associated pneumonia (HAP/VAP) with limited treatment options. Eravacycline (ERA) shows potent in vitro activity and favorable pulmonary pharmacokinetics, but clinical evidence for CRAB HAP/VAP remains scarce. We evaluated the real-world effectiveness and safety of ERA. METHODS:We conducted a retrospective multicenter cohort study at 12 hospitals in China (April 2022-March 2024). Adults with microbiologically confirmed CRAB HAP/VAP treated with ERA or best available therapy (BAT) for ≥ 4 days were propensity scores matched. Primary outcomes were clinical success at end of treatment (EOT) and 28-day mortality. RESULTS:After 3349 patients screened, 382 were included (ERA 204; BAT 178). Baseline characteristics were balanced. Clinical success at EOT was higher with ERA than BAT (63.2% vs 55.1%), while 28-day mortality was similar (40.1% vs 42.0%). ERA achieved higher microbiological eradication at EOT (45.1% vs 32.0%) and day 14 (36.8% vs 22.5%). Adverse events occurred less frequently with ERA. In targeted first-line treatment, ERA showed higher clinical success, greater microbiological eradication and faster clinical response without increased mortality. CONCLUSION:Eravacycline improved microbiological clearance, clinical response, and safety compared with BAT for CRAB HAP/VAP, particularly when used as targeted first-line therapy.

Many phages encode proteins that specifically inhibit host RNA polymerase activity, thereby sabotaging and, in some cases, hijacking the host transcription machinery to serve their needs. Traditional methods for identifying new phage proteins that inhibit bacterial transcription are labor intensive and require access to live phages. To overcome these limitations, we develop a highly efficient pipeline for AlphaFold 3-guided discovery of phage proteins that inhibit bacterial transcription initiation. Using this pipeline, three phage proteins are identified and characterized. Structural and biochemical analyses demonstrate that these phage proteins bind to distinct sites on RNA polymerase and inhibit transcription initiation via different mechanisms. This study showcases the power of AlphaFold 3 in discovering novel binders of large protein complexes, and the pipeline developed here could be readily adapted to screen modulators of other large targets, such as the ribosome, proteasome, and CRISPR-Cas systems.

BACKGROUND:This study investigate the molecular epidemiology and population characteristics of 184 Acinetobacter baumannii clinical isolates collected in Zhejiang (2015-2020), shedding light on the persistence and success of ST208-KL2 lineage. METHODS:Whole-genome sequencing and bioinformatic analysis characterized the population structure and transmission dynamics of 184 A. baumannii. Plasmid analysis identified the surrounding structures of blaOXA-58 and blaNDM-1 genes. Serum inhibition assays and Galleria mellonella virulence experiments, combined with global epidemiological analysis, explored the persistence and dominance of ST208-KL2. RESULTS:Of the isolates, approximately 78.8% (145/184) exhibited carbapenem resistance, primarily driven by blaOXA-23, blaOXA-72, blaOXA-58, and blaNDM-1. The A. baumannii population was divided into 2 clusters, distinguished by differences in ST types and resistance islands profiles. Cluster2 accounted for 71 department-time transmission events, mainly involving long-term interdepartmental spread, particularly in the rehabilitation medicine department and intensive care unit. Recombination analysis revealed that recombination occurred mainly in capsular locus (KL) regions, prophage regions, and predicted protein regions. Phenotypic experiments found that ST208-KL2 isolates displayed increased virulence and greater resistance to serum inhibition compared to ST208-KL7 isolates. This finding likely explains its persistently higher prevalence throughout the study period, except in 2019. CONCLUSIONS:Cluster2 isolates demonstrated significant advantages in ARG prevalence, resistance rates, and transmission capacity. ST208-KL2, with its superior serum resistance and virulence compared to ST208-KL7, has emerged as a dominant lineage with global implications for infection control and public health.

BACKGROUND:Cefiderocol (CFDC) is a novel and potent antimicrobial agent; however, the emergence of CFDC resistance is increasingly concerning. This study investigated the mechanisms underlying CFDC resistance in ST11 carbapenem-resistant Klebsiella pneumoniae (CRKP) and explored potential therapeutic strategies against CFDC-resistant strains. METHODS:Two isogenic ST11 CRKP isolates, KP399 (CFDC-susceptible) and KP400 (CFDC-resistant), were obtained from a single patient. Whole-genome sequencing, genetic knockout, plasmid curing, complementation assays, real-time quantitative PCR, and antimicrobial susceptibility testing (AST) were performed to elucidate the resistance mechanisms. AST of CFDC in combination with β-lactamase inhibitors (BLIs), along with computational structural modeling, was conducted to evaluate BLI activity against SHV-12. RESULTS:An ISKpn74 insertion sequence in the cirA promoter significantly reduced cirA transcription and was associated with decreased CFDC susceptibility. SHV-12 was identified as the predominant β-lactamase conferring CFDC resistance. High-level resistance required the combined effects of reduced cirA expression (<0.5 relative units) and SHV-12 production. In silico analyses predicted that relebactam exhibits the strongest binding affinity to SHV-12, attributable to its bulky C2-linked piperidine ring that enables additional interactions with the Asp104 residue, together with its slow deacylation kinetics. Consistently, AST showed that combining CFDC with relebactam markedly enhanced antimicrobial activity against SHV-12-producing CRKP compared with other BLIs. CONCLUSIONS:This study identifies a novel CFDC resistance mechanism and underscores the importance of assessing regulatory regions when evaluating resistance determinants. It also supports the potential use of relebactam as an effective adjunct to CFDC against SHV-12-producing, CFDC-resistant CRKP.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a persistent nosocomial pathogen, posing a major global health threat owing to limited treatment options. Although tigecycline is still effective against CRAB, resistance emergence has become a critical concern. This study aimed to elucidate the in vivo evolutionary mechanisms underlying tigecycline resistance in CRAB. A total of 11 STpas2-SToxf540 CRAB strains were recovered from rectal swabs, sputum samples, and the surrounding environmental specimens of an intensive care unit (ICU) hospitalized patient who had received tigecycline treatment. CgSNP analysis confirmed environment-to-patient transmission among these CRAB isolates. Comparative genomic analysis indicated that mutations in adeS, pgaA, and gbsA may be associated with tigecycline resistance. In situ mutagenesis and antimicrobial susceptibility testing verified that the adeS N125S mutation mediates tigecycline resistance and collateral sensitivity to cefoperazone/sulbactam. Growth curve assays demonstrated that the adeS N125S mutation imposes a fitness cost on CRAB. Transcriptional analysis showed that the adeS N125S mutation drives the development of tigecycline resistance by upregulating the expression of the AdeABC efflux pump. By examining 45,377 publicly available global A. baumannii genome sequences, we characterized the molecular epidemiology of the STpas2-SToxf540 lineage and the distribution of adeS mutations. Our findings underscore the need to strengthen surveillance and rational antimicrobial use to prevent CRAB dissemination and antimicrobial resistance evolution, particularly in ICUs.

Abstract Tailocins are phage tail-like bacteriocins (PTLBs) thought to be remnants of prophages that have lost the ability to package viral genomes while retaining the ability to kill closely-related bacterial strains, thereby mediating bacterial competition. Tailocins produced by Pseudomonas aeruginosa are referred to as pyocins. Apart from their contribution to ecological fitness, they also have the potential to be harnessed as highly-specific antimicrobials to treat antibiotic resistant bacterial infections. Although pyocins lack the genetic components to package viral genomes, pyocin-encoding gene clusters share a high degree of genetic homology to phage tail genes, attributed to their shared ancestry. This poses a significant annotation-based challenge, as current prophage prediction tools, which rely on phage homology for prediction, can misclassify pyocins or tailocins as prophages. Pyocins unknowingly being misannotated as prophages is not only a bioinformatic issue, but can certainly confound experiments examining bacterial competition and prophage induction, if the experimental setup is based on this unintentional misannotation. In this study, we present “TattleTail”, the first version of a bioinformatic tool designed to accurately identify tailocins in genome sequences, with a focus on identifying phage tail-derived pyocin-encoding gene clusters in P. aeruginosa in its first iteration. The tool leverages conserved pyocin gene cluster markers and accounts for the absence of canonical phage features, such as capsid, terminase and integrase genes, thereby distinguishing pyocins from intact and cryptic prophages. Validation in P. aeruginosa and non- P. aeruginosa genomes confirmed the presence of pyocin regions in all P. aeruginosa genomes, while none were detected in any non- P. aeruginosa genomes. Notably, TattleTail enabled the identification of representative pyocin-encoding gene clusters in clinical P. aeruginosa isolates. The identified pyocins in the clinical isolates were induced using mitomycin C, visualized via transmission electron microscopy, processed via tangential flow filtration and demonstrated bactericidal activity, thereby confirming TattleTail predictions. TattleTail aims to complement existing prophage prediction tools during genomic analyses involving phage-derived elements in bacterial genomes, allowing more accurate identification of these elements facilitated by robust discrimination between prophages and tailocins.

Acinetobacter baumannii is a formidable nosocomial pathogen whose multidrug resistance and immune evasion capabilities present a major therapeutic challenge. This study identifies the type VI secretion system (T6SS) effector VgrG2 as a critical virulence regulator in A. baumannii. VgrG2 employs a two-pronged strategy to undermine host innate immunity. First, it transcriptionally represses the major adhesin Csu pilus, reducing bacterial recognition, phagocytic uptake, and neutrophils recruitment. Second, VgrG2 directly targets and hyperactivates the small GTPase Rac1 within phagocytes. This leads to actin cytoskeletal disarray, which drives excessive macropinocytosis, resulting in compromised phagocytosis and methuosis-a non-apoptotic, vacuole-dependent death specific to phagocytes. Thus, VgrG2 selectively eliminates key immune sentinels and alters pulmonary inflammation, promoting bacterial persistence and dissemination. These findings extend the role of T6SS effectors beyond interbacterial competition to include sophisticated eukaryotic-directed attacks, and highlight the VgrG2-Rac1 axis as a promising therapeutic target.

β-Lactam antibiotics are the cornerstone of therapy for Pseudomonas aeruginosa infections. Their effectiveness, however, has been increasingly compromised by antimicrobial resistance. This article presents a perspective on the evolving challenges and strategies associated with β-lactam resistance in P. aeruginosa, with a particular focus on China. The perspective was developed through a focused narrative review of recent clinical, epidemiological, and mechanistic studies. We first outline the current epidemiology of β-lactam-resistant P. aeruginosa, highlighting regional trends and the dissemination of high-risk clones. We then review the evolving β-lactam resistance mechanisms, emphasizing the prevalence and variety of carbapenemases. Finally, we discuss therapeutic strategies for managing resistant infections. This includes diagnostic advances, rational selection of novel β-lactam agents, dosing optimization, and combination regimens. Together, these insights provide a structured framework for understanding and managing β-lactam-resistant P. aeruginosa in contemporary clinical practice.

IntroductionCarbapenem-resistant Acinetobacter baumannii (CRAB) poses a global health threat due to limited therapeutic options. Cefiderocol, a novel siderophore-conjugated cephalosporin, demonstrates potent in vitro activity against a variety of Gram-negative bacteria, including CRAB. However, cefiderocol's recent introduction into clinical practice means that while several resistance mechanisms have been identified, their full characterization remains incomplete, and novel mechanisms are likely to emerge as clinical use expands.MethodsIn this study, in vitro evolution experiments were performed under cefiderocol pressure using a clinical CRAB isolate as the parental strain to investigate cefiderocol resistance mechanisms.ResultsGenomic comparison and mutant reconstruction revealed that mutations in pbp1b (G271R) and the intergenic region upstream of piuA (-6_-8del) conferred a 4-fold increase in cefiderocol MIC. In silico predictions and EMSA confirmed that the intergenic mutation occurred within the piuA promoter region. qRT-PCR analysis indicated that the piuA promoter mutation significantly reduced piuA expression. This downregulation of piuA expression impaired cefiderocol transport, leading to reduced susceptibility to cefiderocol. A search across publicly deposited genome sequences identified ten piuA promoter mutations in A. baumannii. Among these, the−-6_-8del was the most commonly observed variant, which was primarily associated with ST2Pas/ST457Oxf, ST2Pas/ST208Oxf, and ST499Pas. Functional characterization of the−-6_-8del variant and three additional promoter mutations (-18del, A-17T&-18del, A-17T&-14del) confirmed suppression of piuA expression and reduction of cefiderocol susceptibility.DiscussionCombining in vitro evolution with publicly available A. baumannii genome sequences, this study demonstrates promoter-mediated piuA downregulation as a cefiderocol resistance mechanism in A. baumannii, providing a genomic target for predicting cefiderocol resistance.

Background: The transmission of carbapenem-resistant Pseudomonas aeruginosa (CRPA) between hospital environment and patients poses significant challenges for clinical management. The COVID-19 pandemic may have influenced bacterial transmission dynamics in intensive care units (ICU). This study aimed to prospectively investigate the temporal and spatial spread of P. aeruginosa after a COVID-19 upsurge period in China. Methods: We routinely screened for P. aeruginosa in both the environment and patients in a newly-opened 21-bed tertiary teaching hospital ICU in eastern China from October 2022 to April 2023, during which a COVID-19 upsurge occurred from December 2022 to January 2023. Whole-genome sequencing and antibiotic susceptibility testing were performed on all non-repetitive P. aeruginosa isolates. Results: Among 1694 environmental samples, 40 (2.36%) samples were CRPA. In 1576 nasopharyngeal and rectal samples (from 353 patients), 108 samples (6.86%) were CRPA. Sequence type (ST) 463 was the most prevalent clone in both patient and environmental samples. Spatiotemporal distribution and genomic data revealed sporadic patients-related transmission before COVID-19 upsurge period, while high-risk ST463 clone transmission was detected during COVID-19 upsurge period. However, there was no strong evidence to show that antibiotic consumption significantly influenced CRPA transmission in this study. Additionally, the evolution events of blaKPC (from blaKPC-2 to blaKPC-71) were observed, resulting in multi-sites CRPA colonization in one patient. Conclusion: Our prospective study demonstrates that COVID-19 upsurge is associated with increased P. aeruginosa transmission. These findings provide valuable insights into nosocomial infection management during future public health crisis. We also reported carbapenemase mutation from blaKPC-2 to blaKPC-71 in P. aeruginosa, which provides reference for further antibiotic usage.

Carbapenem-resistant Acinetobacter baumannii (CRAB) poses significant challenges in intensive care units (ICU) due to its multidrug resistance and environmental persistence. To date, most studies have focused on clinical isolates or patient-environment transmission. However, high-touch mobile surfaces have been less extensively characterized. We collected 49 CRAB isolates in 2019 and 2021 from ICU patients (n = 32) and trolley surfaces (n = 17). 91.84

Multidrug-resistant Acinetobacter baumannii has emerged as one of the most antibiotic-resistant bacterial pathogens associated with nosocomial infection, with its resistance highly depending on multiple multidrug efflux pumps. Here, we report the cryoelectron microscopy (cryo-EM) structure of Acinetobacter drug efflux G (AdeG), the inner membrane component of one of three important resistance-nodulation-cell division (RND) pump family members in A. baumannii, which is involved in drug resistance to chloramphenicol, trimethoprim, ciprofloxacin, and clindamycin. We systematically compare the structures and substrate binding specificities of AdeG, AdeB, and AdeJ multidrug efflux pumps via molecular docking, revealing potential determinants for drug binding. Knockout experiments demonstrate a functional complementarity between AdeABC, AdeFGH, and AdeIJK. Our study provides a structural understanding of A. baumannii multidrug efflux pump AdeG and reveals complementary drug efflux activity between AdeG and other RND efflux pumps, which may promote further rational drug discovery efforts targeting multidrug efflux pumps.

The extensively drug-resistant (XDR) Pseudomonas aeruginosa ST463 strains, which co-harbor plasmid-associated metallo-β-lactamase (MBL) and blaKPC-2 genes, exhibit significant resistance and virulence, posing great clinical treatment challenges. Here, we report on three XDR P. aeruginosa ST463 strains, PA64, PA3117, and PA30, all carrying two plasmid types. One plasmid was a ~450 kb IncP-2-type megaplasmid named pPA64_1, pPA3117_1, and pPA30_1 in strains PA64, PA3117, and PA30, respectively. The other plasmid was a type I plasmid named pPA64_2, pPA3117_2, and pPA30_2 in strains PA64, PA3117, and PA30, respectively, harboring the blaKPC-2 gene in the core genetic platform ISKpn27- blaKPC-2-ISKpn6. The blaKPC-2 gene copies were associated with IS26-mediated inversion or duplication events. Notably, the IncP-2 megaplasmids pPA64_1, pPA3117_1, and pPA30_1 were associated with a variable ~57.3 kb Tn1403-like transposon named Tn6485g, Tn6485h, and Tn6485f, respectively. Tn6485g carried the MBL gene blaIMP-45, which was located in the class 1 integron In786, followed by an ISCR1-associated armA module and the IS26-composite transposon Tn6309. On this basis, other ISCR1-associated modules (ISCR1-qnrVC6, ISCR1-blaPER-1, and ISCR1-blaAFM-1) were inserted between In786 derivatives and ISCR1-armA, resulting in a novel transposon, Tn6485h, carrying two MBL genes, blaIMP-45 and blaAFM-1. In contrast to Tn6485h, Tn6485f had another inserted copy of ISCR1-qnrVC6. We inferred that the evolution of the Tn1403-like transposon might be driven by the recruitment of ISCR1-associated antimicrobial resistance (AMR) modules under antibiotic pressure in a clinical setting.

Antimicrobial resistance (AMR) is a critical global health challenge, demanding rapid and accurate diagnostics to guide timely antimicrobial therapy. Current diagnosis is hindered by prolonged culturing and difficulties detecting low pathogen loads. Here, we present a culture-free diagnostic platform that integrates microfluidics, Raman micro-spectroscopy, and deep learning to deliver “sample-to-report” testing within 20 min. The microfluidic enrichment system employs dialysis-dielectrophoresis (DEP) technology to rapidly isolate pathogens directly from clinical samples with a detection limit as low as <2 colony forming unit (CFU)/ml. Combining a single-cell Raman fingerprint database of 342 clinical isolates from 29 bacterial and 7 fungal species with a 1D ResNet deep learning model, our approach achieved 95.1% accuracy in lab settings. Validated in a 305-patient clinical study involving primary urine and other clinical samples, it demonstrated 95.4% agreement with traditional culture methods and 98.5% sensitivity in diagnosing infections. While broader validation is needed for clinical implementation, the integrated, rapid diagnosis pipeline, as well as broad-spectrum detection, offer a promising solution for next-generation diagnostics for combating AMR.

DNA-damage chemicals, including many antibiotics, often induce prophage induction and phage outbreaks within microbial communities, posing a significant threat to bacterial survival. Moraxellaceae strains are clinically relevant due to their remarkable resistance to antibiotics and radiation. However, the cellular-level regulation mechanisms that underlie their DNA damage response and anti-phage defense remain extensively unexplored. Here, we report a WYL family protein, DdaA, that has replaced the ubiquitous SOS system during the evolution of Moraxellaceae. DdaA functions as an activator and directly regulates the transcriptional networks of both DNA damage response and anti-phage defense genes under conditions of DNA damage stress. Our findings elucidate a pathway that shows how these bacteria enhance their immunity under DNA damage and shed light on controlling the resistance of Moraxellaceae strains in clinical practice.