Candida infections are among the most common fungal diseases worldwide and are associated with notable morbidity and mortality, particularly in immunocompromised and critically ill patients. Moreover, the emergence and spread of antifungal resistance pose an increasing threat to global public health. Therefore, evidence-based recommendations are urgently needed to guide the surveillance and control of Candida infections. A multidisciplinary guidance development group comprising experts of infectious diseases, clinical microbiology, pharmacology, infection control, and guideline methodology jointly developed the present clinical practice guidance. The process followed internationally recognized grading of recommendations, assessment, development and evaluation (GRADE) framework. Clinical questions were formulated based on priority issues or areas of uncertainty in current practice, and corresponding recommendations were developed after systematic review and synthesis of the best available evidence. The strength of recommendations was classified as strong or conditional (weak), and the certainty of evidence was graded from high to low. The guidance provides recommendations on diagnostic strategies, antifungal therapy, and infection prevention and control measures for Candida infections, integrating both global evidence and national clinical experience. The target users include clinicians, microbiologists, infection control practitioners, and policymakers involved in the management of fungal diseases. The guidance aims to support optimized clinical decision-making, improve antifungal stewardship, and strengthen infection control strategies to address the growing challenge posed by Candida infections.

AIMS:This study aims to identify predictive plasma protein biomarkers for anti-tuberculosis drug-induced liver injury (ATB-DILI) and develop machine learning models for pre-treatment risk stratification. METHODS:In this retrospective nested case-control study, proteomic profiling of pre-treatment plasma from 24 patients (12 ATB-DILI, 12 controls) identified differentially expressed proteins, which were validated by ELISA in an independent cohort (35 ATB-DILI, 37 controls). Multiple machine learning algorithms were implemented to develop clinical prediction models and evaluate the prognostic value of the identified protein biomarkers. RESULTS:Proteomic analysis of pre-treatment samples from the exploratory cohort identified five significantly differentially expressed proteins: antithrombin III, apolipoprotein D, carboxypeptidase B2, Chromogranin-A, and Retinol-binding protein 4. These proteins are functionally implicated in inflammatory responses, oxidative stress, and drug metabolism pathways. Validation using baseline plasma from an independent cohort confirmed consistent expression patterns for all five proteins (p < 0.01), with directional changes matching the discovery phase findings. The random forest model, built on these pre-treatment biomarkers, demonstrated robust predictive performance in the test set (AUC = 0.94, sensitivity = 90.0%, specificity = 90.0%, accuracy = 0.90). Importantly, consensus across multiple machine learning approaches (GBDT, SVM, GBM, etc.) confirmed predictive stability and generalizability of this protein signature (inter-model AUC range: 0.85-0.96). CONCLUSION:This study has successfully identified five pre-treatment plasma protein signature that, when incorporated into machine learning models, may enable the prediction of ATB-DILI risk, offering potential for early intervention in tuberculosis therapy.

The ESX-3 secretion system serves as a core component in maintaining metal ion homeostasis in mycobacteria, playing an indispensable role in the acquisition of essential elements such as iron and zinc. As a critical virulence determinant, its functional scope extends to immune modulation, cell wall integrity, and antibiotic resistance. This review synthesizes current knowledge on the genetic architecture, evolutionary trajectory, and structural composition of ESX-3, revealing its complex evolutionary history involving both vertical inheritance and horizontal gene transfer via plasmids. We explore its multifaceted biological functions in pathogenesis and its emerging link to antibiotic susceptibility. We also detail its sophisticated regulatory network, governed by metal-dependent transcription factors (IdeR, Zur, MntR), toxin-antitoxin systems, and oxidative stress pathways. Furthermore, we explore its multifaceted biological functions in pathogenesis and its emerging, complex link to antibiotic susceptibility. By integrating existing literature with our preliminary findings, this work provides a comprehensive overview of ESX-3, highlighting its potential as a novel therapeutic target and outlining future research directions to unravel its full functional and mechanistic spectrum.

Two carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates were collected, which lacked known carbapenemases but produced a β-lactamase CAE-1. CAE-1 was recently found in Comamonas aquatica and conferred resistance to penicillins and cephalosporins. In this study, we aim to know whether CAE-1 was responsible for the carbapenem resistance in the CRPA isolates and its enzyme hydrolyzing characteristics. Both CRPA clinical isolates exhibited minimal inhibitory concentrations (MICs) of 8 µg/mL for imipenem and meropenem, with a positive result in the modified Hodge test for meropenem. The blaCAE-1 and blaKPC-2 were cloned and expressed in P. aeruginosa PAO1 and Escherichia coli DH5α, respectively. The blaCAE-1-carrying PAO1 transformant also tested positive using the modified carbapenem inactivation method and was resistant to piperacillin-tazobactam, ticarcillin-clavulanate, and cefoperazone-sulbactam, but susceptible to ceftazidime-avibactam. Expression of blaCAE-1 in P. aeruginosa PAO1 elicited a 64- to 128-fold increase in MICs for piperacillin, ceftazidime, cefepime, and aztreonam, and an eightfold increase for meropenem, exhibiting a broader resistance profile than in E. coli DH5α. Steady-state kinetic assays showed that CAE-1 had catalytic efficiency against all β-lactams tested, with comparatively lower efficiency against three carbapenems relative to KPC-2, while demonstrating approximately equivalent efficiency for the other β-lactam antibiotics tested. Whole-genome sequencing revealed that blaCAE-1 was a class A β-lactamase and encoded on an integrative and conjugative element, which might facilitate its horizontal transfer. The class A β-lactamase CAE-1 is a carbapenemase posing a high risk for horizontal dissemination. Enhanced surveillance for blaCAE-1-harboring isolates is needed.

BACKGROUND:Klebsiella pneumoniae is a key opportunistic pathogen, and its emerging hypervirulent strains pose a growing public health threat. An association exists between the phospholipid transporter MlaFEDCB and bacterial virulence; however, its regulatory role and underlying mechanisms remain elusive. Herein, we focused on K. pneumoniae virulence regulation via mlaFEDCB under in vitro and in vivo conditions. METHODS AND RESULTS:Homology analysis showed that mlaFEDCB gene cluster is highly conservative among gram-negative bacterial strains and is contiguously arranged and co-transcribed within the genome. Experiment involving murine intraperitoneal infection revealed that mice infected with KP-ΔmlaFEDCB strain showed substantially prolonged survival (P = .0005). Furthermore, transcriptomic analysis showed altered expression of virulence-associated genes, especially fimH and fimD, which are involved in fimbrial structure and host cell adherence. Scanning electron and transmission electron microscopy showed that the WT-KP strain demonstrated a complex fibrous fimbrial network, with several long, thin structures interwoven with those of neighboring bacteria, whereas the KP-ΔmlaFEDCB strain showed markedly fewer fimbriae and lacked the fimbrial network. Furthermore, bladder epithelial cells adhesion assay showed an apparent reduction in the KP-ΔmlaFEDCB strain compared with the WT-KP strain. The growth of the KP-ΔmlaFEDCB strain was significantly compromised in comparison to the wild-type strain under stress conditions. CONCLUSIONS:Thus, mlaFEDCB gene cluster increases the adhesion, invasion, and environmental adaptability of K. pneumoniae by modulating virulence-related gene expression, pilus synthesis, and growth under stress conditions.

Background:Infections due to multidrug- and extensively drug-resistant bacteria can be difficult to treat and are associated with high mortality and burden of disease. Ceftazidime-avibactam (CVA) is used in patients with multidrug-resistant Gram-negative bacterial infections. Objective:To describe the real-world usage, effectiveness, and antimicrobial features of CVA in clinical practice in China. Methods:This multicenter, prospective observational study collected medical record data from adult patients (≥18 years) who had been hospitalized and treated with ≥1 dose of CVA. Clinical and microbiological outcomes were evaluated at the end of treatment, with clinical cure defined as resolution of infection following treatment with CVA. Results:Data were obtained from 220 adult patients receiving ≥1 dose of CVA in China. Infections indicated for CVA included pneumonia (64.5%), complicated intra-abdominal infection (16.8%) and bloodstream infection (7.3%). Klebsiella pneumoniae (129/227 isolates, 56.8%) was the most commonly identified pathogen, followed by Pseudomonas aeruginosa (33 isolates, 14.5%). Of 87 K. pneumoniae isolates, 76 (87.4%) were meropenem resistant and the same proportion carried cabapenemase genes. Low levels of resistance to CVA were observed across all isolates. Three-quarters of patients received CVA as definitive therapy. Clinical cure at end of treatment was achieved in 66.4% of patients and microbiological success in 69.6% of patients. Conclusion:Findings from this study provide important real-world data on treatment patterns, microbiological features, and clinical and microbiological outcomes for CVA in routine clinical practice in China.

A novel fosfomycin-resistant glutathione S-transferase (FR-GST) gene, fosZ, was investigated, and its structural characteristics were characterized in Pseudomonas species. The fosZ gene was cloned and expressed in P. aeruginosa PAO1 and HS355, where it displayed reduced susceptibility to fosfomycin (8- to 64-fold) and its inhibitor sodium phosphonoformate (PPF). FosZ shares less than 70% amino acid identity with known FosA proteins. Bioinformatics analyzes revealed that fosZ was a transposable passenger gene within ISPa75, likely captured from Pseudomonas species. A total of 159 fosZ-bearing Pseudomonas strains were identified in GenBank over the past 22 years, sharing ten target site duplications (TSDs) associated with ISPa75. Among them, 34 strains were fully sequenced. ISPa75-fosZ was found on at least two chromosomes and 33 plasmids from four incompatibility groups, including the megaplasmids IncP-2 (24/33) and IncpJBCL41 (8/33), which also carried carbapenem and other antibiotic resistance genes. The most common TSDs were TSD6 (82/156) and TSD10 (48/156), predominantly in carbapenem-resistant P. aeruginosa of various sequence types, especially ST1076, ST1418, and ST463. Mobilization of ISPa75 between plasmid and chromosome was observed in P. aeruginosa MAS152. Structure prediction and analysis of FR-GST proteins revealed distinct features in the dimer interface loop. In the FosZ structure, an expanded K+-binding loop caused a deviation at residue S95, which participated in binding both fosfomycin and PPF. In conclusion, fosZ encodes a distinct FR-GST exhibiting reduced susceptibility to inhibition by PPF and has been acquired by transferable ISPa75 on multidrug-resistant megaplasmids, disseminating fosfomycin resistance in Pseudomonas species, particularly across China.

BACKGROUND:Previous research indicated that physicians possess limited knowledge of the diagnosis and treatment of invasive fungal disease (IFD). OBJECTIVE:This study aimed to assess the efficacy of training via video or PDF formats in increasing physicians' knowledge of IFD. METHODS:This was a multicentre, cluster-randomised controlled trial involving 18 tertiary hospitals in China. Physicians specialising in IFD clinical diagnosis and treatment from four departments were randomised 1:1 into a video training group or PDF training group, and questionnaires were completed before and after training. The primary outcome was the change in total questionnaire score before and after training. RESULTS:Of the 294 participants, 146 were assigned to the video group and 148 to the PDF group. Engagement with the training materials was observed among 127 participants from the video group and 135 from the PDF group. In the per-protocol set (PPS), post-training score improvements for total scores (p = 0.008), invasive candidiasis scores (p < 0.001), and invasive aspergillosis scores (p = 0.044) in the video group were significantly greater than those in the PDF group. Overall, 161 (61.5%) physicians in the PPS exhibited enhanced total scores post-training, with the video group outperforming the PDF group (70.9% vs. 52.6%, p = 0.002). The findings in the FAS were largely consistent with those observed in the PPS. CONCLUSION:Both video and PDF training modules are appealing to physicians. Further, the video training module displayed superior efficacy in improving physicians' knowledge of IFD.

Despite targeting by CRISPR-Cas system, antimicrobial resistance plasmids are prevalent in clinical isolates of carbapenem-resistant Klebsiella pneumoniae which represent a major public health threat. A stable co-existence of plasmids and CRISPR-Cas systems is mediated by anti-CRISPR (Acr) proteins. Here, we report that previously identified AcrIE10 encoded by a resistance plasmid combines two functions: it inhibits CRISPR immunity by directly binding Cas7* subunit through its Acr domain, and acts as an Acr-associated (Aca) protein that self-represses the transcription of Acr locus. AcrIE10 is an example of an Aca protein that utilizes N-terminal ribbon-helix-helix (RHH) domain to specifically recognize the inverted repeat (IR) region in its own promoter. Crucially, a dimerization of AcrIE10 dimers is required for the effective binding to the IR and self-repression, while stoichiometry-dependent interaction with Cas7* facilitates transition to de-repressed state. These findings elucidate molecular mechanisms by which AcrIE10 operates as a dual functionAcr-Aca protein to achieve a delicate balance between host CRISPR-Cas immunity and plasmid anti-defense.

MBL-producing Enterobacterales pose an urgent global healthcare problem with limited treatment options. We aimed to gain a greater understanding of international perspectives on the management of serious infections due to MBL-producing Enterobacterales, using a modified Delphi process to assess expert-based opinion and establish consensus. Per Figure 1, a Delphi panel comprising 10 infectious disease experts with experience in treatment of antimicrobial-resistant bacterial infections completed a survey on epidemiology, disease, testing, and optimal management of infections caused by MBL-producing Enterobacterales. Draft consensus statements developed from the survey results were reviewed by the panel and either endorsed or edited to achieve unanimous consensus during a virtual meeting. The consensus statements showed that all panelists identified MBL-producing Enterobacterales as a major source of antimicrobial resistance and infections in most countries. All panelists agreed that these infections are associated with high morbidity and mortality, potential need for critical-level care, increased length of hospitalization, and significant healthcare costs. In addition, the adverse impact of MBL was exacerbated by inappropriate use of multidrug antimicrobial regimens; hence, an MBL-targeting agent was deemed preferable. When aztreonam-avibactam (ATM-AVI) and cefiderocol are available, based on in vitro data showing activity of ATM-AVI against > 90% of New Delhi MBL-producing Enterobacterales, the preferred first-line treatment should be ATM-AVI. Where ATM-AVI and cefiderocol are not available, treatment options are limited to the co-administration of antimicrobials. The panel also highlighted key factors to guide earlier initiation of appropriate treatment: patient factors, local epidemiology, available testing, and drug characteristics. This Delphi panel achieved consensus on optimal management of MBL-producing Enterobacterales, finding preference for targeted MBL treatment, with monotherapy preferred where available. Study sponsored by Pfizer Inc. A genAI tool (Pfizer) developed the 1st draft; authors assume content responsibility. Yehuda Carmeli, MD, Basilea: Advisor/Consultant|Enlivex: Advisor/Consultant|Omnix: Advisor/Consultant|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Roche: Advisor/Consultant Mark A. Price, MA, MEd, Pfizer: Advisor/Consultant George L. Daikos, MD, MSD: Honoraria|Pfizer: Honoraria|Pfizer: Travel expenses to attend ESCMID Global 2025|Roche: Advisor/Consultant Marco Falcone, MD, Gilead: Grant/Research Support|Infecto Pharm: Honoraria|Menarini: Honoraria|Pfizer: Honoraria|Shionogi: Honoraria|ViiV: Grant/Research Support Ana C. Gales, MD, BioMerieux: Advisor/Consultant|BioMerieux: Speaker|MSD: Advisor/Consultant|Pfizer: Advisor/Consultant|Pfizer: Speaker Stefen Hagel, MD, Gilead: Honoraria|Pfizer: Advisor/Consultant|Pfizer: Honoraria|Shionogi: Advisor/Consultant|Shionogi: Honoraria Ali S. Omrani, FRCP, FRCPath, BioMérieux: Advisor/Consultant|BioMérieux: Honoraria|Cepheid: Advisor/Consultant|Cepheid: Honoraria|Gilead: Advisor/Consultant|Gilead: Honoraria|GSK: Advisor/Consultant|GSK: Honoraria|Hikma: Advisor/Consultant|Hikma: Honoraria|MSD: Advisor/Consultant|MSD: Honoraria|Mundi Pharma: Advisor/Consultant|Mundi Pharma: Honoraria|Pfizer: Advisor/Consultant|Pfizer: Honoraria Maria Gheorghe, PhD, Pfizer: Stocks/Bonds (Public Company) Brett Hauber, PhD, Pfizer Inc: Stocks/Bonds (Private Company) Christopher Little, PhD, Pfizer: Stocks/Bonds (Public Company) Andrew Ian Townsend, PhD, Pfizer: Stocks/Bonds (Public Company) Nathalie Baillon-Plot, MD, Pfizer: Stocks/Bonds (Public Company)

BACKGROUND:Antimicrobial resistance (AMR) poses a major global health threat, with health-care-associated infections contributing substantially to mortality. Attributable disease burden remains poorly quantified by relying on cross-sectional and microbiology data. We aimed to characterise AMR epidemiology and disease burden associated with ventilator-associated pneumonia and bloodstream infection in a network of hospitals in Asia. METHODS:This large-scale, prospective, multinational, multicentre cohort study consecutively enrolled patients of any age with microbiologically-confirmed ventilator-associated pneumonia, hospital-acquired bloodstream infections, or health-care-associated bloodstream infections from 41 selected hospitals capable of standardised prospective data collection in 19 Asian countries and regions. Patients with pathogens typically associated with community-acquired infection or not recognised causes of health-care-associated infection were excluded according to predefined protocol criteria, and patients were followed for 28 days. The primary outcome was 28-day mortality from infection onset. Pathogen profiles, antimicrobial prescriptions, attributable mortality, and health-related quality-of-life outcomes were analysed. FINDINGS:Between Sept 1, 2022, and Feb 28, 2025, 10 111 patients were enrolled, and after exclusions, 9496 patients were included in the final analysis. 9642 infection episodes were analysed, comprising 6597 (68·4%) bloodstream infections and 3045 (31·6%) ventilator-associated pneumonia. Of these infections, 7102 (73·7%) of 9642 infection episodes were associated with AMR bacteria, and Gram-negative bacteria were predominant (7599 [78·8%]). Crude 28-day mortality was 2674 (37·7%) of 7102 AMR infection episodes and 1900 (40·6%) of 4683 multidrug resistance infection episodes, with the highest for carbapenem-resistant Acinetobacter spp (CRA; 51·3%) and carbapenem-resistant Enterobacterales (CRE; 48·4%). AMR-attributable mortality was the highest in ventilator-associated pneumonia (16·9%, 95% CI 13·0-20·9), individuals aged 5-14 years (11·7%, 95% CI 1·6-21·8), individuals aged 15-49 years (11·2%, 95% CI 7·2-15·2), and lower-middle-income countries (10·7%, 95% CI 7·0-14·4). By pathogens, CRA and CRE had the highest attributable mortality (19·4%, 95% CI 14·1-24·7 vs 16·2%, 95% CI 12·8-19·6) and also had the poorest quality-of-life outcomes. Most carbapenem-resistant Gram-negative infections were treated with carbapenems (57·8%) or polymyxins (35·6%). INTERPRETATION:AMR bacterial bloodstream infections and ventilator-associated pneumonia were common and associated with high mortality and reduced quality of life in Asia, particularly in infections due to carbapenem-resistant Acinetobacter spp and CRE, and among children and younger adults. The widespread use of suboptimal antimicrobial regimens highlights the urgent need for equitable access to effective therapies and context-specific evidence to guide antimicrobial stewardship. FUNDING:Wellcome Trust, National University of Singapore, Yong Loo Lin School of Medicine, Duke-NUS Medical School, Nanyang Technological University (Lee Kong Chian School of Medicine), Singapore National Centre for Infectious Diseases, and Singapore Medical Research Council.

Despite growing reports of virulence gene acquisition in carbapenem-resistant Klebsiella pneumoniae (CRKP) strains, the clinical implications of these infections remain poorly understood. In this study, 246 hospitalized patients with clinical CRKP infections were enrolled from six hospitals across China between 2017 and 2019 (CRACKLE-2 China Cohort). All CRKP isolates underwent whole-genome sequencing to determine sequence types and the presence of virulence genes, including iuc (aerobactin), iro (salmochelin), and mucoid phenotype regulators (rmpADC and rmpA2). Clinical, microbiological, and outcome data were compared by iuc status, and multivariable logistic regression was used to assess factors associated with 30-day mortality. Kaplan-Meier analysis was performed to evaluate 30-day survival. Among the 246 CRKP isolates, ST11 (78%, 192/246) and ST15 (14%, 34/246) were predominant. iuc was detected in 63% (154/246) of the isolates. rmpA2 (Pearson coefficient r=0.9; P< 0.01) and rmpA (r=0.7; P< 0.01) were highly correlated with iuc. In only 6/246 isolates (2%) iro was detected, five of which were iuc-positive. Demographics and clinical characteristics did not differ between patients infected with iuc-positive and iuc-negative isolates (Table 1). Clinical outcomes were worse in iuc-positive CRKP infections; unadjusted 30-day mortality was higher in the iuc-positive group compared to the iuc-negative group (16% vs. 5%, 24/154 vs. 5/92; P< 0.05). Additionally, the 30-day DOOR outcomes were significantly different, and the presence of iuc was associated with less desirable outcomes (DOOR probability 37.8%; 95% Halperin confidence interval: 31.3%-44.7%, Table 2). After adjusting for potential confounders, iuc-positive CRKP infections remained independently predictive of increased 30-day all-cause mortality (adjusted odds ratio [aOR], 4.10; 95% CI, 1.31-12.86; P=0.016) (Table 3). Kaplan-Meier survival analysis further demonstrated significantly poorer survival among patients with iuc-positive infections (log-rank p=0.018) (Figure 1). These findings suggest that the presence of iuc contributes to increased mortality in CRKP infections and highlight the need for its surveillance in clinical settings. Angelique E. Boutzoukas, MD, MPH, Elion Therapeutics: Advisor/Consultant|Entasis Therapeutics Inc., an affiliate of Innoviva Specialty Therapeutics, Inc.: Board Member David van Duin, MD, PhD, AbbVie Inc: Advisor/Consultant|Merck & Co., Inc.: Advisor/Consultant|Merck & Co., Inc.: Grant/Research Support|Parexel International: DSMB|Pfizer, Inc.: Advisor/Consultant|Pfizer, Inc.: Honoraria|Roche Pharmaceuticals: Advisor/Consultant|TEVA: Advisor/Consultant

Treatment options for Carbapenem-resistant (CR) Gram-negative infections due to metallo-beta-lactamase (MBL) enzymes are limited. The clinical impact of MBLs vs. other mechanisms of carbapenem resistance in Enterobacterales and non-fermenting bacteria remains unclear.Table 1.Demographics, isolate characteristics, and outcomes of patients with carbapenem-resistant Gram-negative infections, stratified by MBL statusFigure 1.30-day desirability of outcome ranking (DOOR) outcomes of patients with carbapenem-resistant Gram-negative infections, stratified by MBL statusCRPA – carbapenem-resistant Pseudomonas aeruginosa, CRAb – carbapenem-resistant Acinetobacter baumannii, CRE – carbapenem-resistant Enterobacterales, DOOR – desirability of outcome ranking. MBL – metallo-beta-lactamase. DOOR events assessed at 30 days include: unsuccessful discharge, lack of clinical response, and C. difficile infection and/or renal failure. Not all rows total to 100 due to rounding. P-value calculated using Wilcoxon test. We conducted a matched cohort study of patients enrolled in one of three MDRO Network studies, POP (CR Pseudomonas aeruginosa [CRPA]), SNAP (CR Acinetobacter baumannii [CRAb]), or CRACKLE-2 (CR-Enterobacterales [CRE]) with isolates that met infection criteria. Patients with MBL-producing isolates (blaVIM, blaIMP, or blaNDM present) were matched 1:2 to patients with non-MBL CR isolates (a different carbapenemase or CR without a carbapenemase) based on study, region, and anatomical source. We compared baseline characteristics, 30- and 90-day mortality, and 30-day desirability of outcome ranking (DOOR) scores.30-day Desirability of Outcome Ranking (DOOR) Probability by MBL StatusLegend: CI – confidence interval, CRPA – carbapenem-resistant Pseudomonas aeruginosa, CRAb – carbapenem-resistant Acinetobacter baumannii, CRE – carbapenem-resistant Enterobacterales, DOOR – desirability of outcome ranking. MBL – metallo-beta-lactamase. The DOOR probability was calculated as the probability of a more desirable result in the presence of MBL as compared to non-MBL isolate. Confidence intervals were calculated using the method in Halperin et al (Biometric 1989; 45:500-521), and CI’s that do not include 50% are considered statistically significant. Estimates less than 50% signify a less favorable outcome for the MBL group, while estimates greater than 50% signify more favorable outcomes for the MBL group. In total, 170 MBL isolates were matched to 340 non-MBL isolates from 10 countries (Table 1). The cohort included 42% CRPA (216/510), 5% CRAb (24/510), and 53% CRE (270/510). Demographics were balanced between groups; median age at culture was 61 (Q1, 44, Q3 73) years. Common infection sources were respiratory (151/510, 30%), urine (141/510, 28%), and blood (105/510, 21%). Of the MBL isolates, 92/170 harbored blaNDM (54%), 62/170 harbored blaVIM (36%), and 20/170 harbored blaIMP (12%); four isolates co-harbored two distinct MBL enzymes. All-cause 30-day mortality was 19% (33/170) for MBL vs 18% (61/340) for non-MBL (p=0.69); MBL presence was not associated with 30- or 90-day mortality. DOOR outcomes at 30-days (Figure 1) did not differ by MBL status in the full cohort or the CRE subgroup, but did differ in the CRPA/CRAb subgroup (p=0.037). Among CRPA/CRAb infections, MBL presence was associated with less desirable outcomes (DOOR probability 42.1%; 95% Halperin confidence interval: 35.0%-49.5%, Figure 2). MBL presence was not associated with increased 30- or 90-day mortality compared to matched non-MBL isolates. However, in non-fermenter infections (CRPA/CRAb), MBL presence was linked to less desirable outcomes, an association not seen in CRE. These findings may inform prioritization of anti-MBL agents in future drug development. Angelique E. Boutzoukas, MD, MPH, Elion Therapeutics: Advisor/Consultant|Innoviva Speciality Therapeutics: DSMB Participant Souha S. Kanj, MD, Menarini: Honoraria|pfizer: Honoraria Vance G. Fowler, MD, MHS, Affinergy, Janssen, Contrafect: Advisor/Consultant|AstraZeneca; EDE; Basilea: Grant/Research Support|Debiopharm, GSK; Affinium, Basilea,: Advisor/Consultant|Destiny, Amphliphi, Armata, Akagera: Advisor/Consultant|Merck; Contrafect; Karius; Janssen: Grant/Research Support|UpToDate: Royalties|Valanbio: Stock options Yohei Doi, MD, PHD, GSK: Advisor/Consultant|Meiji Seika Pharma: Advisor/Consultant|Shionogi: Advisor/Consultant|Shionogi: Honoraria Michael Satlin, MD, MS, AbbVie: DSMB Participant|bioMerieux: Grant/Research Support|Merck: Grant/Research Support|SNIPRBiome: Grant/Research Support Robert A. Bonomo, MD, Merck: Grant/Research Support|Shinogi: Grant/Research Support|VenatoRx: Grant/Research Support David van Duin, MD, PhD, British Society for Antimicrobial Chemotherapy: Editor stipend|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Roche: Advisor/Consultant|Shionogi: Advisor/Consultant

Background and objectives:Metallo-β-lactamase (MBL)-producing Enterobacterales infections are an urgent global healthcare problem with limited treatment options. To obtain greater understanding of international perspectives on optimal management of serious infections due to MBL-producing Enterobacterales, we used a modified Delphi process to elicit experts' opinion and establish consensus. Methods:A Delphi panel comprising 10 expert physicians experienced in antimicrobial-resistant bacterial infection management were surveyed on epidemiology, disease, testing and management of MBL-producing Enterobacterales infections. Consensus statements were based on survey responses and reviewed by the panel for endorsement or revisions to achieve consensus during a virtual meeting. Results:All panellists acknowledged MBL-producing Enterobacterales as a key source of antimicrobial resistance and infection in most countries. All agreed these infections have high morbidity and mortality, leading to the need for critical-level care, longer hospital stays and significant healthcare costs. The negative clinical and economic consequences of MBL-producing Enterobacterales are compounded by inappropriate use of multidrug antimicrobial regimens. Therefore, a specific MBL-targeting agent is considered desirable. When aztreonam-avibactam and cefiderocol are available, the preferred first-line treatment is aztreonam-avibactam, per in vitro data showing aztreonam-avibactam activity against >90% of New Delhi MBL-producing Enterobacterales. If aztreonam-avibactam or cefiderocol are unavailable, treatment options are limited to co-administration of antimicrobials. Regional differences made it difficult to achieve unanimous consensus across all survey questions due to differences in patient factors, local epidemiology and testing availability. Conclusions:The Delphi panel reached consensus on optimal management of MBL-producing Enterobacterales, confirming need for targeted MBL treatment, with monotherapy preferred where available.

ABSTRACTThe ESKAPE pathogens - Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. - are designated by the World Health Organization as critical-priority multidrug-resistant organisms. These bacteria are leading contributors to the global antimicrobial resistance crisis, significantly increasing morbidity, mortality, and healthcare costs worldwide. Their capacity to evade conventional antibiotics continues to complicate clinical management and undermine infection control efforts. Tackling the global threat of ESKAPE pathogens demands coordinated and sustained interventions. This mini review summarizes recent evidence on the burden and prevalence of ESKAPE infections and assesses emerging strategies to combat resistance. Progress in surveillance and promising preclinical and clinical studies of novel therapies underscore that integrated approaches are crucial. Moving forward, a balanced emphasis on prevention, surveillance, and therapeutic innovation will be essential to mitigating the threat posed by ESKAPE pathogens over the coming decade.

CRISPR-Cas adaptive immunity systems provide defense against mobile genetic elements and are often countered by diverse anti-CRISPR (Acr) proteins. The type IE CRISPR-Cas of Escherichia coli K12 has been a model for structural and functional studies and is a part of the species' core genome. However, this system is transcriptionally silent, which has fueled questions about its true biological function. To clarify the role of this system in defense, we carried out a census of Acr proteins in Enterobacterales and identified AcrIE9 as a potent inhibitor of the E. coli K12 type IE CRISPR-Cas system. While sharing little sequence identity, AcrIE9 proteins from Pseudomonas and Escherichia both interact with the Cas7 subunit of the Cascade complex, thus preventing its binding to DNA. We further show that AcrIE9 is genetically linked to AcrIE10, forming the most widespread anti-CRISPR cluster in Enterobacterales; this module often co-occurs with an AcrIE13 protein with an unusual HTH-like architecture.

Risk factors for mortality among patients with carbapenem-resistant (CR) Gram-negative infections have been extensively studied, however children and younger adults with CR Gram-negative infections are underrepresented in these studies.Table 1.Demographics for included patients aged 2-30 years with carbapenem-resistant isolates (N=434)Figure 1.4-category desirability of outcome ranking (DOOR) outcomes in patients aged 2-30 years with carbapenem resistant isolates at 30-days from index culture, by region The figure shows the distribution of 4-category desirability of outcome ranking at 30 days from the index culture date, by region. The most desirable outcome is to be alive without any events. The least desirable outcome is to be dead at 30 days. The following events were assessed: 1) Lack of clinical response at 30 days, where clinical response is defined as a symptomatic response, no anti-CRE antibiotic, and no relapse within 30 days; 2) not discharged within 30 days or readmitted within 30 days; and 3) renal failure post-culture or C. difficile infection. Here, children and young adults 2-30 years old enrolled in one of three MDRO Network studies with a qualifying culture positive for a CR organism (Enterobacterales - CRACKLE/CRACKLE-2; Acinetobacter baumannii – SNAP; or Pseudomonas aeruginosa – POP) were included. We assessed the relationship between patient characteristics and 30-day mortality (or discharge to hospice) using univariate and multivariable logistic regression models in the full cohort and in the subgroup of patients with isolates that met criteria for infection.Table 2.Patient and isolate characteristics by 30-day mortality outcomesTable 3.Logistic regression models evaluating predictors of 30-day mortality in 2-30 year old patients with carbapenem-resistant Gram-negative isolates Across the MDRO Network, 434 eligible patients from 52 sites and 10 countries were included, with a median age of 24 years (Q1 19, Q3 27, Table 1). Of those, 258 (59%) met infection criteria. 30-day outcomes varied by region (p< 0.001, Figure 1). The mortality rate at 30 days was 16% (68/434, including discharge to hospice). On univariate analysis, higher Pitt score, immunocompromised status, admission from a non-home location, pathogen (A. baumannii versus others), and source (blood and/or respiratory source versus others) were associated with increased 30-day mortality (Table 2). In multivariable analysis, Pitt score and immunocompromised status were predictors of 30-day mortality (Table 3). For the infection-only subgroup, Pitt score was the sole identified predictor of mortality (Table 3); each one-point increase increased the odds of 30-day mortality by 56% (adjusted odds ratio [aOR] 1.56 [95% CI 1.35, 1.79]). Acuity of illness, as measured by the Pitt bacteremia score, was identified as a risk factor for mortality in 2–30-year-old patients with CR Gram-negative infections; notably, age was not a predictor of mortality. This contrasts with known predictors of mortality in older adults, including age, organism, region, immunocompromise, and source of infection. Pitt scores may be useful for prognostication of mortality risk in young patients with highly resistant infections. Angelique E. Boutzoukas, MD, MPH, Elion Therapeutics: Advisor/Consultant|Innoviva Speciality Therapeutics: DSMB Participant Michael J. Satlin, MD, MS, AbbVie: DSMB participant|bioMerieux: Grant/Research Support|Merck: Grant/Research Support|SNIPRBiome: Grant/Research Support Yohei Doi, MD, PHD, GSK: Advisor/Consultant|Meiji Seika Pharma: Advisor/Consultant|Shionogi: Advisor/Consultant|Shionogi: Honoraria Vance G. Fowler, MD, MHS, Affinergy, Janssen, Contrafect: Advisor/Consultant|AstraZeneca; EDE; Basilea: Grant/Research Support|Debiopharm, GSK; Affinium, Basilea,: Advisor/Consultant|Destiny, Amphliphi, Armata, Akagera: Advisor/Consultant|Merck; Contrafect; Karius; Janssen: Grant/Research Support|UpToDate: Royalties|Valanbio: Stock options David van Duin, MD, PhD, British Society for Antimicrobial Chemotherapy: Editor stipend|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Roche: Advisor/Consultant|Shionogi: Advisor/Consultant

BACKGROUND AND OBJECTIVES:Canonical hypervirulent Klebsiella pneumoniae sequence type 23 (ST23) causes severe infections while it generally maintains susceptibility to a broad spectrum of antimicrobials, yet the molecular basis of this phenotypic characteristic remains poorly understood. The purpose of this study was to investigate whether the presence of the DISARM system prevents antimicrobial-resistant plasmid invasion in K. pneumoniae. METHODS:Comparative genomic analysis was performed to detect the distribution of DISARM among K. pneumoniae lineages. PacBio single-molecule real-time sequencing was employed to identify DISARM-associated DNA methylation motifs. Transformation and conjugation assays were conducted to evaluate the impact of DISARM on plasmid defence. RESULTS:Class 1 DISARM system was selectively enriched in ST23 K. pneumoniae strains. The DISARM methylase modified host 5'-GRACRAC-3' motifs, which were distributed in all conjugative plasmids in K. pneumoniae. The transformation efficiency of plasmid pCOLADuet-MTmotif containing the methylation cognate site was reduced 2.9-fold compared with that of the plasmid pCOLADuet-1 in a ST23 DISARM-positive strain KP2613. Conjugation of carbapenemase-encoding plasmids (carrying blaKPC-2, blaNDM-1 and blaNDM-5) decreased 12- to 117-fold in KP2613 and 4.5- to 15.7-fold in Escherichia coli BL21-DISARM(+) compared with DISARM-negative strains KP2613ΔDISARM and BL21-DISARM(-), respectively, indicating that Class 1 DISARM could effectively hinder the antimicrobial resistance plasmid invasion. Systematic deletion of individual DISARM genes revealed that disruption of any single gene did not fully abolish the DISARM-mediated defence or cause substantial growth defects. CONCLUSIONS:Class 1 DISARM provides robust protection against antimicrobial-resistant plasmids, potentially contributing to the high antimicrobial susceptibility observed in ST23 hypervirulent K. pneumoniae.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of antimicrobial-resistant infection worldwide. The spectrum of diseases caused by MRSA range from uncomplicated skin and soft-tissue infections to life-threatening invasive infections such as bacteraemia, pneumonia, osteomyelitis and endocarditis. MRSA commonly colonizes the skin and mucosal surfaces as an asymptomatic commensal but can transition to an invasive pathogen when host barriers are disrupted or immune defences are compromised. The shift from commensal to pathogen is facilitated by a broad repertoire of virulence factors that promote adhesion, immune evasion, tissue invasion and persistence within host tissues. Despite the development of several new antibiotics with activity against MRSA, clinical outcomes during invasive infections are poor, with many patients experiencing persistent or recurrent infection. Strategies to prevent MRSA infection include infection control measures, screening and targeted decolonization approaches; however, the effectiveness of these interventions varies and often depends on local MRSA prevalence and epidemiological context. Understanding the epidemiology, pathogenesis and clinical management of MRSA remains essential to guide effective prevention and treatment strategies.