Cefiderocol (CFDC) is a siderophore-conjugated cephalosporin that hijacks bacterial iron uptake pathways to traverse the outer membrane, offering potent activity against carbapenem-resistant Acinetobacter baumannii (CRAB). Although mutations in pirA, a TonB-dependent siderophore receptor, have been linked to CFDC resistance, the broader genetic basis remains poorly defined. Using Himar1 transposon mutagenesis in a CFDC-susceptible sequence type 2 (ST2) CRAB strain, we identified ten genes whose disruption reduced CFDC susceptibility, spanning siderophore-mediated uptake (pirA and puiA), oxidative and redox stress responses (oxyR, nfuA, aarF, cyoA, and bfmRS), and cell envelope morphogenesis (mreB). Most mutants retained wild-type susceptibility to other β-lactams, indicating that reduced CFDC susceptibility can arise independently of target modification. Quantification of cellular iron revealed modest reductions in several mutants, with the largest decreases observed in strains with disruptions in TonB-dependent receptors. Inactivation of pirA or puiA altered the expression of several alternative TonB-dependent siderophore receptors. Whole-genome sequencing of ST2 clinical isolates with reduced CFDC susceptibility uncovered mutations in TonB-dependent receptors, porins, and PBP3, along with increased β-lactamase expression. Importantly, the β-lactamase inhibitor avibactam restored CFDC susceptibility in isolates with β-lactamase upregulation and intact uptake pathways, whereas strains with concurrent uptake defects remained resistant, underscoring the interplay between permeability and enzymatic drug inactivation. These findings define a multifactorial resistance landscape integrating impaired uptake, redox and envelope stress adaptation, and β-lactamase-mediated drug inactivation.IMPORTANCECefiderocol (CFDC) is one of the few remaining antibiotics with activity against carbapenem-resistant Acinetobacter baumannii (CRAB), an urgent global health threat. Yet, resistance to CFDC is increasingly reported, and the underlying mechanisms remain incompletely defined. Most prior studies have examined single pathways, such as loss of TonB-dependent receptors. Here, we used genome-wide transposon mutagenesis together with genomic and phenotypic analysis of CFDC-resistant clinical isolates to generate a more comprehensive view of how resistance emerges. Our findings show that CFDC resistance is multifactorial, involving disrupted siderophore uptake, alterations in oxidative and envelope-stress responses, porin and cell-wall changes, and β-lactamase activity. By defining how these pathways converge, this work provides a broader mechanistic framework for interpreting emerging resistance in clinical settings. These insights underscore the need for integrated surveillance strategies and highlight the biological complexity that must be considered to preserve the effectiveness of this last-line antibiotic.

ABSTRACT Klebsiella quasipneumoniae subsp. similipneumoniae is an important member of the K. pneumoniae species complex (KpSC) and is increasingly reported as multidrug-resistant (MDR) in healthcare- and community-associated infections. Since clinical laboratories do not routinely distinguish K. quasipneumoniae subsp. similipneumoniae from K. pneumoniae , national prevalence estimates, particularly for MDR, are lacking. In this study, a total of 2,006 community-associated MDR KpSC isolates were collected from 42 U.S. states, with 30 K. quasipneumoniae subsp. similipneumoniae isolates originating from 12 states identified using whole genome sequencing. All isolates were resistant to ceftriaxone and exhibited high rates of resistance to other antimicrobial agents, including ampicillin-sulbactam (56.7%, 17/30), levofloxacin (75.9%, 22/29), and trimethoprim-sulfamethoxazole (53.3%, 16/30). Notably, five isolates were also carbapenem-resistant. Genomic analysis resolved 10 sequence types (STs), with ST2059 ( n = 13) and ST414 ( n = 9) predominating. Ceftriaxone resistance in most isolates (90%, 27/30) was conferred by an extended-spectrum β-lactamase gene, predominantly bla CTX-M-15 (73.3%, 22/30); the remaining isolates carried either a carbapenemase ( bla KPC-3 ) or an AmpC β-lactamase ( bla CMY-2 ). Nanopore sequencing identified bla CTX-M-15 harbored on two types of IncFIB(Kpn3) antimicrobial resistance (AMR) plasmids, either with or without the conjugative tra gene cluster. Interestingly, the KL1 locus, associated with canonical hypervirulent K. pneumoniae strains, was detected in all ST2059 isolates. Further analysis of public genomic data showed that the KL1 locus is widely distributed across KpSC. KL1 phylogenetic analyses indicated frequent intrasubspecies recombination but limited intersubspecies exchange of KL1. The identification of the dominant MDR K. quasipneumoniae subsp. similipneumoniae KL1-ST2059 clone in the U.S. underscores the importance of ongoing genomic surveillance. IMPORTANCE Klebsiella quasipneumoniae subsp. similipneumoniae is an underrecognized member of the Klebsiella pneumoniae species complex that is frequently misidentified in clinical laboratories, leading to an incomplete understanding of its role in antimicrobial resistance. In this study, we used large-scale genomic surveillance of community-associated multidrug-resistant isolates across the U.S. to identify this subspecies as a reservoir of clinically relevant resistance plasmids. Notably, we detected a widely distributed ST2059 lineage carrying the K1 capsular locus, a feature traditionally associated with hypervirulent K. pneumoniae . These findings highlight the convergence of resistance and virulence-associated traits in an overlooked species and underscore the need for genomic surveillance to monitor emerging high-risk lineages in community settings.

Escherichia coli bloodstream infections are common and associated with high mortality. A key feature of E. coli is the lipopolysaccharide (LPS) O-antigen, which contributes to immune evasion during invasive infection. We analyzed serial isolates from patients with relapsed E. coli bacteremia and identified frequent disruption of O-antigen synthesis due to mutations in wbbL , resulting in a rough LPS phenotype. Rough LPS isolates were more serum sensitive and less pathogenic in mice. Despite this apparent attenuation, 11 of 61 (18%) E. coli sequence type 131 bloodstream isolates in our cohort harbored disruptive wbbL mutations and were associated with significantly worse clinical outcomes, including septic shock and mortality. Using a murine model of recurrent bacteremia, we show that rough LPS isolates partially evade protective immunity generated against smooth LPS E. coli , highlighting the importance of host immune context in invasive disease.

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

Traditionally viewed as a hospital pathogen, multidrug-resistant (MDR) Klebsiella pneumoniae is emerging in community settings, yet its population structure and spread are currently undefined. A nationwide genomic survey of 2006 ceftriaxone-nonsusceptible outpatient isolates from 42 US states reveals that the spread of resistance is primarily driven by the blaCTX-M-15 extended-spectrum-β-lactamase (ESBL) gene across diverse K. pneumoniae lineages. This gene, harbored by a family of IncFIB(Kpn3) plasmids, has spread to hundreds of lineages, coupling antibiotic resistance with stress- and metal-tolerance traits that may enhance survival beyond human hosts. Nearly half of isolates formed transmission clusters spanning multiple states, underscoring active community dissemination. The majority originated from elderly women with urinary tract infections, where oral treatment options are increasingly ineffective. Capsule and O-antigen profiling reveal broad antigenic diversity, yet a manageable subset of serotypes could offer wide coverage for vaccine development. Our findings expose a rapidly evolving, plasmid-driven epidemic of community-associated MDR K. pneumoniae across the United States.

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/Objectives: The escalating threat of drug-resistant Neisseria gonorrhoeae underscores the urgent need for novel therapeutic agents. Zoliflodacin, a first-in-class spiropyrimidinetrione antibiotic that targets bacterial DNA gyrase and topoisomerase IV, represents a promising candidate for gonorrhea treatment. Methods: From 2020 to 2023, a total of 876 urogenital N. gonorrhoeae isolates were collected from 35 hospitals across Shanghai, China. In vitro susceptibilities to zoliflodacin and six conventional antibiotics (penicillin, tetracycline, ciprofloxacin, azithromycin, ceftriaxone, and spectinomycin) were determined using the agar dilution method. Whole-genome sequencing was conducted to identify sequence types (STs) and amino-acid substitutions in GyrA, GyrB, ParC, ParE, and MtrR. Results: Zoliflodacin exhibited potent in vitro activity, with minimum inhibitory concentrations (MICs) ranging from ≤0.004 to 0.25 mg/L (MIC50 = 0.06 mg/L; MIC90 = 0.125 mg/L), all below the breakpoint (0.5 mg/L). Notably, zoliflodacin maintained high activity against isolates resistant to ceftriaxone, azithromycin, ciprofloxacin, penicillin, and tetracycline. Although all isolates were susceptible to zoliflodacin, elevated MIC values were observed in ST7363 and ST8123 compared with other clones. Genomic analysis identified no substitutions associated with increased zoliflodacin MICs, and most GyrB sequences, the key gene associated with zoliflodacin resistance, remained intact. Conclusions: These findings demonstrate that zoliflodacin possesses robust activity against circulating multidrug-resistant N. gonorrhoeae lineages in Shanghai and support its potential clinical use for the treatment of gonorrhea. Continued genomic and phenotypic surveillance is warranted to preserve the long-term efficacy of this novel agent.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a critical global health threat, often causing life-threatening infections with limited therapeutic options. Polymyxins are often used as the last-line agent, but its efficacy is limited by heteroresistance-where resistant subpopulations emerge during treatment. To combat this, we evaluated the synergistic activity of colistin combined with nitroxoline against polymyxin heteroresistant (PHR) CRKP subpopulations. The prevalence of PHR was 90.9

ABSTRACT Although gepotidacin is a promising oral candidate for treating multidrug-resistant Neisseria gonorrhoeae , its resistance mechanisms and clinical implications remain poorly understood. In this study, we collected 989 clinical N. gonorrhoeae isolates from 33 hospitals in Shanghai, China (2022–2024). Antimicrobial susceptibility testing showed that gepotidacin exhibited high in vitro activity with MIC 50 and MIC 90 of 0.5 and 1 μg/mL, respectively. Further investigation identified that elevated gepotidacin MICs were significantly associated with substitutions at GyrA position 92/95 and ParC position 86/87, including GyrA A92P (odds ratio [OR], 4.25; 95% confidence interval [CI], 2.61–6.94) and D95Y (OR, 4.61; 95% CI, 2.82–7.54), as well as ParC D86N (OR, 3.12; 95% CI, 1.99–4.90) and S87N (OR, 5.92; 95% CI, 3.63–9.64). Substitution combinations analyses revealed that GyrA D95A plus ParC D86N, GyrA A92P/D95Y plus ParC S87N, GyrA D95A plus ParC S87N, and GyrA D95G plus ParC D86N were significantly associated with elevated gepotidacin MICs. Notably, these high-risk substitutions were enriched in international clones ST7363 and ST8123. In vitro induction experiments demonstrated a stepwise resistance trajectory: from initial diverse QRDR mutations to an intermediate state with GyrA A92P/D95Y, followed by ParC D86N and GyrA A92T/D95A, culminating in high-level resistance, with MICs all reaching 64 μg/mL. We also found that the strain with preexisting GyrA D95A and ParC D86N more readily acquired A92T and developed high-level resistance under gepotidacin exposure. Our study highlights key mutational patterns and clonal backgrounds that promote gepotidacin resistance, emphasizing the need for optimized dosing strategies as well as targeted molecular surveillance to preserve its effectiveness.

This study provides the first prospective, genomically validated dataset integrated into the global MultiDrug-Resistant Organism (MDRO) Network from the Middle East and North Africa region. To address the substantial gap in antimicrobial resistance (AMR) data, the American University of Beirut Medical Center (AUBMC) investigated carbapenem-resistant Enterobacterales (CRE), Acinetobacter baumannii (CRAB), and Pseudomonas aeruginosa (CRPA). Consecutive, hospitalized patients whose clinical cultures were positive for CRE, CRAB, or CRPA were prospectively enrolled between June 2018 and November 2019. Data on demographics, hospitalization, outcomes, and antimicrobial susceptibility were collected. Whole-genome sequencing (WGS) characterized the isolates. The primary outcome was the Desirability of Outcome Ranking (DOOR). Ninety-four patients were included: 35 with CRE, 19 with CRAB, and 40 with CRPA. Overall, 30

BACKGROUND:Ceftriaxone-resistant Escherichia coli infections are increasingly common, partially because of the emergence of E. coli sequence type 131 (ST131), including its subclade C2/H30Rx, which produce extended-spectrum β-lactamases. METHODS:A prospective cohort including 14 US sites, which enrolled monomicrobial ceftriaxone-resistant and susceptible E. coli bloodstream infection (BSI) cases in a 1:1 ratio, was used to compare ST131 versus non-ST131 E. coli BSI, with specific attention to E. coli ST131 C2/H30Rx. Desirability of outcome ranking was determined at 30 days after infection onset. RESULTS:This analysis included 282 patients with E. coli BSI; 43% (121/282) were E. coli ST131 and 23% (66/282) belonged to the C2/H30Rx subclade. Resistance to ceftriaxone was present in 79% (96/121) ST131, 86% (57/66) E. coli ST131 C2/H30Rx, and 27% (43/161) E. coli non-ST131. Compared to patients with non-ST131 E. coli BSI, patients with ST131 BSI were older (median 70 years, [Q1 62, Q3 76] years vs 65 years, [51, 74]; P = .005) and more often admitted from long-term care facilities (21/121 [17%] vs 7/161 [4%], P < .001). Overall and empiric carbapenem use was more frequent in the treatment of patients with ST131 BSI compared with non-ST131 BSI (overall 89/121 [74%] vs 50/161 [31%]; empiric: 58/121 [48%] vs 31/161 [19%], P < .001). Desirability of outcome ranking outcomes were similar between groups. CONCLUSIONS:Most ceftriaxone-resistant E. coli from US patients with E. coli BSI belong to ST131, particularly E. coli ST131 C2/H30Rx, serving as an important driver of carbapenem use.

BACKGROUND:Despite the global public health threat posed by carbapenem-resistant Enterobacter spp, clinical and molecular epidemiological studies on international isolates remain scarce. Historically, the taxonomy of Enterobacter has been challenging, limiting our understanding of the clinical characteristics and outcomes of carbapenemase-producing Enterobacter spp infections. METHODS:Hospitalized patients enrolled in the CRACKLE-2 study (ClinicalTrials.gov, NCT03646227) from 2016 to 2018 with cultures positive for carbapenemase-producing Enterobacter spp were included. Clinical and microbiologic data were collected from health records. Whole genome sequencing was performed, and the population structures of selected predominant clones were analyzed. RESULTS:We enrolled 136 hospitalized patients with carbapenemase-producing Enterobacter spp from 30 hospitals in 7 countries. Among the 136 isolates, 11 Enterobacter spp were identified, with most isolates belonging to E xiangfangensis (n = 81 [60%]) and E hoffmannii (n = 17 [13%]) and carrying blaKPC (n = 106 [78%]) and blaNDM (n = 12 [9%]). Clinical characteristics and outcomes were similar among patients with E xiangfangensis, E hoffmannii, or the other Enterobacter spp. Thirty-day mortality was 20%, and older age at enrollment (adjusted odds ratio, 1.42 [95% confidence interval, 1.08-1.87]) was associated with increased mortality. Sequence type (ST) 171 E xiangfangensis, ST78 E hoffmannii, and ST93 E xiangfangensis were the predominant clones, and the acquisition of fluoroquinolone resistance-associated mutations and carbapenemase-encoding plasmids contributed to their formation and global dissemination. CONCLUSIONS:Our findings demonstrate that E xiangfangensis and E hoffmannii are common species among international carbapenemase-producing Enterobacter spp, potentially linked to the clonal spread of a few predominant clones that have acquired fluoroquinolone resistance and carbapenemase-encoding plasmids.

Summary: Background: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) has been increasingly reported worldwide, posing a severe challenge to public health; however, the mechanisms driving its emergence and global dissemination remain unclear. Methods: We analysed CR-hvKp strains derived from canonical hvKp backgrounds, and acquired a carbapenemase-encoding gene. These strains were identified from 485 CRKp isolates in the CRACKLE-2 China cohort, 259 CRKp isolates from a multi-centre study, and 67,631 K. pneumoniae genomes available in GenBank. Clinical isolates harbouring the IncFIIK34 KPC-2 plasmid were selected for genome sequencing, RNA-Seq, conjugation assays, in vivo, ex vivo, and in vitro phenotypic characterisation. Findings: Analysis of clinical CR-hvKp isolates and the 414 genomes from 24 countries available in GenBank identified an IncFIIK34 KPC-2 plasmid as the prevalent KPC plasmid (detected in 25%, 45/178 of KPC-producing CR-hvKp). Compared with the epidemic IncFIIK2 KPC-2 plasmid, the IncFIIK34 KPC-2 plasmid exhibited a 100- to 1000-fold increase in conjugation frequency (10−4–10−5 vs. 10−7) and an in vitro growth advantage under meropenem challenge–likely due to the overexpression of conjugation-related genes and an increased blaKPC copy number and expression. CR-hvKp isolates and hvKp transconjugants carrying this plasmid often exhibited reduced mucoviscosity, while retaining hypervirulence in both murine models and human neutrophil assays. Interpretation: The IncFIIK34 plasmid may be a key factor driving the global dissemination of CR-hvKp, underscoring the urgent need for enhanced molecular surveillance of this emerging pathogen. Funding: National Natural Science Foundation of China and National Institutes of Health.

Klebsiella pneumoniae is one of the most prevalent pathogens responsible for multiple infections in healthcare settings and the community. K. pneumoniae CG147, primarily including ST147 (the founder ST), ST273, and ST392, is one of the most globally successful MDR clone linked to various carbapenemases. One hundred and one CG147 strains were sequenced and additional 911 publicly available CG147 genome sequences were included for analysis. The molecular epidemiology, population structure, and time phylogeny were investigated. The virulome, resistome, and mobilome were analyzed, and the recombination in the capsular region was studied. The CRISPR-Cas and anti-CRISPR were identified. The interplay between CRISPR-Cas, anti-CRISPR, and carbapenemase-encoding plasmids was analyzed and experimentally validated. We analyzed 1012 global CG147 genomes, with 80.4

Abstract Background Drug-resistant E. coli is a leading cause of antimicrobial resistance-associated deaths globally. Specifically, resistance to ceftriaxone (CRO-R) is increasing in E. coli. High-risk clonal group ST131 and its pandemic H30 subclone are of high concern yet studies characterizing these infections are limited. We evaluated baseline characteristics and clinical outcomes associated with H30 ST131, non-H30 ST131 and non-ST131 E. coli bloodstream infections (BSI). Methods Patients with monomicrobial carbapenem-susceptible E. coli BSI that were matched 1:1 by study site (CRO-R and CRO-susceptible community-acquired and hospital onset cases) were prospectively enrolled from 14 United States hospitals between November 12, 2020 to April 28, 2021 in the multicenter Study of Highly Resistant E. coli (SHREC). Isolates underwent whole genome sequencing. The primary outcome was a 30-day Desirability of Outcome Ranking (DOOR) after index culture including clinical response to treatment and all-cause mortality. Results There were 92 (33%) H30 ST131, 29 (10%) non-H30 ST131, and 161 (57%) non-ST131 isolates in 282 E. coli BSI (Table 1). Most ceftriaxone resistance was conferred by CTX-M-15 produced by H30 ST131 isolates (Figure 1, Table 1). H30 ST131 BSI patients were older (median age [IQR] 70.5 [63,76] vs. 67 [56,77] vs 65 [51,74] years, p = 0.017), had higher Charlson comorbidity indices (3 [2,5] vs. 2 [1,4] vs. 2 [1,4], p=0.009), and were more often admitted from long-term care facilities (18/92 [20%] vs. 3/29 [10%] vs. 7/161 [4%], p = 0.003) compared to non-H30 ST131 and non-ST131 BSI patients. Among H30 ST131 isolates, high rates of antibiotic resistance were observed to cephalosporins and fluoroquinolones, resulting in significantly more carbapenem use compared with non-H30 ST131 and non-ST131 isolates (75/92 [82%] vs. 14/29 [48%] vs. 50/161 [31%], p < 0.001) (Figure 2). 30-day DOOR and hospital length of stay did not differ between groups (Table 2). Conclusion Compared with non-H30 ST131 and non-ST131 E. coli BSI, H30 ST131 E. coli BSI have a unique epidemiology with more healthcare exposures, comorbidities and antibiotic resistance and are more likely to be treated with carbapenems, though no significant difference in clinical outcomes was observed. Disclosures Yohei Doi, MD, PHD, AbbVie: Honoraria|Entasis: Grant/Research Support|Gilead: Advisor/Consultant|GSK: Advisor/Consultant|Meiji Seika: Advisor/Consultant|Moderna: Advisor/Consultant|Pfizer: Advisor/Consultant|Shionogi: Advisor/Consultant|Shionogi: Honoraria Elie Saade, MD, MPH, FIDSA, Janssen Global Services: Advisor/Consultant|Janssen Global Services: Advisor/Consultant|Janssen Research and Development: Advisor/Consultant|Janssen Research and Development: Advisor/Consultant Loren G. Miller, MD MPH, Armata: Grant/Research Support|Contrafect: Grant/Research Support|GSK: Grant/Research Support|Merck: Grant/Research Support|Paratek: Grant/Research Support Michael J. Satlin, MD, AbbVie: DSMB participant|bioMerieux: Grant/Research Support|Merck: Grant/Research Support|Selux Diagnostics: Grant/Research Support|SNIPRBiome: Grant/Research Support W. Charles Huskins, MD, MSc, ADMA Biologics: Advisor/Consultant|Bristol Myers Squibb: Stocks/Bonds (Public Company)|Pfizer: Advisor/Consultant|Pfizer: Stocks/Bonds (Public Company)|Zimmer Biomet: Stocks/Bonds (Public Company) Carol Hill, PhD, Glaxo SmithKline: Retirement Health, Cash Balance Plan|Glaxo SmithKline: Stocks/Bonds (Public Company) Robin Patel, MD, a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, a: See above|MicuRx Pharmaceuticals and BIOFIRE: Grant/Research Support|PhAST, Day Zero Diagnostics, Abbott Laboratories, Sysmex, DEEPULL DIAGNOSTICS, S.L., Netflix, Oxford Nanopore Technologies and CARB-X: Advisor/Consultant|Up-to-Date and the Infectious Diseases Board Review Course.: Honoraria Vance G. Fowler, MD, MHS, Affinergy: Advisor/Consultant|ArcBio: Stocks/Bonds (Private Company)|Armata: Advisor/Consultant|Astra Zeneca: Advisor/Consultant|Astra Zeneca: Grant/Research Support|Basilea: Advisor/Consultant|Basilea: Grant/Research Support|ContraFect: Advisor/Consultant|ContraFect: Grant/Research Support|Debiopharm: Advisor/Consultant|Destiny: Advisor/Consultant|EDE: Grant/Research Support|Genentech: Advisor/Consultant|Genentech: Grant/Research Support|GSK: Advisor/Consultant|Janssen: Advisor/Consultant|Karius: Grant/Research Support|MedImmune: Grant/Research Support|Merck: Grant/Research Support|sepsis diagnostics: Patent pending|UptoDate: Royalties|Valanbuio: Stocks/Bonds (Private Company)|Valanbuio: Stocks/Bonds (Private Company) David van Duin, MD, PhD, Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Qpex: Advisor/Consultant|Roche: Advisor/Consultant|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support

Klebsiella pneumoniae is a significant human pathogen in both hospital and community settings; however, limited data exist regarding its prevalence in district-level hospitals. This study aimed to characterize the drug resistance mechanisms, molecular epidemiology, and virulence profiles of K. pneumoniae in a district hospital in Chengdu, China. A total of 114 clinical isolates were collected between May 2023 and May 2024. Antimicrobial susceptibility testing using the broth microdilution method revealed resistance rates of 14.0–21.1% to third-generation cephalosporins, and 5.3% to carbapenems. Whole-genome sequencing showed that 18.4% (21/114) of the isolates carried ESBL genes, with blaCTX–M–15 (n = 7) being the most common. Six carbapenem-resistant isolates were identified, of which four produced carbapenemases: three harbored blaNDM–5 and one carried blaKPC–2. MLST analysis identified 67 sequence types, with ST23 (n = 9) being the most prevalent. The 21 ESBL-producing isolates were distributed across 15 sequence types, while the six carbapenem-resistant isolates were assigned to four distinct sequence types. Virulence-associated genes were detected at high frequencies, including ybt (39.5%), clb (12.3%), iuc (50.0%), iro (60.5%), rmpA (59.6%), and rmpA2 (36.8%), and were commonly found in ST23 (n = 9), ST412 (n = 6), ST25 (n = 5), ST268 (n = 5), and ST375 (n = 4). In conclusion, K. pneumoniae isolates from this district hospital showed low resistance rates but a worrying high prevalence of virulence genes. This highlights the urgent need for continuous surveillance and early intervention strategies to prevent the emergence of highly virulent and multidrug-resistant strains in healthcare settings.

Abstract Background Recently, comparative phylogenetics dictated the formerly named Enterobacter aerogenes be separated from the genus Enterobacter and renamed Klebsiella aerogenes (KA); however, it remains unclear whether the genotypic differences responsible for the reclassification translate into clinical differences. We aimed to evaluate clinical characteristics and outcomes of patients colonized or infected with carbapenem-resistant (CR) Enterobacter cloacae complex (ECC) or CR KA. Methods We conducted a nested, case-control study of patients hospitalized from May 9th, 2016 until November 11th, 2019 and enrolled in The Consortium on Resistance Against Carbapenems in Klebsiella and other Enterobacterales II (CRACKLE-2). Cases were patients with an index culture with CR KA and controls were patients with an index culture positive for CR ECC, matched to each case by: 1) country, 2) anatomical source, 3) infection vs. colonization, and 4) availability/result of whole genome sequencing for the isolate. Where WGS was available the pan-genome Ortholog Clustering Tool was used to identify flexible genomic islands (FGI) associated with KA (n=65) versus ECC (n=66). Results Cases with CR KA (n=95) were matched with 95 controls with CR ECC. 49% of cultures met infection criteria. When compared with control patients, cases with KA were less likely to have liver disease (6% vs. 19%,p=0.009) but more likely to have been admitted for longer at the time of culture (median [IQR] 16[1,32] vs. 7[1,23]), have a Pitt score ≥ 4 (53% vs. 38%, p=0.041), and to have been in an intensive care unit (ICU) at the time of culture (54% vs. 45%, p=0.246) (Table 1). 30-day mortality (21% vs. 25%, p=0.492), 90-day mortality (26% vs. 35% p=0.208), and 30-day DOOR outcomes (p=0.727) did not differ between cases and controls for all patients (Figure 1, 2). There were 94 unique FGI containing 216 putative virulence factors for KA versus 77 and132 for ECC. Conclusion Despite significant differences in baseline characteristics and in the putative virulence genes present, clinical outcomes are similar in patients with CR KA and CR ECC colonization and infection (alive without events 37% vs. 36%). Disclosures Michael J. Satlin, MD, AbbVie: DSMB participant|bioMerieux: Grant/Research Support|Merck: Grant/Research Support|Selux Diagnostics: Grant/Research Support|SNIPRBiome: Grant/Research Support Keith S. Kaye, MD, MPH, Allecra: Advisor/Consultant|CARB-X: Advisor/Consultant|GSK: Advisor/Consultant|Merck: Advisor/Consultant|Shionogi: Advisor/Consultant|Spero: Advisor/Consultant Carol Hill, PhD, Glaxo SmithKline: Retirement Health, Cash Balance Plan|Glaxo SmithKline: Stocks/Bonds (Public Company) David van Duin, MD, PhD, Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Qpex: Advisor/Consultant|Roche: Advisor/Consultant|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support