Citations

ABSTRACT Novel β-lactam/β-lactamase inhibitors (βL/βLIs) are important therapies for carbapenem-resistant Pseudomonas aeruginosa (CRPA). However, the global extent of resistance to these agents and the impact of resistance on patient outcomes are unclear. We therefore evaluated patients with CRPA isolates at 35 hospitals (nine countries) from December 2018 to November 2019. Antimicrobial susceptibility testing was performed at a central laboratory by agar dilution for ceftolozane-tazobactam (C/T) and ceftazidime-avibactam (CZA) and by broth microdilution for imipenem-relebactam (I/R). Characteristics and outcomes, including desirability of outcome rankings (DOOR), were compared between patients infected with isolates not susceptible vs susceptible to each agent. Of 800 CRPA isolates, susceptibility to C/T, CZA, and I/R was 69%, 67%, and 33%, respectively. USA isolates ( n = 526) were more frequently susceptible to these agents than isolates from other countries ( n = 274; C/T: 83% vs 42%; CZA: 77% vs 47%; I/R: 37% vs 23%; P < 0.001 for each comparison) and isolates with carbapenemases ( n = 157) were less frequently susceptible than isolates without carbapenemases ( n = 643; C/T: 7% vs 84%; CZA: 24% vs 77%; I/R: 6% vs 39%; P < 0.001 for each comparison). Thirty-day mortality and DOOR were similar overall in patients infected with isolates not susceptible vs susceptible to each βL/βLI. However, the adjusted probability of a better DOOR outcome for a randomly selected patient with bacteremia due to a C/T-not susceptible vs -susceptible isolate was 38.2% (95% confidence interval, 25.6%–52.7%). Resistance to novel βL/βLIs, especially I/R, is common in CRPA, particularly outside the USA and in carbapenemase-producing isolates. Additional treatment options are needed for CRPA infections.

ABSTRACT Serine–aspartate repeat-containing protein D (SdrD) is a Staphylococcus aureus cell wall–anchored, calcium-binding adhesin member of the MSCRAMM Sdr subfamily that may contribute to bacterial adhesion and virulence. S. aureus is the most common cause of periprosthetic joint infection (PJI). Population-level distribution and sequence diversity of SdrD among clinical PJI isolates have not been systematically characterized, and the SdrD binding mechanism is still not well understood. To address these gaps, sdrD alleles were queried across 156 newly sequenced PJI isolates and compared to publicly available S. aureus genomes, and nucleotide– and protein-level phylogenies of the sdrCDE locus constructed. The SdrD crystal structure from S. aureus JH1 was determined, with solution small-angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, and assessment of conformational changes with calcium depletion. Three dominant sdrD subtypes were defined, associating with USA300, JH1, and TCH60; the JH1 sdrD subtype was predominant among PJI isolates. Structural studies showed that the conformation of individual domains and interdomain organization of the multidomain SdrD have limited flexibility in solution, and that the calcium-binding B domain retains its core fold under conditions of calcium depletion. Together, the findings presented support functional diversification among Sdr family members in mediating host attachment and inform a re-evaluation of the ligand-binding mechanism previously proposed for SdrD. AUTHOR SUMMARY Staphylococcus aureus is the leading cause of infections that develop around joint implants (periprosthetic joint infection, PJI). This bacterium has a large arsenal of surface proteins that allow it to stick to human tissues and implanted devices. This work focused on one such protein, SdrD, which has been linked to implant-associated infections but the structure and diversity of which among patients with PJI had not been well characterized. The genetic sequences of SdrD were analyzed across thousands of bacterial genomes, including those from patients with PJI. Distinct genetic variants of the protein were found, one of which was particularly common with PJI. The three-dimensional structure of SdrD was determined at atomic resolution and solution small-angle X-ray scattering (SAXS) and molecular dynamics used to study how it moves and responds to changes in its environment. Contrary to what was previously described, SdrD was shown to be relatively rigid. These findings change how SdrD’s mechanism of action should be considered, potentially informing design strategies to block bacterial attachment before infection takes hold.

Importance Novel blood culture diagnostics provide rapid antimicrobial susceptibility testing (AST) results for bacteria causing bloodstream infections (BSIs) but have unclear clinical impact. Objective To evaluate clinical outcomes of blood culture testing using a rapid AST method compared with standard AST in patients with BSIs caused by gram-negative bacilli in regions with high prevalence of antimicrobial resistance. Design, Setting, and Participants Open-label randomized clinical trial that enrolled participants from December 2023 to May 2025 (final follow-up, June 18, 2025) at 7 medical centers in Greece (n = 2), India (n = 1), Israel (n = 3), and Spain (n = 1). Hospitalized patients (adults and children) with BSIs caused by gram-negative bacilli were eligible. Statistical analysis was conducted from August 2025 to January 2026. Intervention Patients were randomized to undergo blood culture evaluation using rapid, phenotypic AST directly from positive blood cultures plus standard susceptibility testing (n = 413) vs standard susceptibility testing alone (n = 437). Local antimicrobial stewardship teams reviewed all patients and provided treatment recommendations. Main Outcomes and Measures The primary outcome was a desirability of outcome ranking (DOOR) at day 30, with 3 categories (alive without deleterious events, alive with deleterious events, and death). The difference between rapid and standard testing was summarized as the probability that the DOOR outcomes were more desirable in the rapid testing group. Superiority was concluded if the lower limit of the 95% CI exceeded 50%. Secondary outcomes included 30-day mortality, length of hospitalization, intensive care unit admission, acquisition of hospital-acquired infections, time to effective antibiotic therapy within 3 days, and time to antibiotic escalation or deescalation within 3 days. Results Of the 899 patients randomized, 850 were included in the primary outcome analysis (median [IQR] age, 72 [21] years; 43% female). The probability that DOOR outcomes were more favorable in the rapid testing group was 48.8% (95% CI, 45.3%-52.4%). Median time to effective antibiotic therapy was not different between the groups in the as-randomized population. Median time to antibiotic escalation or deescalation was faster in the rapid testing group by 14 hours (95% CI, 6-22) in the as-randomized population. There were no differences between the groups in other secondary outcomes. In the prespecified subgroup with carbapenem-resistant infections, median time to effective therapy was 9.5 hours in the rapid testing group vs 28 hours in the standard testing group (difference, −18 hours [95% CI, −42 to 6]). Conclusions and Relevance Among patients with gram-negative bacilli BSIs, rapid blood culture AST was not superior to standard testing by DOOR. When considered with other efficacy and safety outcomes, these findings may help inform the use of rapid susceptibility testing in clinical practice. Trial Registration ClinicalTrials.gov Identifier: NCT06174649

ABSTRACT Intrinsic Acinetobacter -derived cephalosporinases (ADCs) in Acinetobacter baumannii are AmpC-type β-lactamases that confer resistance to β-lactam agents, typically through insertion sequence (IS) element-driven overexpression. However, the contribution of ADCs to resistance against advanced β-lactam agents has not been systematically investigated. Given the increasing clinical use of these agents, including cefiderocol, we analyzed the diversity and function of ADC variants in a global collection of carbapenem-resistant A. baumannii (CRAb) isolates. We identified 52 distinct ADC variants in a collection of 428 CRAb clinical isolates from the United States. Among these, variants carrying both a valine substitution at position 292 in the R2 loop and an alanine duplication (ADUP) in the Ω loop consistently conferred stable resistance to cefiderocol. Biochemical and crystallographic analyses demonstrated that ADC-227, which harbors a V292W substitution with an ADUP at position 218a in the Ω loop, exhibits enhanced catalytic efficiencies for ceftazidime and cefiderocol and moderately reduced inhibition by avibactam, leading to resistance not only to cefiderocol but also to ceftazidime–avibactam. Structural studies revealed conformational flexibility of the R2 loop, allowing dynamic accommodation of substrates. Collectively, the findings identify Val292, in combination with an ADUP in the Ω loop, as a mutational “hot spot” for ADCs evolution that may undermine the efficacy of newer β-lactams, including cefiderocol. These results underscore the need for ongoing molecular surveillance of A. baumannii isolates to detect and track the emergence of such variants in clinical settings. IMPORTANCE Carbapenem-resistant Acinetobacter baumannii (CRAb) has been designated as a critical priority pathogen by the World Health Organization (WHO). Cefiderocol has been introduced as a novel therapy against CRAb; however, recent clinical data highlight concerning treatment failures and excess mortality. Understanding resistance mechanisms is therefore essential to preserve the clinical utility of this agent. This study addresses a critical knowledge gap by investigating the role of intrinsic Acinetobacter -derived cephalosporinases (ADCs), which are ubiquitous in A. baumannii and diverse in sequence. By defining specific mutational patterns that endanger cefiderocol activity, this work highlights how chromosomally encoded enzymes can evolve to erode the effectiveness of newer β-lactams such as cefiderocol. These insights underscore the importance of integrating molecular surveillance into clinical practice and antimicrobial stewardship to ensure timely detection of emerging resistance in clinical A. baumannii isolates, ultimately informing treatment strategies and guiding future drug development.

ABSTRACT The increasing prevalence of carbapenem-resistant Klebsiella pneumoniae represents a serious global health challenge. Rapid and accurate detection methods are essential to inform early and correct use of antimicrobial therapy to idealize patient outcomes and limit the spread of resistant isolates. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been evaluated for resistance detection, but its clinical application remains limited. In this study, MALDI-TOF MS was integrated with machine learning (ML) to improve detection of Klebsiella pneumoniae carbapenemase (KPC) among K. pneumoniae isolates harboring KPC carbapenemase. Ensemble learning strategies were applied to 435 clinical isolates from different geographic locations (Spain, United States, South America, and Asia), with validation performed using external data sets. By constructing two peak matrices and applying four ML algorithms, as well as combinations of two and three in ensemble models, 92 different classifiers were tested. Ensemble combinations increased the specificity of classifiers to over 95%, while sensitivity reached 72%, being significantly higher than that of the MALDI Biotyper KPC module. Low sensitivity may be affected by technical variability during spectral acquisition. The first annotated MALDI-TOF MS spectrum for K. pneumoniae by in silico prediction of protein masses was developed to enable peak identification, including peaks potentially related to antimicrobial resistance. Overall, the results of this study show that combining MALDI-TOF MS with ensemble learning can enhance KPC detection performance. IMPORTANCE Rapid and accurate detection of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae informs early and correct use of antimicrobial therapy to idealize patient outcomes and limit the spread of antimicrobial resistance. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and ensemble machine learning were employed to detect KPC-producing isolates, using diverse isolate collections, obtaining excellent specificity. An annotated K. pneumoniae MALDI-TOF MS profile is provided with peak annotation, providing a resource for peak analysis.

Abstract 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 &lt; .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 &lt; .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.

ABSTRACT Lytic phages are a promising consideration to combat recalcitrant bacterial infections. To advance the possibility of using phage therapy in the clinical routine, standardized phage susceptibility testing methods are needed. Here, staphylococcal phage K was tested against 154 periprosthetic joint infection-associated Staphylococcus aureus isolates using a double overlay spot assay in technical and biological triplicates. Individual plaques were assessed using a morphology scoring system ranging from 0 (no plaques) to 4 (large, clear plaques), with scores ≥2 considered indicative of infectivity. Overall, 107 isolates exhibited scores ≥2. The intra-day and inter-day coefficients of variation were 0.9 and 2.5, respectively. Plaque-forming unit per milliliter (PFU/mL) titers correlated with morphology scores (Spearman’s ρ = 0.8, P < 0.0001). The results suggest high precision of the described spot assay assessing S. aureus susceptibility to phage K. IMPORTANCE Standardized laboratory assays for assessing phage susceptibility are a prerequisite for clinical implementation of phage therapy. No assay has been denoted a reference standard. Criteria that can be used to assess assay precision and suitability for routine testing are intermediate precision and repeatability. Here, staphylococcal phage K was tested using the double overlay spot assay against 154 periprosthetic joint infection-associated Staphylococcus aureus isolates in technical and biological triplicates. In addition to measuring phage titers, plaque-forming units were assessed with a scoring system. Results indicate high intermediate precision and repeatability of the method described.

Abstract Background Legionella pneumonia is an important cause of severe pneumonia, yet contemporary data describing its clinical phenotype, diagnostic patterns, and outcomes in the modern era are limited. Methods We conducted a retrospective multicenter cohort study of adults with laboratory-confirmed Legionella pneumonia across an integrated health system between January 2019 and September 2025. The primary outcome was 30-day mortality. Severe disease was defined as having a requirement for high-flow nasal cannula, noninvasive ventilation, mechanical ventilation, or extracorporeal membrane oxygenation (ECMO). Univariate and multivariable logistic regression were used to identify predictors of mortality and severe disease. Results There were 344 patients with laboratory-confirmed Legionella pneumonia during the study period. The median age was 66.6 years, and 45.1% were immunocompromised. Most patients required hospital admission (94.2%), with 36.1% admitted to the intensive care unit; 22.7% required mechanical ventilation and 1.5% ECMO. Thirty- and 90-day mortality were 11.9% and 16.6%, respectively. In multivariable analysis, cirrhosis (OR 10.2, 95% CI 2.15–48.3, P = .003), immunocompromised status (odds ratio [OR] 2.24, 95% CI 1.05–4.77, P = .036), age (OR 1.03, 95% CI 1.00–1.06, P = .031), and lymphopenia at presentation (OR 2.09, 95% CI 1.02–4.24, P = .043) were independently associated with increased 30-day mortality. Among patients who were polymerase chain reaction (PCR) or culture positive, urinary antigen testing was positive in only 25.6%. Conclusions Legionella pneumonia was associated with substantial short-term mortality. Cirrhosis, age, immunocompromised status, and lymphopenia at presentation emerged as independent predictors of 30-day mortality, underscoring the importance of host vulnerability in determining outcomes.

Abstract Background Identification of the microorganisms causing infection in children with central nervous system infection is important. Conventional cerebrospinal fluid (CSF) tests may provide limited pathogen detection. CSF metagenomic next-generation sequencing (mNGS) may provide broader pathogen detection in a single assay. Methods Results of CSF mNGS testing performed at Mayo Clinic on patients and nonpatients between January 2024 and June 2025 were retrospectively analyzed. The subcohort of children treated at Mayo Clinic was analyzed to assess test performance as compared with conventional CSF diagnostics and potential clinical impact. Results In total 134 patients (138 CSF mNGS tests) from 22 US states and 2 international sites were included. Overall positivity was 16% with viruses representing most (59%) detections. In the Mayo Clinic subcohort (n = 15), test positivity was 27%: organisms included single detections of Streptococcus mitis group and HIV type 1, as well as 2 detections of Epstein-Barr virus, all confirmed by conventional tests. Positive results were associated with CSF pleocytosis (P = .026). Higher yield was observed in participants whose care was overseen by pediatric infectious diseases (PID) specialists (P = .026) and when a specimen hold strategy was applied (P = .033). Most results (87%) did not alter antimicrobial therapy, with 2 negative results contributing to antimicrobial de-escalation. Conclusions CSF mNGS demonstrated higher diagnostic yield in patients whose specimens showed CSF pleocytosis and in those who had PID consultation. Diagnostic stewardship strategies were applied, including specimen hold approaches and PID consultation.

Abstract Background New insights into community-acquired pleural infection microbiology may enable development of more rapid and accessible diagnostics that can streamline treatment. This study systematically compared molecular approaches for pathogen detection in pleural fluid from patients with such infections. Considering the need for rapid results with minimum hands-on time and test complexity, off-label use of the cassette-based, random-access BIOFIRE Joint Infection Panel (BF-JIP, bioMérieux) with and without supplementary in-house polymerase chain reaction (PCR) assays was evaluated. Methods Pleural fluids from 162 subjects from the Mayo Clinic (Rochester, Minnesota) and Haukeland University Hospital (Bergen, Norway), where molecular tests are routinely used for analyzing pleural fluid, were studied. The performance of the BF-JIP was compared to 16S rRNA gene PCR followed by Sanger and/or next-generation sequencing (16S rRNA gene PCR/sequencing), an in-house PCR panel specifically designed for pleural space infections, and culture. Results The BF-JIP supplemented with 3 in-house PCR assays targeting Fusobacterium nucleatum group, Streptococcus intermedius/constellatus, and Aggregatibacter aphrophilus detected 96.8% of pleural infections, providing complete microbial profiles in 70.1%. In comparison, the detection rate of 16S rRNA gene PCR/sequencing was 92.2%, providing complete microbial profiles in 87.0%. Culture detected bacteria in only 31.9% of pleural fluids, providing complete microbial profiles in 15.3%. Conclusions These results underscore the importance of molecular approaches in diagnosing pleural space infections and demonstrate the feasibility of panel-based PCR-based diagnostics for these sometimes-complex infections. The BF-JIP, designed for testing synovial fluid, could be modified to provide a useful diagnostic for testing pleural fluid.