Patel, Robin

ABSTRACT Periprosthetic joint infection (PJI) is a devastating complication of total joint arthroplasty that can be challenging to diagnose and treat. Neutrophils are a prominent feature in PJI, with neutrophil extracellular traps (NETs) known to be present; little is known, however, about the physiology of PJI-associated NETs. Here, the proteomes of PJI and non-infectious arthroplasty failure (NIAF), analyzed using liquid chromatography tandem mass-spectrometry (LC-MS/MS) and proximity extension assay (PEA)-based profiling of fluids derived from sonication of arthroplasty components removed from patients who had undergone revision arthroplasty, were compared, with a focus on NET formation. In addition, in vitro effects of synovial fluid components on Staphylococcus aureus- induced NET formation were characterized. LC-MS/MS (PJI n = 85, NIAF n = 26) and PEA (PJI n = 95, NIAF n = 39) showed NET formation and neutrophil degranulation to be associated with PJI, with the complement cascade and platelet activation pathways identified as potential contributors. In vitro studies showed delayed NET formation in artificial synovial fluid compared to standard cell culture media conventionally used to evaluate NETs. Together, these results demonstrate the presence of NETs in PJI, identify potential pathways involved in PJI-associated NETosis, and point to an influence of synovial components on NET formation during PJI. IMPORTANCE Periprosthetic joint infection (PJI) is a major complication of joint replacement, placing a significant burden on patients and the healthcare system. Our research reveals that neutrophils and their antimicrobial “traps” are dominant features of the immune response at the infection joint site. Importantly, we found that the unique environment of the joint delays the formation of these traps, potentially contributing to the pathogenesis of PJI. By establishing a new experimental platform tailored for the infected joint, this work provides a foundational platform for probing these mechanisms and supports the development of improved diagnostic and therapeutic strategies.

Background: Infection following extensor mechanism reconstruction (EMR) with Marlex mesh (C.R. Bard) is devastating and limb-threatening. The purpose of this study was to evaluate whether vancomycin-loaded mesh reduces staphylococcal infection in a murine EMR model. Methods: Mouse-sized, 8-layer mesh constructs were fabricated to simulate clinical reconstruction and soaked in a vancomycin solution (50 mg/mL) for in vitro and in vivo testing. Three methicillin-resistant clinical isolates from human periprosthetic Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus lugdunensis knee infections were evaluated. In vitro testing assessed vancomycin loading capacity, mechanical properties, antibacterial activity, and inhibition of biofilm formation. For in vivo experiments, plain or vancomycin-loaded mesh was implanted into C57BL/6 mouse extensor mechanisms, which were intraoperatively contaminated with 1 of the 3 study isolates (5 per group). Mesh and surrounding tissue were harvested for quantitative culture on postoperative day 9. Results: For all 3 isolates, vancomycin-loaded mesh produced zones of inhibition and exhibited antibacterial activity through at least 3 days in vitro (p < 0.0001). Mechanical testing revealed that loading mesh with vancomycin did not negatively impact tensile strength, hysteresis, or suture pull-through properties. In vitro testing of inhibition of biofilm formation demonstrated reduced colony-forming units 6 hours after inoculation, which remained low throughout the 9-day experimental period (p < 0.05 for all isolates). In vivo, vancomycin-loaded mesh reduced bacterial burden on mesh and in surrounding tissue by >3 log 10 compared with plain mesh for all 3 isolates (p < 0.01 for both mesh and tissue). Conclusions: Vancomycin-loaded mesh provided in vitro antibacterial activity without compromising mesh mechanical properties and reduced staphylococcal infection in a murine EMR model. Further work is needed to evaluate translational potential and safety before clinical application. Clinical Relevance: Mesh-associated infection after EMR is devastating and difficult to treat. These preclinical findings suggest that local vancomycin delivery may serve as an adjunct to systemic prophylaxis, warranting further study before clinical use.

Background Staphylococcus aureus causes a multiplicity of human diseases acquired in community and healthcare settings alike around the globe. While most studies focus on coding changes to assess genome evolution and study genetic adaptation, interrogation of silent mutations in the form of synonymous codon usage bias is less well-studied. As such, understanding of patterns in codon bias at the gene and genome levels, and how codon bias impacts protein expression in S. aureus remains incomplete. Methods The codon bias of 2,565 protein encoding genes from NCTC 8325 was queried against all publicly available closed S. aureus genomes. Using public BioSample data, genomes were sorted by disease state, submitting institution, and collection site. Codon bias was assessed at the level of gene and genome using the codon adaptation index (CAI), calculated using 30S and 50S ribosomal genes. Gene set enrichment analysis was applied to determine associations between physiological functions, CAI gene scores, and interquartile ranges. CAI scores were also compared to an in vitro S. aureus proteomics database to correlate codon bias and protein expression. Results CAI scores varied within and between isolates at the gene and genome levels. Genes with ribosome-associated functions were most enriched among high CAI genes, and had low CAI interquartile ranges (IQR), suggesting selective pressure to maintain high expression of these genes across all S. aureus isolates. Genome sequences submitted by Aga Khan University Hospital, Nairobi, Kenya were most different from others. For the LAC USA 300 strain, CAI and protein expression were moderately positively correlated (cor = 0.534, p < 2.2e-16). Conclusions Codon bias in S. aureus was shown to vary between gene, and to be a source of genetic variation between isolates; CAI and in vitro protein expression were positively correlated.

Aims Extensor mechanism disruption is a major complication following total knee arthroplasty, with treatment often consisting of extensor mechanism reconstruction utilizing monofilament polypropylene mesh (Marlex mesh, C. R. Bard, Inc). Infection following this procedure is devastating, often resulting in revision surgery and mesh removal, with permanent limb dysfunction. This study evaluated the in vitro ability of vancomycin-loaded polypropylene mesh to inhibit growth of isolates of three Staphylococcus species from patients with periprosthetic joint infection. Methods The mesh was prepared in two configurations – single-layer and eight-layer – and soaked for ten minutes in either sterile water (control) or vancomycin solution (50 mg/ml), after which the mesh was placed on tryptic soy agar plates inoculated with staphylococci and incubated at 37°C for 18 to 24 hours. Zones of inhibition were measured daily over seven days, with mesh transferred to freshly inoculated plates daily. Testing was performed in triplicate, with experiments repeated three times for each bacterial isolate. A total of 30 clinical isolates were tested (five methicillin-susceptible Staphylococcus epidermidis , five methicillin-resistant S. epidermidis , ten methicillin-resistant Staphylococcus aureus , and ten Staphylococcus lugdunensis ). Results Across all species, the eight-layer vancomycin-loaded mesh produced statistically significantly larger median zones of inhibition than the single-layer mesh. The antibacterial activity of the eight-layer mesh persisted through to day 7, while the single-layer mesh showed reduced activity by days 4 to 5. Conclusion Compared with single-layer vancomycin-loaded mesh, multilayer vancomycin-loaded mesh, which mimics clinical application, provides enhanced and sustained antibacterial activity in vitro. Cite this article: Bone Joint Res 2026;15(8):998–1004.

ABSTRACT Blood culture contamination can lead to false-positive blood culture results, potentially contributing to increased costs and unneeded antibiotic utilization. Initial specimen diversion devices (ISDD) have been proposed to reduce blood culture contamination rates. A single-center, observational study conducted at Mayo Clinic Rochester compared blood culture contamination rates using Steripath or Kurin ISDDs versus standard blood culture collection protocols. Over 255 phlebotomists were trained in the use of the ISDDs, with 31,215 blood cultures, 410 of which were contaminated, collected from adults in the emergency department and inpatient wards. Baseline contamination rates were 1.29%–2.53% with standard collection methods. The Steripath ISDD with direct-to-media inoculation of blood culture bottles (Steripath-DTM) reduced blood culture contamination compared to standard practice (0.34% vs. 1.43%, respectively, P < 0.001). Follow-up studies with the alternative Steripath configuration with an integrated syringe (Steripath-syringe), and then the Kurin ISDD with syringe collection (Kurin-syringe), did not meet the primary endpoint of <0.5% contamination (Steripath-syringe: 1.28%; Kurin-syringe: 1.76%). All three ISDD configurations failed to meet our goal of >75% utilization, with an average measured utilization of 26% for the Steripath-DTM, 26.4% for the Steripath ISDD (combined DTM and syringe) in a follow-up study, and 41.5% in the Kurin study. Additionally, a survey of phlebotomists revealed low staff satisfaction with both Steripath ISDD configurations. Staff reported multiple ergonomic barriers limiting the Steripath ISDD’s utility, which precluded implementation of the Steripath-DTM in routine clinical practice. IMPORTANCE We conducted a very large, real-world study of two blood diversion devices to determine whether either would meet goals for reduced blood culture contamination and a high rate of utilization by phlebotomists.

Abstract A low-cost, easy-to-adopt noninvasive imaging modality for differentiating bacterial infection from inflammation is desirable to accurately diagnose bacterial infections. Herein, a bacteria-specific PET probe, [68Ga]Ga-BP, was developed and evaluated in rat models of foreign body-associated osteomyelitis induced by methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae and was directly compared with sterile inflammation and uninstrumented uninfected controls. [68Ga]Ga-BP-based imaging of MRSA-infected tibiae was also compared to [68Ga]Ga-citrate and [18F]FDG. After iron depletion, dynamic PET/CT imaging was performed at 15–120 min postinjection of radiotracers, followed by assessment of ex vivo biodistribution. [68Ga]Ga-BP successfully detected tibial infection and demonstrated &gt;6-fold higher uptake in infected tibiae with all three bacterial species versus contralateral tibiae via PET imaging. [68Ga]Ga-BP outperformed [68Ga]Ga-citrate and [18F]FDG in identifying MRSA infection. [68Ga]Ga-BP represents a promising, noninvasive PET imaging agent for direct bacterial detection and bacterial infection treatment monitoring.

ABSTRACT Legionella species are important causes of community-acquired pneumonia. The diagnostic performance and clinical implications of a fluorescence resonance energy transfer (FRET)-based real-time PCR assay targeting the Legionella 5S rRNA gene were evaluated, with emphasis on melting temperature (Tm) analysis to differentiate detected species and associated clinical outcomes. A retrospective multicenter cohort study of adults with laboratory-confirmed Legionella infection between 2019 and 2025 was conducted. PCR crossing point (Cp) and Tm data were analyzed and compared with species identified in culture from respiratory specimens. Associations between Tm and clinical outcomes were assessed using multivariable logistic regression. Among 51,489 PCR tests performed, 823 (1.6%) were positive. Among 376 Legionella infections, PCR demonstrated the highest diagnostic yield. Among patients undergoing all three diagnostic tests, the addition of PCR increased detection from 49% with urine antigen and culture combined to 100% ( P < 0.001). Of 189 PCR-positive patient specimens with available Tm data, Tm showed a bimodal distribution, being either <66°C or ≥66°C. A Tm cutoff of ≥66°C discriminated L. pneumophila / L. longbeachae from other Legionella species with excellent performance (AUC, 1.00). Overall, 29.6% of infections had a Tm <66°C, consistent with non- pneumophila/ non- longbeachae Legionella species. Infections due to L. pneumophila / L. longbeachae , which correspond to high Tm values in the study assay, were independently associated with ICU admission (adjusted odds ratio, 2.85; 95% CI, 1.33–6.11; P = 0.007), but not 90-day mortality. Real-time PCR targeting the Legionella 5S rRNA gene provides superior detection compared to culture and/or urine antigen testing and enables higher-risk species discrimination through Tm analysis. IMPORTANCE Nearly one-third of Legionella infections in this cohort were attributable to non- pneumophila species, highlighting limitations of diagnostic strategies reliant on Legionella urine antigen testing or L. pneumophila PCR. The findings suggest that species-level inference can be derived from melting-temperature data generated during routine PCR testing. Incorporating validated melting-temperature interpretation into reporting workflows may improve epidemiologic surveillance and clinical understanding of characteristics of Legionella pneumonia.

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 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.