Rapid multiplex PCR panel for pneumonia in hospitalised patients with suspected pneumonia in the USA: a single-centre, open-label, pragmatic, randomised controlled trial.

BACKGROUND: The clinical utility of rapid multiplex respiratory specimen PCR panels for pneumonia for patients with suspected pneumonia is undefined. We aimed to compare the effect of the BioFire FilmArray pneumonia panel (bioMérieux, Salt Lake City, UT, USA) with standard of care testing on antibiotic use in a real-world hospital setting. METHODS: We conducted a single-centre, open-label, pragmatic, randomised controlled trial at the Mayo Clinic, Rochester, MN, USA. Hospitalised patients (aged ≥18 years) with suspected pneumonia, from whom expectorated or induced sputum, tracheal secretions, or bronchoalveolar lavage fluid respiratory culture samples (one per individual) could be collected during index hospitalisation, were eligible for inclusion. Samples from eligible participants were randomly assigned (1:1) with a computerised tool to undergo testing with either the BioFire FilmArray pneumonia panel, conventional culture, and antimicrobial susceptibility testing (intervention group) or conventional culture and antimicrobial susceptibility testing alone (control group). Antimicrobial stewardship review in both groups involved an assessment and recommendations for antibiotic modifications based on clinical data and the results from the BioFire FilmArray pneumonia panel, conventional culture, or both. The primary outcome was median time to first antibiotic modification (ie, escalation or de-escalation of antibiotics against Gram-negative and Gram-positive bacteria) within 96 h of randomisation, assessed with the Wilcoxon rank-sum test and analysed in a modified intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT05937126). FINDINGS: Between Sept 15, 2020, and Sept 19, 2022, 1547 patients were screened for eligibility, of whom 1181 (76·3%) were randomly assigned: 582 (49·3%) to the intervention group and 599 (50·7%) to the control group. In total, 1152 participants were included in the modified intention-to-treat analysis, 589 (51·1%) in the control group and 563 (48·9%) in the intervention group. For the modified intention-to-treat population, median time to any first antibiotic modification was 20·4 h (95% CI 18·0-20·4) in the intervention group and 25·8 h (22·0-28·7) in the control group (p=0·076). Median time to any antibiotic escalation was 13·8 h (9·2-19·0) in the intervention group and 24·1 h (19·5-29·6) in the control group (p=0·0022). Median time to escalation of antibiotics against Gram-positive organisms was 10·3 h (6·2-30·9) in the intervention group and 24·6 h (19·5-37·2) in the control group (p=0·044); median time to escalation of antibiotics against Gram-negative organisms was 17·3 h (10·8-23·3) in the intervention group and 27·2 h (21·3-33·9) in the control group (p=0·010). Median time to any antibiotic de-escalation did not differ between groups (p=0·37). Median time to first de-escalation of antibiotics against Gram-positive organisms was 20·7 h (17·8-24·0) in the intervention group and 27·8 h (22·9-33·0) in the control group (p=0·015); median time to first de-escalation of antibiotics against Gram-negative organisms did not differ between groups (p=0·46). INTERPRETATION: Clinical use of the BioFire FilmArray pneumonia panel might lead to faster antibiotic escalations, including for Gram-negative or Gram-positive bacteria, and faster antibiotic de-escalations directed at Gram-positive bacteria. Additional research is needed regarding antimicrobial de-escalation, especially when antibiotics with broad Gram-negative spectrum are being used, by use of rapid diagnostics in patients with lower respiratory tract infection. FUNDING: bioMérieux.

Engineered Bacteriophage-Polymer Nanoassemblies for Treatment of Wound Biofilm Infections.

The antibacterial efficacy and specificity of lytic bacteriophages (phages) make them promising therapeutics for treatment of multidrug-resistant bacterial infections. Restricted penetration of phages through the protective matrix of biofilms, however, may limit their efficacy against biofilm infections. Here, engineered polymers were used to generate noncovalent phage-polymer nanoassemblies (PPNs) that penetrate bacterial biofilms and kill resident bacteria. Phage K, active against multiple strains of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), was assembled with cationic poly(oxanorbornene) polymers into PPNs. The PPNs retained phage infectivity, while demonstrating enhanced biofilm penetration and killing relative to free phages. PPNs achieved 3-log10 bacterial reduction (∼99.9%) against MRSA biofilms in vitro. PPNs were then incorporated into Poloxamer 407 (P407) hydrogels and applied onto in vivo wound biofilms, demonstrating controlled and sustained release. Hydrogel-incorporated PPNs were effective in a murine MRSA wound biofilm model, showing a 1.5-log10 reduction in bacterial load compared to a 0.5 log reduction with phage K in P407 hydrogel. Overall, this work showcases the therapeutic potential of phage K engineered with cationic polymers for treating wound biofilm infections.

Dual action electrochemical bandage operated by a programmable multimodal wearable potentiostat.

We have developed electrochemical bandage (e-bandage) prototypes that generate the reactive oxygen species hypochlorous acid (HOCl) or hydrogen peroxide (H2O2) for potential use to treat biofilm-infected wounds in humans. We have shown that both e-bandage-generated HOCl and H2O2 kill biofilms in vitro and in infected wounds on mice, with the former being more active in vitro. The H2O2-generating e-bandage, more so than the HOCl-generating e-bandage, was associated with improved healing of infected wounds. Here, a strategy in which H2O2 and HOCl are alternately generated-for dual action-was explored. The goal was to develop a programmable multimodal wearable potentiostat [PMWP] that can generate HOCl or H2O2, as needed. An ultralow-power microcontroller unit was developed to manage operation of the PMWP. The system was operated with a 260-mAh capacity coin battery and weighed 4.6 g, making it suitable for future small animal experiments (and ultimately, potential evaluation in humans). As assessed using electrochemical parameters, the device functioned comparably to a commercial benchtop potentiostat. To confirm antimicrobial activity, PMWP-controlled e-bandages were tested in vitro against clinical isolates of methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus faecium, and Candida auris. When programmed to deliver HOCl followed by H2O2, PMWP-controlled e-bandages exhibited activity against biofilms of all study isolates tested. Finally, we demonstrated the PMWP's usability in a murine wound infection model.

Increased mortality in hospital- compared to community-onset carbapenem-resistant enterobacterales infections.

BACKGROUND: The CDC reported a 35% increase in hospital-onset (HO) carbapenem-resistant Enterobacterales (CRE) infections during the COVID-19 pandemic. We evaluated patient outcomes following HO and community-onset (CO) CRE bloodstream infections (BSI). METHODS: Patients prospectively enrolled in CRACKLE-2 from 56 hospitals in 10 countries between 30 April 2016 and 30 November 2019 with a CRE BSI were eligible. Infections were defined as CO or HO by CDC guidelines, and clinical characteristics and outcomes were compared. The primary outcome was desirability of outcome ranking (DOOR) 30 days after index culture. Difference in 30-day mortality was calculated with 95% CI. RESULTS: Among 891 patients with CRE BSI, 65% were HO (582/891). Compared to those with CO CRE, patients with HO CRE were younger [median 60 (Q1 42, Q3 70) years versus 65 (52, 74); P < 0.001], had fewer comorbidities [median Charlson comorbidity index 2 (1, 4) versus 3 (1, 5); P = 0.002] and were more acutely ill (Pitt bacteraemia score ≥4: 47% versus 32%; P < 0.001). The probability of a better DOOR outcome in a randomly selected patient with CO BSI compared to a patient with HO BSI was 60.6% (95% CI: 56.8%-64.3%). Mortality at 30-days was 12% higher in HO BSI (192/582; 33%) than CO BSI [66/309 (21%); P < 0.001]. CONCLUSION: We found a disproportionately greater impact on patient outcomes with HO compared to CO CRE BSIs; thus, the recently reported increases in HO CRE infections by CDC requires rigorous surveillance and infection prevention methods to prevent added mortality.

A comparison of phage susceptibility testing with two liquid high-throughput methods.

Phage therapy is a promising antibacterial strategy, especially given that drug-resistant bacterial infections are escalating worldwide. Because phages are not active against all strains of a given species, phages being considered for therapeutic use would ideally be tested against bacterial isolates from individual patients prior to administration. Standardized, clinically validated phage susceptibility testing (PST) methods are needed for assessing in vitro phage activity. This study compared two high-throughput liquid-based PST assays. The first, using the Biolog Omnilog™, assessed changes in microbial respiration leading to color changes based on a tetrazolium dye. The second, Agilent BioTek Cytation 7, assessed changes in optical density. Both used 96-well microtiter plate formats. A total of 55 diverse phages with activity against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, or Enterococcus faecalis were studied against their respective susceptible bacterial hosts and non-susceptible controls, with susceptibility defined based on plaque assay. PST was performed by both assays in replicates, with results compared in terms of hold times (time through which bacterial growth is inhibited by phage compared to controls). Coefficients of variance and interclass correlation coefficients were used to assess inter- and intra-assay reproducibility. Based on a ≤50% coefficient of variance cutpoint, 87% of Biolog and 84% of Agilent assays were considered valid for susceptible bacteria, with 100% considered valid for non-susceptible bacteria by both systems. Using a 8 h hold time cutpoint, 100% of the results matched between the two assays. The interclass correlation coefficient showed 26% excellent agreement, 35% good agreement, and 17% moderate agreement between the two assays for susceptible isolates and 100% excellent agreement for non-susceptible isolates. Overall, the assays compared provided good/fair statistical reproducibility for the assessment of phage susceptibility.

ARGONAUT-III and -V: susceptibility of carbapenem-resistant Klebsiella pneumoniae and multidrug-resistant Pseudomonas aeruginosa to the bicyclic boronate ß-lactamase inhibitor taniborbactam combined with cefepime.

Taniborbactam, a bicyclic boronate ß-lactamase inhibitor with activity against Klebsiella pneumoniae carbapenemase (KPC), Verona integron-encoded metallo-ß-lactamase (VIM), New Delhi metallo-ß-lactamase (NDM), extended-spectrum beta-lactamases (ESBLs), OXA-48, and AmpC ß-lactamases, is under clinical development in combination with cefepime. Susceptibility of 200 previously characterized carbapenem-resistant K. pneumoniae and 197 multidrug-resistant (MDR) Pseudomonas aeruginosa to cefepime-taniborbactam and comparators was determined by broth microdilution. For K. pneumoniae (192 KPC; 7 OXA-48-related), MIC90 values of ß-lactam components for cefepime-taniborbactam, ceftazidime-avibactam, and meropenem-vaborbactam were 2, 2, and 1 mg/L, respectively. For cefepime-taniborbactam, 100% and 99.5% of isolates of K. pneumoniae were inhibited at ≤16 mg/L and ≤8 mg/L, respectively, while 98.0% and 95.5% of isolates were susceptible to ceftazidime-avibactam and meropenem-vaborbactam, respectively. For P. aeruginosa, MIC90 values of ß-lactam components of cefepime-taniborbactam, ceftazidime-avibactam, ceftolozane-tazobactam, and meropenem-vaborbactam were 16, >8, >8, and >4 mg/L, respectively. Of 89 carbapenem-susceptible isolates, 100% were susceptible to ceftolozane-tazobactam, ceftazidime-avibactam, and cefepime-taniborbactam at ≤8 mg/L. Of 73 carbapenem-intermediate/resistant P. aeruginosa isolates without carbapenemases, 87.7% were susceptible to ceftolozane-tazobactam, 79.5% to ceftazidime-avibactam, and 95.9% and 83.6% to cefepime-taniborbactam at ≤16 mg/L and ≤8 mg/L, respectively. Cefepime-taniborbactam at ≤16 mg/L and ≤8 mg/L, respectively, was active against 73.3% and 46.7% of 15 VIM- and 60.0% and 35.0% of 20 KPC-producing P. aeruginosa isolates. Of all 108 carbapenem-intermediate/resistant P. aeruginosa isolates, cefepime-taniborbactam was active against 86.1% and 69.4% at ≤16 mg/L and ≤8 mg/L, respectively, compared to 59.3% for ceftolozane-tazobactam and 63.0% for ceftazidime-avibactam. Cefepime-taniborbactam had in vitro activity comparable to ceftazidime-avibactam and greater than meropenem-vaborbactam against carbapenem-resistant K. pneumoniae and carbapenem-intermediate/resistant MDR P. aeruginosa.

HOCl-producing electrochemical bandage is active in murine polymicrobial wound infection.

Wound infections, exacerbated by the prevalence of antibiotic-resistant bacterial pathogens, necessitate innovative antimicrobial approaches. Polymicrobial infections, often involving Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA), present challenges due to biofilm formation and antibiotic resistance. Hypochlorous acid (HOCl), a potent antimicrobial agent, holds promise as an alternative therapy. An electrochemical bandage (e-bandage) that generates HOCl in situ via precise polarization controlled by a miniaturized potentiostat was evaluated for the treatment of murine wound biofilm infections containing both P. aeruginosa with "difficult-to-treat" resistance and MRSA. Previously, HOCl-producing e-bandage was shown to reduce murine wound biofilms containing P. aeruginosa alone. Here, in 5-mm excisional skin wounds containing 48-h biofilms comprising MRSA and P. aeruginosa combined, polarized e-bandage treatment reduced MRSA by 1.1 log10 CFU/g (P = 0.026) vs non-polarized e-bandage treatment (no HOCl production), and 1.4 log10 CFU/g (0.0015) vs Tegaderm only controls; P. aeruginosa was similarly reduced by 1.6 log10 CFU/g (P = 0.0032) and 1.6 log10 CFU/g (P = 0.0015), respectively. For wounds infected with MRSA alone, polarized e-bandage treatment reduced bacterial load by 1.1 log10 CFU/g (P = 0.0048) and 1.3 log10 CFU/g (P = 0.0048) compared with non-polarized e-bandage and Tegaderm only, respectively. The e-bandage treatment did not negatively impact wound healing or cause tissue toxicity. The addition of systemic antibiotics did not enhance the antimicrobial efficacy of e-bandages. This study provides additional evidence for the HOCl-producing e-bandage as a novel antimicrobial strategy for managing wound infections, including in the context of antibiotic resistance and polymicrobial infections. IMPORTANCE: New approaches are needed to combat the rise of antimicrobial-resistant infections. The HOCl-producing electrochemical bandage (e-bandage) leverages in situ generation of HOCl, a natural biocide, for broad-spectrum killing of wound pathogens. Unlike traditional therapies that may exhibit limited activity against biofilms and antimicrobial-resistant organisms, the e-bandage offers a potent, standalone solution that does not contribute to further resistance or require adjunctive antibiotic therapy. Here, we show the ability of the e-bandage to address polymicrobial infection by antimicrobial resistant clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa, two commonly isolated, co-infecting wound pathogens. Effectiveness of the HOCl-producing e-bandage in reducing pathogen load while minimizing tissue toxicity and avoiding the need for systemic antibiotics underscores its potential as a tool in managing complex wound infections.

Clinical outcomes in patients infected with ertapenem-only-resistant Enterobacterales versus multi-carbapenem-resistant Enterobacterales.

BACKGROUND: Use of anti-carbapenem-resistant Enterobacterales (anti-CRE) agents such as ceftazidime/avibactam has been associated with improved clinical outcome in cohorts that primarily include patients infected with CRE that are resistant to meropenem (MCRE). OBJECTIVES: To clarify whether patients with CRE resistant to ertapenem but susceptible to meropenem (ertapenem-only-resistant Enterobacterales; EORE) benefit from therapy with anti-CRE agents. METHODS: Patients treated for CRE infection in hospitals in the USA between 2016 and 2019 and enrolled in the CRACKLE-2 study were included. The primary outcome was the desirability of outcome ranking (DOOR) assessed at 30 days after index cultures. RESULTS: The EORE group included 213 patients and the MCRE group included 643. The demographics were similar between the groups except for the patients' race and origin before admission. The MCRE group received anti-CRE agents for definitive therapy significantly more frequently compared with the EORE group (30% versus 5% for ceftazidime/avibactam). We did not observe a significant difference between the groups in the adjusted DOOR probability of a more desirable outcome for a randomly selected patient in the EORE group compared with the MCRE group (52.5%; 95% CI, 48.3%-56.7%). The MCRE group had a similar proportion of patients who died at 30 days (26% versus 21%) and who were discharged to home (29% versus 40%), compared with the EORE group. CONCLUSIONS: Patients with clinical EORE infection rarely received anti-CRE agents, but attained similar outcomes compared with patients with MCRE infection. The findings support current IDSA treatment guidance for meropenem- or imipenem-based therapy for treatment of EORE infections.

Nature-Derived Gelatin-Based Antifungal Nanotherapeutics for combatting Candida albicans Biofilms.

Infections caused by fungi are emerging global health challenges that are exacerbated by the formation of fungal biofilms. Further challenges arise from environmental contamination with antifungal agents, which promotes environmental acquisition of antifungal resistance. We report the generation of an efficient, sustainable, all-natural antifungal nanotherapeutic based on the integration of an antimicrobial natural essential oil into a gelatin-based nanoemulsion platform. Carvacrol-loaded gelatin nanoemulsions penetrated Candida albicans biofilms, resulting in death of C. albicans cells in biofilms, and displayed selective biofilm elimination without harmful effects on fibroblast cells in a fungal biofilm-mammalian fibroblast co-culture model. Furthermore, the nanoemulsions degraded in the presence of physiologically relevant biomolecules, reducing the potential for environmental pollution and ecotoxicity. Overall, the sustainability, and efficacy of the described gelatin nanoemulsion formulation provides an environmentally friendly strategy for treating biofilm-associated fungal infections, including those caused by drug-resistant fungi.

Genotypic and phenotypic characterization of Enterococcus faecalis isolates from periprosthetic joint infections.

Over 2.5 million prosthetic joint implantation surgeries occur annually in the United States. Periprosthetic joint infections (PJIs), though occurring in only 1-2% of patients receiving replacement joints, are challenging to diagnose and treat and are associated with significant morbidity. The Gram-positive bacterium Enterococcus faecalis, which can be highly antibiotic-resistant and is a robust biofilm producer on indwelling medical devices, accounts for 2-11% of PJIs. E. faecalis PJIs are understudied compared to those caused by other pathogens, such as Staphylococcus aureus. This motivates the need to generate a comprehensive understanding of E. faecalis PJIs to guide future treatments for these infections. To address this, we describe a panel of E. faecalis strains isolated from the surface of prosthetic joints in a cohort of individuals treated at the Mayo Clinic in Rochester, MN. Here, we present the first complete genome assemblage of E. faecalis PJI isolates. Comparative genomics shows differences in genome size, virulence factors, antimicrobial resistance genes, plasmids, and prophages, underscoring the genetic diversity of these strains. These isolates have strain-specific differences in in vitro biofilm biomass, biofilm burden, and biofilm morphology. We measured robust changes in biofilm architecture and aggregation for all isolates when grown in simulated synovial fluid (SSF). Finally, we evaluated the antibiotic efficacy of these isolates and found strain-specific changes across all strains when grown in SSF. Results of this study highlight the existence of genetic and phenotypic heterogeneity among E. faecalis PJI isolates which will provide valuable insight and resources for future E. faecalis PJI research. IMPORTANCE: Periprosthetic joint infections (PJIs) affect ~1-2% of those who undergo joint replacement surgery. Enterococcus faecalis is a Gram-positive opportunistic pathogen that causes ~10% of PJIs in the United States each year, but our understanding of how and why E. faecalis causes PJIs is limited. E. faecalis infections are typically biofilm-associated and can be difficult to clear with antibiotic therapy. Here, we provide complete genomes for four E. faecalis PJI isolates from the Mayo Clinic. These isolates have strain-specific differences in biofilm formation, aggregation, and antibiotic susceptibility in simulated synovial fluid. These results provide important insight into the genomic and phenotypic features of E. faecalis isolates from PJI.

Broad-range amplification and sequencing of the rpoB gene: a novel assay for bacterial identification in clinical microbiology.

The rpoB gene has been proposed as a promising phylogenetic marker for bacterial identification, providing theoretically improved species-level resolution compared to the 16S rRNA gene for a range of clinically important taxa. However, its utility in diagnostic microbiology has been limited by the lack of broad-range primers allowing for its amplification from most species with a single PCR assay. Here, we present an assay for broad-range partial amplification and Sanger sequencing of the rpoB gene. To reduce cross-reactivity and allow for rpoB amplification directly from patient samples, primers were based on the dual priming oligonucleotide principle. The resulting amplicon is ~550 base pairs in length and appropriate for species-level identification. Systematic in silico evaluation of a wide selection of taxa demonstrated improved resolution within multiple important genera, including Enterococcus, Fusobacterium, Mycobacterium, Streptococcus, and Staphylococcus species and several genera within the Enterobacteriaceae family. Broad-range rpoB amplification and Sanger sequencing of 115 bacterial isolates provided unambiguous species-level identification for 97 (84%) isolates, as compared to 57 (50%) using a clinical 16S rRNA gene assay. Several unresolved taxonomic matters disguised by the low resolution of the 16S rRNA gene were revealed using the rpoB gene. Using a collection of 33 clinical specimens harboring bacteria and assumed to contain high concentrations of human DNA, the rpoB assay identified the pathogen in 29 specimens (88%). Broad-range rpoB amplification and sequencing provides a promising tool for bacterial identification, improving discrimination between closely related species and making it amenable for use in culture-based and culture-independent diagnostic approaches.

Evaluation of treatment of methicillin-resistant Staphylococcus aureus biofilms with intermittent electrochemically generated H2O2 or HOCl.

Chronic wound infections can be difficult to treat and may lead to impaired healing and worsened patient outcomes. Novel treatment strategies are needed. This study evaluated the effects of intermittently produced hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), generated via an electrochemical bandage (e-bandage), against methicillin-resistant Staphylococcus aureus biofilms in an agar membrane biofilm model. By changing the working electrode potential, the e-bandage generated either HOCl (1.5 VAg/AgCl) or H2O2 (-0.6 VAg/AgCl). The degree of biocidal activity of intermittent treatment with HOCl and H2O2 correlated with HOCl treatment time; HOCl treatment durations of 0, 1.5, 3, 4.5, and 6 hours (with the rest of the 6-hour total treatment time devoted to H2O2 generation) resulted in mean biofilm reductions of 1.36 ± 0.2, 2.22 ± 0.16, 3.46 ± 0.38, 4.63 ± 0.74, and 7.66 ± 0.5 log CFU/cm2, respectively, vs. non-polarized controls, respectively. However, application of H2O2 immediately after HOCl treatment was detrimental to biofilm removal. For example, 3 hours HOCl treatment followed by 3 hours H2O2 resulted in a 1.90 ± 0.84 log CFU/cm2 lower mean biofilm reduction than 3 hours HOCl treatment followed by 3 hours non-polarization. HOCl generated over 3 hours exhibited biocidal activity for at least 7.5 hours after e-bandage operation ceased; 3 hours of HOCl generation followed by 7.5 hours of non-polarization resulted in a biofilm cell reduction of 7.92 ± 0.12 log CFU/cm2 vs. non-polarized controls. Finally, intermittent treatment with HOCl (i.e., interspersed with periods of e-bandage non-polarization) for various intervals showed similar effects (approximately 6 log CFU/cm2 reduction vs. non-polarized control) to continuous treatment with HOCl for 3 hours, followed by 3 hours of non-polarization. These findings suggest that timing and sequencing of HOCl and H2O2 treatments are crucial for maximizing biofilm control when using an e-bandage strategy.

Evaluation of Sonicate Fluid Culture Cutoff Points for Periprosthetic Joint Infection Diagnosis.

Introduction: Implant sonication is useful for recovery of periprosthetic joint infection (PJI) pathogens in culture, but exact cutoff points for definition of clinically significant sonicate fluid culture results vary from study to study. The aim of this study was to define ideal sonicate fluid culture cutoff points for PJI diagnosis. Methods: Sonicate fluid cultures from hip and knee prosthesis components removed between February 2007 and December 2020 were studied. Prosthesis components were placed in solid containers in the operating room; in the clinical microbiology laboratory, 400 mL Ringer's solution was added, and containers subjected to vortexing, sonication and then vortexing, followed by centrifugation. Concentrated sonicate fluid was plated on aerobic and anaerobic solid media, and culture results reported semiquantitatively, as no growth, <20, 20-50, 51-100, or >100 CFU/10 mL sonicate fluid. Sonicate cultures from cement spacers and cultures yielding more than 1 microorganism were excluded. Sensitivity and specificity of each cutoff point was evaluated. Results: A total of 1448 sonicate fluid cultures were evaluated, 68% from knees and 32% from hips. PJI was present in 644 (44%) cases. Sensitivity of sonicate culture was 75.0% at <20 CFU/10 mL, 55.3% at ≥20 CFU/10 mL, 46.9% at >51 CFU/10 mL, and 39.8% at >100 CFU/10 mL. Specificity was 78.2%, 99.8%, 100%, and 100%, at the 4 cutoff points, respectively. Conclusions: A cutoff point for sonicate fluid culture positivity of ≥20 CFU/10 mL is suitable for PJI diagnosis.

Phage K exposure generates phage-neutralizing activity in a rabbit model-humoral receptiveness may impact efficacy of phage therapy.

Phage therapy has not been established in the clinical routine, in part due to uncertainties concerning efficacy and immunogenicity. Here, three rabbits were immunized against staphylococcal phage K to assess viral potency in the presence of immunized serum. Three rabbits received weekly intramuscular injections of ~1010±1 pfu/mL phage K. Phage K-specific IgG formation was measured by an enzyme-linked immunosorbent assay (ELISA); phage inactivation was assessed by calculating K-rates. Using transmission electron microscopy (TEM) and immunogold labeling, antibody binding to phage K was visualized. This was numerically assessed by objective imaging analysis comparing the relative distances of each gold particle to the nearest phage head and tail structure. Immunization led to a strong IgG response, plateauing 7 days after the last phage injection. There was no significant correlation between K-rate and antibody titer over time. TEM showed IgG binding to the head structure of phage K. Image analysis showed a significant reduction in relative distances between antibodies and phage head structures when comparing samples from day 0 and day 28 (P < 0.0001). These results suggest that while individual serum analysis for antibodies against therapeutic phage bears consideration prior to and with prolonged therapy, during phage application, the formation of specific antibodies against phage may only partially explain decreased phage potency in the presence of immunized serum. Instead, other factors may contribute to an individual's "humoral receptiveness" to phage therapy. Future investigations should be directed toward the identification of the humoral factors that have the most significant predictive value on phage potency in vivo.

Duration of cefazolin prophylaxis did not impact infection risk in a murine model of joint arthroplasty.

To minimize periprosthetic joint infection (PJI) risk, some clinicians prescribe extended antibiotic prophylaxis (EAP) following total joint arthroplasty (TJA). Given the limited evidence supporting EAP, we sought to evaluate impact of prophylactic antibiotic duration on PJI risk in a murine TJA model. A titanium prosthesis was implanted into the proximal tibia of 89 mice and inoculated with 102 colony forming units (cfu) of Staphylococcus aureus Xen36. Control mice (n = 20) did not receive antibiotics. Treated mice received either 24 h (n = 35) or 4 days (n = 34) of cefazolin prophylaxis. Cultures were obtained from the prostheses, tibia, femur, and knee tissues 3 weeks after surgery. All mice in the control group developed PJI. Both prophylaxis regimens reduced the rate of PJI relative to the control, with only 2/35 mice in the 24-h cohort (p < 0.0001) and 1/34 in 4-day cohort developing PJI (p < 0.0001). CFU counts from the prostheses, bone and knee tissues were reduced for the 24-h and 4-day prophylaxis cohorts relative to the control (p < 0.0001 for both). There was no difference in rates of PJI or CFU counts between the two prophylaxis cohorts (p = 0.58). Prophylactic cefazolin profoundly reduced rates of PJI in a murine model of TJA in which all control animals developed PJI. Extending cefazolin prophylaxis duration from 24 h to 4 days did not result in improved PJI rates or decreased bacterial loads in infected cases. While these results strongly support use of antibiotic prophylaxis for TJA, EAP did not appear to add benefit in the described mouse model.

Evaluation of Treatment of Methicillin-Resistant Staphylococcus aureus Biofilms with Intermittent Electrochemically-Generated H2O2 or HOCl.

Chronic wound infections can be difficult to treat and may lead to impaired healing and worsened patient outcomes. Novel treatment strategies are needed. This study evaluated effects of intermittently produced H2O2 and HOCl, generated via an electrochemical bandage (e-bandage), against methicillin-resistant Staphylococcus aureus biofilms in an agar membrane biofilm model. By changing the working electrode potential, the e-bandage generated either HOCl (1.5 VAg/AgCl) or H2O2 (-0.6 VAg/AgCl). The degree of biocidal activity of intermittent treatment with HOCl and H2O2 correlated with HOCl treatment time; HOCl treatment durations of 0, 1.5, 3, 4.5, and 6 hours (with the rest of the 6 hour total treatment time devoted to H2O2 generation) resulted in mean biofilm reductions of 1.36±0.2, 2.22±0.16, 3.46±0.38, 4.63±0.74 and 7.66±0.5 log CFU/cm2, respectively vs. non-polarized controls, respectively. However, application of H2O2 immediately after HOCl treatment was detrimental to biofilm removal. For example, 3-hours HOCl treatment followed by 3-hours H2O2 resulted in a 1.90±0.84 log CFU/cm2 lower mean biofilm reduction than 3-hours HOCl treatment followed by 3-hours non-polarization. HOCl generated over 3-hours exhibited biocidal activity for at least 7.5-hours after e-bandage operation ceased; 3-hours of HOCl generation followed by 7.5-hours of non-polarization resulted in a biofilm cell reduction of 7.92±0.12 log CFU/cm2 vs. non polarized controls. Finally, intermittent treatment with HOCl (i.e., interspersed with periods of e-bandage non-polarization) for various intervals showed similar effects (approximately 6 log CFU/cm2 reduction vs. non-polarized control) to continuous treatment with HOCl for 3-hours, followed by 3-hours of non-polarization. These findings suggest that timing and sequencing of HOCl and H2O2 treatments are crucial for maximizing biofilm control.

Comparative transcriptomic analysis of Staphylococcus epidermidis associated with periprosthetic joint infection under in vivo and in vitro conditions.

Staphylococcus epidermidis is part of the commensal microbiota of the skin and mucous membranes, though it can also act as a pathogen in certain scenarios, causing a range of infections, including periprosthetic joint infection (PJI). Transcriptomic profiling may provide insights into mechanisms by which S. epidermidis adapts while in a pathogenic compared to a commensal state. Here, a total RNA-sequencing approach was used to profile and compare the transcriptomes of 19 paired PJI-associated S. epidermidis samples from an in vivo clinical source and grown in in vitro laboratory culture. Genomic comparison of PJI-associated and publicly available commensal-state isolates were also compared. Of the 1919 total transcripts found, 145 were from differentially expressed genes (DEGs) when comparing in vivo or in vitro samples. Forty-two transcripts were upregulated and 103 downregulated in in vivo samples. Of note, metal sequestration-associated genes, specifically those related to staphylopine activity (cntA, cntK, cntL, and cntM), were upregulated in a subset of clinical in vivo compared to laboratory grown in vitro samples. About 70% of the total transcripts and almost 50% of the DEGs identified have not yet been annotated. There were no significant genomic differences between known commensal and PJI-associated S. epidermidis isolates, suggesting that differential genomics may not play a role in S. epidermidis pathogenicity. In conclusion, this study provides insights into phenotypic alterations employed by S epidermidis to adapt to infective and non-infected microenvironments, potentially informing future therapeutic targets for related infections.

HOCl-producing Electrochemical Bandage is Active in Murine Polymicrobial Wound Infection.

Wound infections, exacerbated by the prevalence of antibiotic-resistant bacterial pathogens, necessitate innovative antimicrobial approaches. Polymicrobial infections, often involving Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA), present formidable challenges due to biofilm formation and antibiotic resistance. Hypochlorous acid (HOCl), a potent antimicrobial agent produced naturally by the immune system, holds promise as an alternative therapy. An electrochemical bandage (e-bandage) that generates HOCl in situ was evaluated for treatment of murine wound biofilm infections containing both MRSA and P. aeruginosa with "difficult-to-treat" resistance. Previously, the HOCl-producing e-bandage was shown to reduce wound biofilms containing P. aeruginosa alone. Compared to non-polarized e-bandage (no HOCl production) and Tegaderm only controls, the polarized e-bandages reduced bacterial loads in wounds infected with MRSA plus P. aeruginosa (MRSA: vs Tegaderm only - 1.4 log10 CFU/g, p = 0.0015, vs. non-polarized - 1.1 log10 CFU/g, p = 0.026. P. aeruginosa: vs Tegaderm only - 1.6 log10 CFU/g, p = 0.0015, vs non-polarized - 1.6 log10 CFU/g, p = 0.0032), and MRSA alone (vs Tegaderm only - 1.3 log10 CFU/g, p = 0.0048, vs. non-polarized - 1.1 log10 CFU/g, p = 0.0048), without compromising wound healing or causing tissue toxicity. Addition of systemic antibiotics did not enhance the antimicrobial efficacy of e-bandages, highlighting their potential as standalone therapies. This study provides additional evidence for the HOCl-producing e-bandage as a novel antimicrobial strategy for managing wound infections, including in the context of antibiotic resistance and polymicrobial infections.

DUAL ACTION ELECTROCHEMICAL BANDAGE OPERATED by a PROGRAMMABLE MULTIMODAL WEARABLE POTENTIOSTAT.

Electrochemical bandages (e-bandages) can be applied to biofilm-infected wounds to generate reactive oxygen species, such as hypochlorous acid (HOCl) or hydrogen peroxide (H 2 O 2 ). The e-bandage-generated HOCl or H 2 O 2 kills biofilms in vitro and in infected wounds on mice. The HOCl-generating e-bandage is more active against biofilms in vitro , although this distinction is less apparent in vivo . The H 2 O 2 -generating e-bandage, more than the HOCl-generating e-bandage, is associated with improved healing of infected wounds. A strategy in which H 2 O 2 and HOCl are generated alternately-for dual action-was explored. The goal was to develop a programmable multimodal wearable potentiostat (PMWP) that could be programmed to generate HOCl or H 2 O 2 , as needed. An ultralow-power microcontroller unit managed operation of the PMWP. The system was operated with a 260-mAh capacity coin battery and weighed 4.6 grams, making it suitable for small animal experiments or human use. The overall cost of a single wearable potentiostat was $6.50 (USD). The device was verified using established electrochemical systems and functioned comparably to a commercial potentiostat. To determine antimicrobial effectiveness, PMWP-controlled e-bandages were tested against clinical isolates of four prevalent chronic wound bacterial pathogens, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Acinetobacter baumannii , and Enterococcus faecium , and one fungal pathogen of emerging concern, Candida auris . PMWP-controlled e-bandages exhibited broad-spectrum activity against biofilms of all study isolates tested when programmed to deliver HOCl followed by H 2 O 2 . These results show that the PMWP operates effectively and is suitable for animal testing.

Practical Guidance for Clinical Microbiology Laboratories: Microbiologic diagnosis of implant-associated infections.

SUMMARYImplant-associated infections (IAIs) pose serious threats to patients and can be associated with significant morbidity and mortality. These infections may be difficult to diagnose due, in part, to biofilm formation on device surfaces, and because even when microbes are found, their clinical significance may be unclear. Despite recent advances in laboratory testing, IAIs remain a diagnostic challenge. From a therapeutic standpoint, many IAIs currently require device removal and prolonged courses of antimicrobial therapy to effect a cure. Therefore, making an accurate diagnosis, defining both the presence of infection and the involved microorganisms, is paramount. The sensitivity of standard microbial culture for IAI diagnosis varies depending on the type of IAI, the specimen analyzed, and the culture technique(s) used. Although IAI-specific culture-based diagnostics have been described, the challenge of culture-negative IAIs remains. Given this, molecular assays, including both nucleic acid amplification tests and next-generation sequencing-based assays, have been used. In this review, an overview of these challenging infections is presented, as well as an approach to their diagnosis from a microbiologic perspective.