Greenwood-Quaintance, Kerryl

Background: Phages are being assessed for treatment of periprosthetic joint infection (PJI). Staphylococci are the most common cause of PJI. Phages are traditionally considered to be strain-specific and restricted to specific species. We aimed to assess the in vitro activity of a collection of staphylococcal phages against a selection of clinical Staphylococcus isolates recovered from PJI over a 20-year period. Methods: The activity of 37 staphylococcal phages was evaluated against 204 staphylococcal isolates, including 102 Staphylococcus aureus, 81 Staphylococcus epidermidis , and 21 Staphylococcus lugdunensis , using a tetrazolium dye–based metabolic assay. Standardized inocula of bacteria and phage were incubated over 48 hours. Growth inhibition ≥8 hours was considered indicative of phage activity. Results: A total of 7,548 phage-isolate pairs were assessed. Overall, 31 of 37 phages had activity against at least 1 isolate. The most broadly active phage showed activity against 50 of 195 isolates (26%) tested. Twenty-three phages showed cross-species activity, with 11 phages active against all 3 Staphylococcus species, and 12 phages active against 2 species. Of all S. aureus isolates tested, 61% were susceptible to at least 1 phage, while 31% and 76% of S. epidermidis and S. lugdunensis isolates were susceptible to at least 1 phage, respectively. Conclusions: This is the largest library of phages evaluated against staphylococci from PJI. Results show that staphylococcal phages can have cross-species activity, suggesting that phage libraries may offer cross-species coverage of clinically encountered staphylococcal isolates from PJI. Level of Evidence: Level V . Mechanism-based reasoning. See Instructions for Authors for a complete description of levels of evidence.

ABSTRACT 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.

ABSTRACT Standardized approaches to phage susceptibility testing (PST) are essential to inform selection of phages for study in patients with bacterial infections. There is no reference standard for assessing bacterial susceptibility to phage. We compared agreement between PST performed at three centers: two centers using a liquid assay standardized between the sites with the third, a plaque assay. Four Pseudomonas aeruginosa phages: PaWRA01ø11 (EPa11), PaWRA01ø39 (EPa39), PaWRA02ø83 (EPa83), PaWRA02ø87 (EPa87), and a cocktail of all four phages were tested against 145 P . aeruginosa isolates. Comparisons were made within measurements at the two sites performing the liquid assay and between these two sites. Agreement was assessed based on coverage probability (CP 8 ), total deviation index, concordance correlation coefficient (CCC), measurement accuracy, and precision. For the liquid assay, there was satisfactory agreement among triplicate measurements made on different days at site 1, and high agreement based on accuracy and precision between duplicate measurements made on the same run at site 2. There was fair accuracy between measurements of the two sites performing the liquid assay, with CCCs below 0.6 for all phages tested. When compared to the plaque assay (performed once at site 3), there was less agreement between results of the liquid and plaque assays than between the two sites performing the liquid assay. Similar findings to the larger group were noted in the subset of 46 P . aeruginosa isolates from cystic fibrosis. Results of this study suggest that reproducibility of PST methods needs further development.

AbstractPurposeThis pilot study aimed to use proteomic profiling of sonicate fluid samples to compare host response during Staphylococcus aureus‐associated periprosthetic joint infection (PJI) and non‐infected arthroplasty failure (NIAF) and identify potential novel biomarkers differentiating the two.Experimental designIn this pilot study, eight sonicate fluid samples (four from NIAF and four from S. aureus PJI) were studied. Samples were reduced, alkylated, and trypsinized overnight, followed by analysis using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) on a high‐resolution Orbitrap Eclipse mass spectrometer. MaxQuant software suite was used for protein identification, filtering, and label‐free quantitation.ResultsPrincipal component analysis of the identified proteins clearly separated S. aureus PJI and NIAF samples. Overall, 810 proteins were identified based on their detection in at least three out of four samples from each group; 35 statistically significant differentially abundant proteins (DAPs) were found (two‐sample t‐test p‐values ≤0.05 and log2fold‐change values ≥2 or ≤−2). Gene ontology pathway analysis found that microbial defense responses, specifically those related to neutrophil activation, to be increased in S. aureus PJI compared to NIAF samples.Conclusion and clinical relevanceProteomic profiling of sonicate fluid using LC‐MS/MS differentiated S. aureus PJI and NIAF in this pilot study. Further work is needed using a larger sample size and including non‐S. aureus PJI and a diversty of NIAF‐types.

Background: Although cellularity is traditionally assessed morphologically, deep sequencing approaches being used for microorganism detection may be able to provide information about cellularity. We hypothesized that cellularity predicted using CIBERSORTx (Stanford University), a transcriptomic-based cellular deconvolution tool, would differentiate between infectious and non-infectious arthroplasty failure. Methods: CIBERSORTx-derived cellularity profiles of 93 sonicate fluid samples, including 53 from subjects who underwent failed arthroplasties due to periprosthetic joint infection (PJI) (abbreviated for the purpose of this study as PJIF) and 40 from subjects who had undergone non-infectious arthroplasty failure (abbreviated NIAF) that had been subjected to bulk RNA sequencing were evaluated. Results: Samples from PJIF and NIAF subjects were differentially clustered by principal component analysis based on the cellularity profile. Twelve of the 22 individual predicted cellular fractions were differentially expressed in the PJIF cases compared with the NIAF cases, including increased predicted neutrophils (mean and standard error, 9.73% ± 1.06% and 0.81% ± 0.60%), activated mast cells (17.12% ± 1.51% and 4.11% ± 0.44%), and eosinophils (1.96% ± 0.37% and 0.42% ± 0.21%), and decreased predicted M0 macrophages (21.33% ± 1.51% and 39.75% ± 2.45%), M2 macrophages (3.56% ± 0.52% and 8.70% ± 1.08%), and regulatory T cells (1.57% ± 0.23% and 3.20% ± 0.34%). The predicted total granulocyte fraction was elevated in the PJIF cases (32.97% ± 2.13% and 11.76% ± 1.61%), and the samples from the NIAF cases had elevated predicted total macrophage and monocyte (34.71% ± 1.71% and 55.34% ± 2.37%) and total B cell fractions (5.89% ± 0.30% and 8.62% ± 0.86%). Receiver operating characteristic curve analysis identified predicted total granulocytes, neutrophils, and activated mast cells as highly able to differentiate between the PJIF cases and the NIAF cases. Within the PJIF cases, the total granulocyte, total macrophage and monocyte, M0 macrophage, and M2 macrophage fractions were differentially expressed in Staphylococcus aureus compared with Staphylococcus epidermidis -associated samples. Within the NIAF cases, the predicted total B cell, naïve B cell, plasma cell, and M2 macrophage fractions were differentially expressed among different causes of failure. Conclusions: CIBERSORTx can predict the cellularity of sonicate fluid using transcriptomic data, allowing for the evaluation of the underlying immune response during the PJIF and NIAF cases, without a need to phenotypically assess cell composition.

Chronic wound biofilm infections represent a major clinical challenge which results in a substantial burden to patients and healthcare systems. Treatment with topical antibiotics is oftentimes ineffective as a result of antibiotic‐resistant microorganisms and biofilm‐specific antibiotic tolerance. Use of biocides such as hypochlorous acid (HOCl) has gained increasing attention due to the lack of known resistance mechanisms. An HOCl‐generating electrochemical bandage (e‐bandage) is designed that delivers HOCl continuously at low concentrations targeting infected wound beds in a similar manner to adhesive antimicrobial wound dressings. A battery‐operated wearable potentiostat is developed that controls the e‐bandage electrodes at potentials suitable for HOCl generation. It is demonstrated that e‐bandage treatment is tunable by changing the applied potential. HOCl generation on electrode surfaces is verified using microelectrodes. The developed e‐bandage shows time‐dependent responses against in vitro Acinetobacter baumannii and Staphylococcus aureus biofilms, reducing viable cells to nondetectable levels within 6 and 12 h of treatment, respectively. The developed e‐bandage should be further evaluated as an alternative to topical antibiotics to treat wound biofilm infections.

AbstractThe activity of a hypochlorous acid‐producing electrochemical bandage (e‐bandage) in preventing methicillin‐resistant Staphylococcus aureus infection (MRSA) infection and removing biofilms formed by MRSA was assessed using a porcine explant biofilm model. e‐Bandages inhibited S. aureus infection (p = 0.029) after 12 h (h) of exposure and reduced 3‐day biofilm viable cell counts after 6, 12, and 24 h exposures (p = 0.029). Needle‐type microelectrodes were used to assess HOCl concentrations in explant tissue as a result of e‐bandage treatment; toxicity associated with e‐bandage treatment was evaluated. HOCl concentrations in infected and uninfected explant tissue varied between 30 and 80 µM, decreasing with increasing distance from the e‐bandage. Eukaryotic cell viability was reduced by an average of 71% and 65% in fresh and day 3‐old explants, respectively, when compared to explants exposed to nonpolarized e‐bandages. HOCl e‐bandages are a promising technology that can be further developed as an antibiotic‐free treatment for wound biofilm infections.

Drug-resistant Gram-positive bacterial infections are still a substantial burden on the public health system, with two bacteria ( Staphylococcus aureus and Streptococcus pneumoniae ) accounting for over 1.5 million drug-resistant infections in the United States alone in 2017. In 2019, 250,000 deaths were attributed to these pathogens globally. We have developed a preclinical glycopeptide antibiotic, MCC5145, that has excellent potency (MIC 90 ≤ 0.06 μg/ml) against hundreds of isolates of methicillin-resistant S. aureus (MRSA) and other Gram-positive bacteria, with a greater than 1000-fold margin over mammalian cell cytotoxicity values. The antibiotic has therapeutic in vivo efficacy when dosed subcutaneously in multiple murine models of established bacterial infections, including thigh infection with MRSA and blood septicemia with S. pneumoniae , as well as when dosed orally in an antibiotic-induced Clostridioides difficile infection model. MCC5145 exhibited reduced nephrotoxicity at microbiologically active doses in mice compared to vancomycin. MCC5145 also showed improved activity against biofilms compared to vancomycin, both in vitro and in vivo, and a low propensity to select for drug resistance. Characterization of drug action using a transposon library bioinformatic platform showed a mechanistic distinction from other glycopeptide antibiotics.

Abstract Aims Effects of H2O2 producing electrochemical-bandages (e-bandages) on methicillin-resistant Staphylococcus aureus colonization and biofilm removal were assessed using a porcine explant biofilm model. Transport of H2O2 produced from the e-bandage into explant tissue and associated potential toxicity were evaluated. Methods and Results Viable prokaryotic cells from infected explants were quantified after 48 h treatment with e-bandages in three ex vivo S. aureus infection models: (1) reducing colonization, (2) removing young biofilms and (3) removing mature biofilms. H2O2 concentration-depth profiles in explants/biofilms were measured using microelectrodes. Reductions in eukaryotic cell viability of polarized and nonpolarized noninfected explants were compared. e-Bandages effectively reduced S. aureus colonization (p = 0.029) and reduced the viable prokaryotic cell concentrations of young biofilms (p = 0.029) with limited effects on mature biofilms (p > 0.1). H2O2 penetrated biofilms and explants and reduced eukaryotic cell viability by 32–44% compared to nonpolarized explants. Conclusions H2O2 producing e-bandages were most active when used to reduce colonization and remove young biofilms rather than to remove mature biofilms. Significance and Impact of Study The described e-bandages reduced S. aureus colonization and young S. aureus biofilms in a porcine explant wound model, supporting their further development as an antibiotic-free alternative for managing biofilm infections.

Aims The aim of this study was to determine the diagnostic accuracy of α defensin (AD) lateral flow assay (LFA) and enzyme-linked immunosorbent assay (ELISA) tests for periprosthetic joint infection (PJI) in comparison to conventional synovial white blood cell (WBC) count and polymorphonuclear neutrophil percentage (PMN%) analysis. Methods Patients undergoing joint aspiration for evaluation of pain after total knee arthroplasty (TKA) or total hip arthroplasty (THA) were considered for inclusion. Synovial fluids from 99 patients (25 THA and 74 TKA) were analyzed by WBC count and PMN% analysis, AD LFA, and AD ELISA. WBC and PMN% cutoffs of ≥ 1,700 cells/mm3 and ≥ 65% for TKA and ≥ 3,000 cells/mm3 and ≥ 80% for THA were used, respectively. A panel of three physicians, all with expertise in orthopaedic infections and who were blinded to the results of AD tests, independently reviewed patient data to diagnose subjects as with or without PJI. Consensus PJI classification was used as the reference standard to evaluate test performances. Results were compared using McNemar’s test and area under the receiver operating characteristic curve (AUC) analysis. Results Expert consensus classified 18 arthroplasies as having failed due to PJI and 81 due to aseptic failure. Using these classifications, the calculated sensitivity and specificity of AD LFA was 83.3% (95% confidence interval (CI) 58.6 to 96.4) and 93.8% (95% CI 86.2 to 98.0), respectively. Sensitivity and specificity of AD ELISA was 83.3% (95% CI 58.6 to 96.4) and 96.3% (95% CI 89.6 to 99.2), respectively. There was no statistically significant difference between sensitivity (p = 1.000) or specificity (p = 0.157) of the two AD assays. AUC for AD LFA was 0.891. In comparison, AUC for synovial WBC count, PMN%, and the combination of the two values was 0.821 (sensitivity p = 1.000, specificity p < 0.001), 0.886 (sensitivity p = 0.317, specificity p = 0.011), and 0.926 (sensitivity p = 0.317, specificity p = 0.317), respectively. Conclusion The diagnostic accuracy of synovial AD for PJI diagnosis is comparable and not statistically superior to that of synovial WBC count plus PMN% combined. Cite this article: Bone Joint J 2021;103-B(6):1119–1126.