Fackler, Joseph

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 Background Methicillin-resistant Staphylococcus aureus (MRSA) biofilms can be resilient to the human immune system and antibiotics, a challenge augmented by acquired antimicrobial resistance. Phage therapy is being evaluated for biofilm-associated infections. Methods Here, model phages SaMD07ø1 and SaRB105030ø5 were evaluated against their hosts SaMD07 and SaRB105030, respectively, grown as biofilms in 96-well plates, as biofilms on glass beads and planktonically. Trypticase soy broth (TSB) and phosphate buffered saline (PBS) were evaluated as testing media (Figure 1). Biofilms were incubated with phages in the presence of media containing tetrazolium dye in 96-well plates and assessed for hold times (delays in reaching bacterial log-phase metabolic activity compared to controls) over 48 hours (h) using a Biolog Omnilog® system and by assessing colony forming units (CFUs). Subsequently, using the beads biofilm assay in TSB medium performed on the Omnilog® (BBTO) (Figure 2), test phage SaMD22ø1 was evaluated in quintuplicate against biofilms formed by MRSA isolates IDRL-6130, IDRL-6927, IDRL-6987, IDRL-7381, IDRL-7542 and IDRL-10947, all recovered from periprosthetic joint infection. Test phage K was evaluated against GFP-labelled MRSA AH1263 using BBTO method and simultaneously by fluorescence microscopy.Figure 1.Biofilm phage susceptibility testing methods evaluated (created with BioRender.com)Figure 2.Biofilm phage susceptibility testing using biofilms grown on beads assessed in TSB medium using the Omnilog® (BBTO) method with phage SaMD22ø1. Results A comparison of result of testing of biofilms in wells and on beads and against planktonic bacteria is shown in Figure 3. Phage SaMD07ø1 showed no growth at 4 h; whereas phage SaRB105030ø5 showed ∼1-log decrease in CFUs at 4 h and ∼3-log decrease in CFUs at 8 h (Figure 3). BBTO hold-times of 37, 28 and 15 h were observed for phages SaMD07ø1, SaRB105030ø5 and phage K respectively. Phage SaMD22ø1 had an average hold time of 9 h and showed hold times >6 hours for all isolates tested (Figure 4). Microscopic inspection showed eradication of GFP-labelled MRSA biofilm by phage K (Figure 5).Figure 3.Results of quantitative culture of biofilms treated with phages SaMD07ø1 and SaRB105030ø5 in TSB medium.Panel 1 shows results of phage SaMD07ø1 with biofilms grown in wells (1a), on beads (1b) and planktonically (1c). Panel 2 shows results of phage SaRB105030ø5 with biofilms grown in wells (2a), on beads (2b) and planktonically (2c).Figure 4.Activity of phage SaMD22ø1 against biofilms formed by six MRSA strains determined using the Omnilog® (BBTO) method. The solid blue line indicates a hold time of 8 hours.Figure 5.Efficacy of Phage K to efficiently eradicate GFP-labelled MRSA strain AH1263 observed under the fluorescence microscope at 40X magnification. The authors are thankful to Dr. Alexander Horswill at University of Colorado for providing the GFP-labelled MRSA strain. Conclusion In conclusion, BBTO is a promising method for testing biofilm phage susceptibility although clinical correlation and testing of larger numbers of phage and bacterial isolates are needed. Disclosures Joseph Fackler, n/a, Adaptive Phage Therapeutics, Inc: Employee|Adaptive Phage Therapeutics, Inc: Stocks/Bonds John M. Sowers, n/a, Adaptive Phage Therapeutics: Employee|Adaptive Phage Therapeutics: Stocks/Bonds Robin Patel, MD, Abbott Laboratories: Advisor/Consultant|Adaptive Phage Therapeutics: Grant/Research Support|Adaptive Phage Therapeutics: Mayo Clinic has a royalty-bearing know-how agreement and equity in Adaptive Phage Therapeutics.|BIOFIRE: Grant/Research Support|CARB-X: Advisor/Consultant|ContraFect: Grant/Research Support|Day Zero Diagnostics: Advisor/Consultant|HealthTrackRx: Advisor/Consultant|Mammoth Biosciences: Advisor/Consultant|Netflix: Advisor/Consultant|Oxford Nanopore Technologies: Advisor/Consultant|PhAST: Advisor/Consultant|See details: Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic|See details: continued, patent on an anti-biofilm substance issued|TenNor Therapeutics Limited: Grant/Research Support|Torus Biosystems: Advisor/Consultant|Trellis Bioscience, Inc.: Advisor/Consultant

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.

Abstract Background Pseudomonas aeruginosa has the ability to exhibit resistance to a broad range of antibiotics, highlighting the importance of identifying alternative or adjunctive treatment options, such as phages. Patients and methods We report the case of a 25-year-old male who experienced an accidental electrocution resulting in exposed calvarium in the left parieto-temporal region, complicated by a difficult-to-treat P. aeruginosa (DTR-P. aeruginosa) infection. Cefiderocol was the sole antibiotic with consistent activity against six bacterial isolates obtained from the infected region over a 38 day period. Results WGS analysis identified a blaGES-1 gene as well as the MDR efflux pumps MexD and MexX in all six of the patient’s ST235 DTR-P. aeruginosa isolates, when compared with the reference genome P. aeruginosa PA01 and a P. aeruginosa ST235 isolate from an unrelated patient. After debridement of infected scalp and bone, the patient received approximately 6 weeks of cefiderocol in conjunction with IV phage Pa14NPøPASA16. Some improvement was observed after the initiation of cefiderocol; however, sustained local site improvement and haemodynamic stability were not achieved until phage was administered. No medication-related toxicities were observed. The patient remains infection free more than 12 months after completion of therapy. Conclusions This report adds to the growing literature that phage therapy may be a safe and effective approach to augment antibiotic therapy for patients infected with drug-resistant pathogens. Furthermore, it highlights the importance of the GES β-lactamase family in contributing to inactivation of a broad range of β-lactam antibiotics in P. aeruginosa, including ceftolozane/tazobactam, ceftazidime/avibactam and imipenem/relebactam.

Abstract Background Prosthetic joint infection (PJI) is a potentially limb-threatening complication of total knee arthroplasty. Phage therapy is a promising strategy to manage such infections including those involving antibiotic-resistant microbes, and to target microbial biofilms. Experience with phage therapy for infections associated with retained hardware is limited. A 62-year-old diabetic man with a history of right total knee arthroplasty 11 years prior who had suffered multiple episodes of prosthetic knee infection despite numerous surgeries and prolonged courses of antibiotics, with progressive clinical worsening and development of severe allergies to antibiotics, had been offered limb amputation for persistent right prosthetic knee infection due to Klebsiella pneumoniae complex. Intravenous phage therapy was initiated as a limb-salvaging intervention. Methods The patient received 40 intravenous doses of a single phage (KpJH46Φ2) targeting his bacterial isolate, alongside continued minocycline (which he had been receiving when he developed increasing pain, swelling, and erythema prior to initiation of phage therapy). Serial cytokine and biomarker measurements were performed before, during, and after treatment. The in vitro anti-biofilm activity of KpJH46Φ2, minocycline and the combination thereof was evaluated against a preformed biofilm of the patient’s isolate and determined by safranin staining. Results Phage therapy resulted in resolution of local symptoms and signs of infection and recovery of function. The patient did not experience treatment-related adverse effects and remained asymptomatic 34 weeks after completing treatment while still receiving minocycline. A trend in biofilm biomass reduction was noted 22 hours after exposure to KpJH46Φ2 (P = .063). The addition of phage was associated with a satisfactory outcome in this case of intractable biofilm-associated prosthetic knee infection. Pending further studies to assess its efficacy and safety, phage therapy holds promise for treatment of device-associated infections.