Karau, Melissa J.

Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of orthopedic implant-associated infection, where S. aureus can survive intracellularly and form biofilms. Vancomycin has poor activity against staphyloccoccal biofilms. Although rifampin is effective against staphylococcoal biofilms and has intracellular activity, resistance may develop when it is used as a single agent. Contezolid is a novel oxazolidinone with reported activity against S. aureus in vitro and in vivo in both murine systemic and thigh infection models. Here, we assessed the in vivo activity of contezolid, with and without rifampin, in a rat model of foreign body osteomyelitis. Figure 1. Quantities of MRSA IDRL-6169 recovered from A. tibia B. K-wire. Circles represent values from individual animals; horizontal lines represent mean values. Compared to no treatment: *p=0.0186 **p<0.0002 Methods The left tibia of 60 male Wister rats were inoculated with arachidonic acid (sclerosing agent) and 106 cfu MRSA IDRL-6169 and a K-wire implanted. After four weeks, rats received either no treatment, contezolid (50 mg/kg orally, every 12 h), rifampin (10 mg/kg intraperitoneally every 12 h), rifampin plus contezolid, or vancomycin (75 mg/kg intraperitoneally, every 12 h) plus rifampin for 21 days. Following treatment, tibiae were harvested and cyropulverized. Tibiae and K-wires were cultured separately, and results reported as log10 cfu/gram of bone or K-wire (limit of detect 0.1 log10), respectively. Wilcoxon rank-sum test and false discovery rates were performed and p< 0.05 considered significant. Results Results are shown in Figure 1. MRSA was recovered from tibiae and K-wires of all untreated animals, at means of 5.1 log10 cfu/bone and 3.0 log10 cfu/K-wire, respectively. There was a 1.0 log10 cfu/g mean reduction in MRSA recovered from the tibiae of contezolid-treated rats compared to untreated rats (p=0.0186), and a 0.2 log10 reduction on K-wires. No MRSA was recovered from the tibiae or K-wires in animals receiving rifampin, whether alone or in combination with contezolid or vancomycin. Conclusion Contezolid was active against MRSA in a rat model of foreign body osteomyelitis, reducing bacterial loads in bone, albeit not on K-wires, unless co-administered with rifampin. Disclosures Robin Patel, MD, a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, a: See above|MicuRx Pharmaceuticals and BIOFIRE: Grant/Research Support|PhAST, Day Zero Diagnostics, Abbott Laboratories, Sysmex, DEEPULL DIAGNOSTICS, S.L., Netflix, Oxford Nanopore Technologies and CARB-X: Advisor/Consultant|Up-to-Date and the Infectious Diseases Board Review Course.: Honoraria

Abstract Background Phage therapy is being evaluated to treat bacterial infections. Host-related factors that might abrogate phage efficacy remain incompletely characterized. Phage aggregation has been hypothesized to protect phages in harsh environments. Herein, effects of immunized rabbit sera on phage aggregation were assessed. Methods Three New Zealand rabbits were immunized weekly for 3 weeks by intramuscular injection of phage K. Serum samples were collected weekly at times 0, 7, 14, 21 and 28. Development of an antibody response was confirmed by ELISA. Phage K was exposed to immunized serum with or without heat-inactivated host bacterium (Staphylococcus aureus ATCC 19685) as follows. 45 µl of phage K (1010±1 plaque forming units/mL [pfu/ml]) was mixed with 5 µl immunized serum diluted 1:1000 with or without heat-inactivated bacterial host in phosphate buffered saline (PBS). After incubation, exposed phage was visualized using transmission electron microscopy (TEM, JEOL) at 80kV. Results Phage K immunization elicited a strong IgG response. Exposure to immunized serum led to a decrease in viable phage over time, abrogated by adding heat-inactivated bacteria (Fig 1). TEM imaging showed phage clustering with exposure to immunized serum (Fig 2A); clusters appeared disorganized with concomitant exposure to heat-inactivated host bacteria (Fig 2B). Exposure of phage to heat-inactivated bacteria without serum yielded evenly distributed phage, with minimal clustering (Fig 2C). Conclusion Phage K exposure elicited a neutralizing immune response associated with decreased phage survival in immunized serum, prolonged by adding heat-inactivated host bacteria. TEM imaging showed phage K clustering in different ways depending on the microenvironment encountered. Biological relevance and mechanistic underpinnings of these phenomena need further characterization. Disclosures Robin Patel, MD, a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, a: See above|MicuRx Pharmaceuticals and BIOFIRE: Grant/Research Support|PhAST, Day Zero Diagnostics, Abbott Laboratories, Sysmex, DEEPULL DIAGNOSTICS, S.L., Netflix, Oxford Nanopore Technologies and CARB-X: Advisor/Consultant|Up-to-Date and the Infectious Diseases Board Review Course.: Honoraria

Abstract Background Chronic periprosthetic joint infection is commonly managed with two-stage exchange. Clinically, evaluation for persistent infection at the second stage of exchange may inform management. The aim of this study was to evaluate the yield of periprosthetic tissue and antimicrobial-containing cement spacer sonicate fluid cultures at the second stage of two-stage exchange surgeries. Methods Periprosthetic tissue and sonicate fluid cultures of hip and knee cement spacers removed at the second stage of two-stage exchange surgeries between January 2010 and December 2020 were studied. Two or more positive tissue cultures yielding the same organism were considered positive. For sonicate culture, ≥20 CFU/10 ml of sonicate fluid was considered positive. Persistent infection was defined by the presence of at least one of the following: intraoperative purulence, sinus tract, histopathology showing acute inflammation, or clinical signs of infection. Cement spacers were removed as part of spacer exchange if persistent infection was suspected or for insertion of a new implant if infection was not suspected. Results 172 cultures were evaluated, 69% from knees and 31% from hips. Median patient age was 64 years; 53% were men. 38/172 (22%) of cement spacers were removed as part of a spacer exchange and 134/172 (78%) for insertion of a new implant. Persistent infection was present in 49/172 (28%). 18/172 (10%) received antibiotics 2 weeks before culture, including 8/18 (44%) undergoing spacer exchange. For persistent infections, tissue and sonicate culture were positive in 11/49 and 5/49, respectively. In the 123 without persistent infection, no tissue or sonicate cultures were positive. When a new implant was placed, persistent infection was observed in 18/134 (13%), and tissue and sonicate cultures were positive in 4/18 and 1/18, respectively. When a new implant was placed (116) and no infection was found, all tissue and sonicate cultures were negative. After spacer exchange, persistent infection was present in 31/38 (81%), and tissue and sonicate cultures were positive in 8/31 and 3/31, respectively; in the 7 without infection, there were no positive cultures. Conclusion Sonication culture of cement spacers and periprosthetic tissue cultures have low sensitivities for persistent infection. Disclosures Matthew Abdel, MD, Stryker: Advisor/Consultant|Stryker: Royalties Nicholas Bedard, MD, Stryker: Advisor/Consultant Robin Patel, MD, a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, a: See above|MicuRx Pharmaceuticals and BIOFIRE: Grant/Research Support|PhAST, Day Zero Diagnostics, Abbott Laboratories, Sysmex, DEEPULL DIAGNOSTICS, S.L., Netflix, Oxford Nanopore Technologies and CARB-X: Advisor/Consultant|Up-to-Date and the Infectious Diseases Board Review Course.: Honoraria

ABSTRACT 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 log 10 CFU/g ( P = 0.026) vs non-polarized e-bandage treatment (no HOCl production), and 1.4 log 10 CFU/g (0.0015) vs Tegaderm only controls; P. aeruginosa was similarly reduced by 1.6 log 10 CFU/g ( P = 0.0032) and 1.6 log 10 CFU/g ( P = 0.0015), respectively. For wounds infected with MRSA alone, polarized e-bandage treatment reduced bacterial load by 1.1 log 10 CFU/g ( P = 0.0048) and 1.3 log 10 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.

AbstractTo 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.

ABSTRACT 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 ~10 10±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 .

Abstract 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, &lt;20, 20–50, 51–100, or &gt;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 &lt;20 CFU/10 mL, 55.3% at ≥20 CFU/10 mL, 46.9% at &gt;51 CFU/10 mL, and 39.8% at &gt;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.

ABSTRACT The growing threat of antibiotic-resistant bacterial pathogens necessitates the development of alternative antimicrobial approaches. This is particularly true for chronic wound infections, which commonly harbor biofilm-dwelling bacteria. A novel electrochemical bandage (e-bandage) delivering low-levels of hypochlorous acid (HOCl) was evaluated against Pseudomonas aeruginosa murine wound biofilms. 5 mm skin wounds were created on the dorsum of mice and infected with 10 6 colony-forming units (CFU) of P. aeruginosa . Biofilms were formed over 2 days, after which e-bandages were placed on the wound beds and covered with Tegaderm. Mice were administered Tegaderm-only (control), non-polarized e-bandage (no HOCl production), or polarized e-bandage (using an HOCl-producing potentiostat), with or without systemic amikacin. Purulence and wound areas were measured before and after treatment. After 48 hours, wounds were harvested for bacterial quantification. Forty-eight hours of polarized e-bandage treatment resulted in mean biofilm reductions of 1.4 log 10 CFUs/g ( P = 0.0107) vs non-polarized controls and 2.2 log 10 CFU/g ( P = 0.004) vs Tegaderm-only controls. Amikacin improved CFU reduction in Tegaderm-only ( P = 0.0045) and non-polarized control groups ( P = 0.0312) but not in the polarized group ( P = 0.3876). Compared to the Tegaderm-only group, there was less purulence in the polarized group ( P = 0.009). Wound closure was neither impeded nor improved by either polarized or non-polarized e-bandage treatment. Concurrent amikacin did not impact wound closure or purulence. In conclusion, an HOCl-producing e-bandage reduced P. aeruginosa in wound biofilms with no impairment in wound healing, representing a promising antibiotic-free approach for addressing wound infection.

Abstract Background Biofilms formed by antibiotic-resistant bacteria in wound beds present unique challenges in terms of treating wound infections. In this work, the in vivo anti-biofilm activity of a novel electrochemical bandage (e-bandage) designed to continuously deliver low amounts of hypochlorous acid (HOCl) was evaluated against wound biofilm infections caused by a clinical isolate of Pseudomonas aeruginosa resistant to all antibiotic classes except aminoglycosides. Methods 5 mm skin wounds were created on the dorsal surface of Swiss-Webster mice and infected with 106 CFU of P. aeruginosa. Biofilms were allowed to form over 2 days, after which e-bandages with Tegaderm™ or Tegaderm only were placed on the wound beds. Specifically, mice were randomized to one of three experimental groups (n = 7-8): 1) Tegaderm only (control); 2) non-polarized e-bandage; 3) polarized e-bandage using a wearable potentiostat. After 48 hours, animals were sacrificed and 10 mm skin biopsies containing the wound and surrounding tissue were harvested for bacterial quantification. Histopathology was performed on H&E-stained sections of wound beds from each treatment group. Blood was screened with an inflammatory cytokine panel. Wound healing (% area reduction; digital measurement) and purulence (observational scoring) were assessed in pre- and post-treatment wounds. Results 48 hours of polarized e-bandage treatment resulted in a mean reduction of 1.86 log10 biofilm CFUs/g (p &lt; 0.0001) vs non-polarized controls, and 2.18 log10 CFU/g reduction (p &lt; 0.0001) vs Tegaderm only controls (polarized, 7.6 ± 0.66 log10 CFU/g; non-polarized, 9.45 ± 0.26 log+10 CFU/g; Tegaderm only, 9.78 ± 0.16 log10 CFU/g). Compared to Tegaderm only, purulence reduction and wound healing were significantly higher for both non-polarized (purulence - p &lt; 0.0001; healing - p &lt; 0.01 ) and polarized treatment (purulence - p &lt; 0.0001; healing - p &lt; 0.0001) groups, but were not significantly different from each other. Histopathology and blood inflammation panel testing showed no significant differences between groups. Conclusion An HOCl-producing e-bandage reduced P. aeruginosa in wound biofilms, representing a potential antibiotic-free strategy for the treatment of recalcitrant wound infections. Disclosures Audrey N. Schuetz, MD, Merck: Advisor/Consultant 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 Background Periprosthetic joint infection (PJI) is a devastating complication following joint replacement, associated with morbidity and mortality. Here, we established a clinically representative murine model of knee arthroplasty and determined the ideal bacterial inoculum that would result in PJI without causing significant limb compromise.Figure 1.An example of an uninfected titanium prosthesis osseointegrated into the tibia of a mouse. Methods A titanium tibial prosthesis designed for the murine knee was 3D printed and surgically implanted into the proximal tibia (Figure 1) of 38 C57BL/6 mice. The prosthesis is designed to osseointegrate into the tibia and has a smooth articular surface to allow for immediate weight bearing and range of motion. At the time of surgery, the arthroplasty was inoculated with five inocula of Staphylococcus aureus Xen36, ranging from 29 to 14,000 colony forming units (CFU) (Table 1). Mice were sacrificed at 3 weeks, or earlier if necessary due to infectious sequelae, and the joint tissue, tibia, femur, and prosthesis quantitatively cultured. Results All mice inoculated with 181 to 14,000 CFU developed PJI as did 90% inoculated with 29 CFU. Bacterial loads from joint tissues and tibial prostheses are shown in Table 1. All mice inoculated with 14,000 and 760 CFU had overt findings of infection (abscesses, sinus tracts, significant limb dysfunction) requiring early euthanasia (Table 2). Mice inoculated with 640 CFU had swelling, redness, and small sores in first 3 days, and 1 death, but otherwise appeared well; only 2/9 had a visible abscess at 3-week sacrifice. 2/10 mice receiving 181 CFU had small sores that resolved in the first week; all limbs functioned well prior to sacrifice. Mice receiving 29 CFU had no external evidence of infection, however 9/10 had purulence within the joint at sacrifice. Conclusion This study demonstrates that inoculum size directly impacts clinical manifestations of infection in a murine model of S. aureus PJI. An inoculum of 181 CFU resulted in universal infection without significantly compromising the limb. The ability of this model to create PJI without overt infectious sequela will allow for evaluation of therapeutics, including those locally delivered. Disclosures 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 Nicholas Bedard, MD, Stryker: Advisor/Consultant