Koscianski, Christina

Three previously uncharacterized bacterial species, designated strains BD586 T , BD613 T and BD626 T , isolated from human clinical specimens, were identified at Mayo Clinic, Rochester, Minnesota, USA. Initial identification efforts using matrix-assisted laser desorption ionization-time-of-flight MS and partial 16S rRNA gene sequencing proved inconclusive. Comprehensive analysis involving phenotypic characterization, biochemical assays and whole-genome sequencing was undertaken. The isolates were Gram-negative, motile, facultatively anaerobic rods, occurring singly, in pairs and in short chains; BD613 T additionally formed small aggregates. The isolates tested positive for catalase and negative for oxidase. Their colonies appeared smooth, white, opaque and non-haemolytic. Growth was observed at 35 °C under aerobic, anaerobic and CO₂-enriched conditions, as well as in media with NaCl concentrations up to 10% and at pH 7-9. Phylogenetic relationships were inferred from core gene alignments, average nucleotide identity and digital DNA–DNA hybridization comparisons. Results confirmed the placement of BD586 T , BD613 T and BD626 T within the recently established Dryocola genus, while also indicating their novelty as distinct species. The major cellular fatty acids were C 16:0 and C 17:0 cyclo. Based on these findings, a formal description of three new species, Dryocola mayonis sp. nov. (type strain BD586 T =TSD 474 T , =NCTC 15089 T , =DSM 119465 T ), Dryocola sharpae sp. nov. (type strain BD613 T =TSD 475 T , =NCTC 15090 T , =DSM 119466 T ) and Dryocola baronae sp. nov. (type strain BD626 T =TSD 476 T , =NCTC 15091 T , =DSM 119479 T ), is proposed, and the genus description of Dryocola is emended to refine its genomic, morphological and chemotaxonomic boundaries.

ABSTRACT Periprosthetic joint infection (PJI) is the most common and difficult to treat form of arthroplasty failure. While treatment with debridement, antibiotics, and implant retention (DAIR) is preferable to one- or two-stage implant exchange based on morbidity and cost, outcomes are not successful in all cases selected for this management strategy. DAIR is currently recommended when infection is perceived to be in an “acute” phase, based on symptom duration; despite this selection strategy, DAIR failure rates are high. A more objective means of predicting DAIR success could improve patient outcomes by limiting DAIR failures. To explore this possibility, advanced proteomic analysis of sonicate fluid samples from 95 subjects with PJI was performed using proximity extension assay (PEA), with 77 samples additionally undergoing complementary proteomics analysis with liquid chromatography with tandem mass spectrometry (LC-MS/MS). Sparse partial least squares differentiation analysis (sPLS-DA) separated samples from patients who underwent DAIR with known outcomes ( n = 19) into successful or unsuccessful groups, predicting DAIR surgical outcome (area under the curve [AUC]: 1; mean “leave one out” AUC: 0.6). Proteins predictive of DAIR surgical outcome were significantly enriched for the “protein repair” Gene Ontology (GO) pathway ( P -adj = 0.02). Additionally, proteomic signatures associated with symptom duration in PJI categorized samples into short and long symptom duration PJI. In summary, an investigation of 7,261 unique human proteins separated PJI-associated samples based on symptom duration and identified a proteomic profile predictive of DAIR success. IMPORTANCE Chronic infections are generally understood to last months to years, with acute infections lasting days to weeks. The transition from acute to chronic infection is, however, poorly understood. Periprosthetic joint infection (PJI) has been considered more challenging to treat when “chronic” than when “acute.” A surgery preferred for its recovery time and cost—debridement, antibiotics, and implant retention (DAIR)—is recommended for PJI management when symptom duration is short; yet, even in this select patient group, DAIR is associated with a high treatment failure rate. A means to better identify those predicted to have a successful outcome if they undergo DAIR is needed. Here, over 7,000 proteins from human clinical PJI samples were measured and shown to be able to separate samples based on symptom duration. Importantly, a proteomic profile predictive of DAIR success was identified.

ABSTRACT Periprosthetic joint infection (PJI) is a serious complication of total joint arthroplasty (TJA), with Staphylococcus aureus being one of the most common causative organisms. Irrigation and debridement with component retention (IDCR) is often performed to manage PJI. Antimicrobial treatment that targets S. aureus biofilms on prosthesis surfaces and within bone may be especially helpful when retaining a prosthesis. Phages, viruses that specifically infect bacteria, are a potential therapeutic consideration. Using a biofilm time kill assay in bovine synovial fluid, bactericidal activity of vancomycin or phage K (32 µg/mL and 10 11 pfu/mL, respectively) as well as the combination of phage K (10 10 pfu/mL) and vancomycin (32 µg/mL) was observed against a methicillin‐resistant S. aureus (MRSA) strain. Using a murine PJI model, mice infected with MRSA underwent IDCR followed by phage administration into the joint space with or without systemically administered vancomycin. Mice receiving both phage and vancomycin had significant reductions in MRSA on the prosthesis surface compared to untreated or vancomycin‐treated mice. Phage K showed bactericidal activity in vitro and was active when locally delivered in combination with systemically administered vancomycin in a murine PJI model managed with IDCR.

Background/Objectives: Staphylococci are the most common causes of periprosthetic joint infection (PJI); new antimicrobials are needed to manage these difficult infections. Nisin A is a lantibiotic peptide derived from Lactococcus lactis that has antimicrobial activity against Gram-positive bacteria, including staphylococci, and is an FDA-approved preservative used in the food and dairy industry. Here, the in vitro nisin A susceptibility of PJI-associated staphylococci was assessed. Methods: The minimum inhibitory concentrations (MICs), minimum biofilm inhibitory concentrations (MBICs), and minimum biofilm bactericidal concentrations (MBBCs) of nisin A were measured by broth microdilution against 106 staphylococcus isolates isolated from PJI. MICs were assessed using 5 × 105 CFU/mL plus nisin A. For MBICs, biofilms were grown on pegged lids for 6 h, followed by 20 h of treatment. For MBBCs, pegged lids were transferred to plates containing media only for 20 h. The results were determined as the lowest concentrations with no visual growth. Two-dimensional MICs with nisin A and vancomycin were assessed for 20 isolates. Fractional inhibitory concentrations (FICs) were calculated to determine synergistic, additive, antagonistic, or indifferent interactions. Results: The MIC that inhibited 90% of S. aureus and S. epidermidis was 4 µg/mL, apart from for the MRSA subset (8 µg/mL). The MBIC that inhibited 90% of isolates was 4 µg/mL. The MBBCs ranged from 4 to 256 µg/mL. When tested together, nisin A and vancomycin yielded an FIC between 1.25 and 1.5, indicative of indifference, except for one isolate each of MRSA and MSSA, for which an additive effect (FIC of 1) was observed. Conclusions: Nisin A showed inhibitory activity against staphylococci that cause PJI.

ABSTRACT Contezolid has activity against methicillin-resistant Staphylococcus aureus (MRSA), a common cause of orthopedic infection. In a rat model of foreign body osteomyelitis, 1.3 log 10 cfu/g bone and 0.5 log 10 cfu/k-wire reduction in MRSA was found in tibiae ( P = 0.0186) and K-wires, respectively, of contezolid-treated compared to untreated rats. No MRSA was recovered from animals receiving rifampin alone or in combination with contezolid or vancomycin. There was no emergence of resistance to contezolid, vancomycin, or rifampin.

A novel Corynebacterium species, strain BD556T, isolated from blood, was identified at Mayo Clinic, Rochester, MN, USA. After failing definitive identification using MALDI-ToF MS and partial 16S rRNA gene sequencing, BD556T was characterized using a polyphasic approach, including phenotypic, biochemical and whole-genome sequencing methods. BD556T was a Gram-positive rod with clubbed ends, facultatively anaerobic, catalase-positive, oxidase-negative and non-motile. Colonies were white, opaque and non-haemolytic with halo-like edges. BD556T grew at 35 °C in room air, with CO2 and under anaerobic conditions. BD556T grew well in 0 and 6% NaCl and weakly in 10% NaCl. The genome size was 2 349 779 bp with a G+C content of 60.39%. Phylogenetic analysis using 16S rRNA gene sequence analysis, average nucleotide identity and digital DNA–DNA hybridization between the genome of BD556T and the closest type strains from the Type Strain Genome Server database yielded separation values well beyond those required for species delineation. Chemotaxonomic analyses of BD556T revealed ribose, arabinose and galactose as whole-cell sugars and an A1γ meso-diaminopimelic acid-direct peptidoglycan type. The major cellular fatty acids were C15 : 0 (21.0%), C16 : 0 (14.8%), C17 : 1 ω9c (26.2%), C17 : 0 (13.3%) and C18 : 1 ω9c (18.3%). Polar lipids included diphosphatidylglycerol, phosphatidylglycerol and unidentified glycolipids and phospholipids. BD556T also contained mycolic acids (32–36 carbons) typical of corynebacteria. The respiratory quinones were dominated by MK-8(H2) (71.2%) and MK-9(H2) (25.9%), with smaller amounts of MK-7(H2) and MK-10(H2). The results presented support the tenet that BD556T (=TSD 427T=NCTC 15078T) is a novel species for which the name Corynebacterium mayonis sp. nov. is proposed.

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 Intestinal colonization with carbapenem-resistant organisms (CRO) may be considered to inform empiric antibiotic selection for symptomatic infection. However, it is unclear how often gut colonizing CRE isolates are related to bloodstream isolates in hospitalized patients with bacteremia, especially in areas with high CRO prevalence. Minimum spanning tree of isolate relatedness Isolates and corresponding patient number (P1-P6) and rectal (R) or blood (B) specimen are noted within circles and allelic differences are numerated on linking lines between the circles. Group 1 and 3 isolates are possibly related to each other. Created with BioRender.com. Methods Patients with paired K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae isolates cultured from surveillance rectal specimens and clinically collected blood specimens collected within 1-8 days of each other between 2019-2023 were identified at 3 Greek hospitals. Isolates were frozen and shipped to a reference laboratory in the U.S for whole genome sequencing (WGS) using Illumina MiSeq. Assembly and core genome multilocus sequence typing (cgMLST) analysis were performed with Ridom SeqSphere+ software. Isolate relatedness was based on the total number of allelic differences between each isolate: < 15, related; 16-50, possibly related; ≥ 51, unrelated. Results One hundred eight paired isolates were identified from 54 unique patients; 6 pairs and 12 total isolates underwent cgMLST. In 5 of 6 pairs (83%), the rectal isolate was collected prior to the bloodstream isolate. Rectal and bloodstream isolates were related to one another in 5 of 6 (83%) pairs and were unrelated in 1 pair. Two pairs collected from 2 distinct patients admitted to the same intensive care unit (patients 2 and 3, Figure) were possibly related (16-50 allelic differences) to each other. Conclusion In this pilot evaluation conducted in a setting of high CRO prevalence, KPC-producing K. pneumoniae isolated from paired rectal and blood specimens from individual patients were often related to each other, suggesting that intestinal colonization by this organism could inform empiric antibiotic treatment for bacteremia. Two patients from the same hospital unit had isolates that were possibly related, suggesting within hospital transmission. WGS is a powerful tool that can inform clinical and infection control practices. WGS of the remaining K. pneumoniae pairs and additional Acinetobacter baumannii pairs is ongoing. Disclosures Robin Patel, MD, I already disclosed so please pull from disclosure for other abstracts: Advisor/Consultant|I already disclosed so please pull from disclosure for other abstracts: Grant/Research Support|I already disclosed so please pull from disclosure for other abstracts: 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 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.