In Vitro and In Vivo Evaluation of Ultrasound-Triggered Release From Novel Spinal Device.

OBJECTIVES: Bacterial infection following spinal fusion is a major clinical concern with up to 20% incidence. An ultrasound-triggered bulk-release system to combat postsurgical bacterial survival was designed and evaluated. METHODS: Polylactic acid (PLA) clips were loaded with vancomycin (VAN) and microbubbles (Sonazoid, GE HealthCare) in vitro. Stability was determined over 14 days. VAN-loaded clips were submerged in water and insonated using a Logiq E10 scanner (GE HealthCare) with a curvilinear C6 probe. Doppler-induced VAN release was quantified using spectrophotometry. For in vivo testing, clips were loaded with methylene blue (MeB) solution and Sonazoid. These clips were implanted into a rabbit along the spine at L2 and L5, as well as a pig at L1 and L3, then insonated in Doppler mode using the C6 probe. RESULTS: Sonazoid microbubbles were better preserved when incubated in VAN compared with distilled water at 4°C, 25°C, and 37°C incubation temperatures (P = .0131). Contrast enhancement was observed from both solutions when incubated at 4°C storage conditions. Insonated clips achieved average cumulative VAN release of 101.8 ± 2.8% (81.4 ± 2.8 mg) after 72 hours. Uninsonated clips had only 0.3 ± 0.1% (0.3 ± 0.1 mg) average cumulative VAN release (P < .0001). Clips retrieved from the rabbit did not rupture with insonation nor produce MeB staining of surrounding tissues. In the pig, the PLA film was visibly ruptured and MeB tissue was observed following insonation, whereas the uninsonated clip was intact. CONCLUSION: These results demonstrate ultrasound-triggered release of an encapsulated prophylactic solution and provide an important proof-of-concept for continuing large animal evaluations for translational merit.

Surgical Loupes Worn by Orthopaedic Surgeons Are a Reservoir for Microorganisms.

BACKGROUND: Surgeons frequently use optical loupes to magnify the surgical field; they are typically unprotected when positioned directly over the wound, where particulate shedding containing microorganisms could potentially lead to surgical site infections (SSIs). SSIs are rare in some orthopaedic subspecialties such as hand surgery; however, in other subspecialties, for example, the spine, where surgeons often use loupes, SSIs can have devastating consequences. QUESTIONS/PURPOSES: (1) What is the degree of bacterial and fungi organism colonization of surgical loupes and storage cases? (2) Is there a difference in the degree of colonization at the beginning and the end of a surgery day? (3) Does an alcohol swab reduce bacterial colonization of surgical loupes? METHODS: The surgical loupes of 21 orthopaedic surgeons from a large, regional orthopaedic practice were cultured over a 3-month period and form the basis of this study. Five loupe storage cases were also cultured. In two different subgroup comparisons, the presence of microorganisms was evaluated just before the start and immediately after the end of the surgical day (n = 9) and before and 1 minute after cleaning with an alcohol swab (n = 6). A total of 36 cultures were evaluated. Surgeons who declined to participate in the study were excluded. The number of loupes selected for all of the analyses were samples of convenience and limited by surgeon availability. The degree of bacterial and fungal presence was graded using a point system: 0 = no growth; 1 = limited growth (meaning few scattered colonies); 2 = moderate growth; 3 = extensive but scattered growth; and 4 = growth consuming the entire plate. Demographic data were assessed using descriptive statistics. Additionally, the Student's t and Wilcoxon signed-rank tests were used to detect differences in categorical bacterial growth between paired samples. A p value of 0.05 represented statistical significance. Kappa statistics of reliability were performed to evaluate interobserver agreement of microorganism growth in the culture plates. RESULTS: Bacteria were present in 19 of 21 (90%) sets of loupes. Five species of bacteria were noted. Fungi were present in 10 of 21 (48%) sets of loupes. Bacterial contamination was identified in two storage cases (40%) and fungi were present in five cases (100%). In a subset of nine loupes tested, the degree of bacterial presence had a median of 2 (range, 1-4; 95% confidence interval [CI], 1.0-2.6) in samples collected before starting the surgical day compared with 3 (range, 2-4; 95% CI, 2.0-3.3) at the end of the day (p = 0.004). In a separate study arm comprised of six loupes, 1 minute after being cleaned with an alcohol swab, bacterial presence on loupes decreased from a median of 2 (range, 2-3; 95% CI, 1.9-2.5) to a median of 1 (range, 0-2; 95% CI, 0.5-1.5; p = 0.012). CONCLUSIONS: Loupes are a common reservoir for bacteria and fungi. Given the use of loupes directly over the surgical field and the lack of a barrier, care should be taken to decrease the bacterial load by cleaning loupes and airing out storage cases, which may decrease the risk of surgical field contamination and iatrogenic wound infections. CLINICAL RELEVANCE: Routine cleaning and disinfecting of optical loupes with alcohol pads can reduce microorganism colonization and should be implemented by surgeons who regularly use loupes in the operating room. Theoretically, particulate shedding from the loupes into the surgical field containing microorganisms could increase the risk of SSI, although this has not been proven clinically.

Cold atmospheric plasma is a viable solution for treating orthopedic infection: a review.

Bacterial infection and antibiotic resistance are major threats to human health and very few solutions are available to combat this eventuality. A growing number of studies indicate that cold (non-thermal) plasma treatment can be used to prevent or eliminate infection from bacteria, bacterial biofilms, fungi and viruses. Mechanistically, a cold plasma discharge is composed of high-energy electrons that generate short-lived reactive oxygen and nitrogen species which further react to form more stable compounds (NO2, H2O2, NH2Cl and others) depending on the gas mixture and plasma parameters. Cold plasma devices are being developed for medical applications including infection, cancer, plastic surgery applications and more. Thus, in this review we explore the potential utility of cold plasma as a non-antibiotic approach for treating post-surgical orthopedic infections.

Effect of biofilms on recalcitrance of staphylococcal joint infection to antibiotic treatment.

The pathogenesis of joint infections is not well understood. In particular, we do not know why these infections respond poorly to antibiotic treatment. Here we show that methicillin-resistant Staphylococcus aureus, a major cause of joint infections, forms exceptionally strong biofilmlike aggregates in human synovial fluid (SF), to an extent significantly exceeding biofilm formation observed in growth medium or serum. Screening a transposon bank identified bacterial fibronectin- and fibrinogen-binding proteins as important for the formation of macroscopic clumps in SF, suggesting an important role of fibrin-containing clots in the formation of bacterial aggregates during joint infection. Pretreatment of SF with plasmin led to a strongly reduced formation of aggregates and increased susceptibility to antibiotics. These results give important insight into the pathogenesis of staphylococcal joint infection and the mechanisms underlying resistance to treatment. Furthermore, they point toward a potential novel approach for treating joint infections.

Role of Phenol-Soluble Modulins in Formation of Staphylococcus aureus Biofilms in Synovial Fluid.

Staphylococcus aureus is a leading cause of prosthetic joint infections, which, as we recently showed, proceed with the involvement of biofilm-like clusters that cause recalcitrance to antibiotic treatment. Here we analyzed why these clusters grow extraordinarily large, reaching macroscopically visible extensions (>1 mm). We found that while specific S. aureus surface proteins are a prerequisite for agglomeration in synovial fluid, low activity of the Agr regulatory system and subsequent low production of the phenol-soluble modulin (PSM) surfactant peptides cause agglomerates to grow to exceptional dimensions. Our results indicate that PSMs function by disrupting interactions of biofilm matrix molecules, such as the polysaccharide intercellular adhesin (PIA), with the bacterial cell surface. Together, our findings support a two-step model of staphylococcal prosthetic joint infection: As we previously reported, interaction of S. aureus surface proteins with host matrix proteins such as fibrin initiates agglomeration; our present results show that, thereafter, the bacterial agglomerates grow to extremely large sizes owing to the lack of PSM expression under the specific conditions present in joints. Our findings provide a mechanistic explanation for the reported extreme resistance of joint infection to antibiotic treatment, lend support to the notions that Agr functionality and PSM production play a major role in defining different forms of S. aureus infection, and have important implications for antistaphylococcal therapeutic strategies.

Staphylococcal persistence due to biofilm formation in synovial fluid containing prophylactic cefazolin.

Antibiotic prophylaxis is standard for patients undergoing surgical procedures, yet despite the wide use of antibiotics, breakthrough infections still occur. In the setting of total joint arthroplasty, such infections can be devastating. Recent findings have shown that synovial fluid causes marked staphylococcal aggregation, which can confer antibiotic insensitivity. We therefore asked in this study whether clinical samples of synovial fluid that contain preoperative prophylactic antibiotics can successfully eradicate a bacterial challenge by pertinent bacterial species. This study demonstrates that preoperative prophylaxis with cefazolin results in high antibiotic levels. Furthermore, we show that even with antibiotic concentrations that far exceed the expected bactericidal levels, Staphylococcus aureus bacteria added to the synovial fluid samples are not eradicated and are able to colonize model implant surfaces, i.e., titanium pins. Based on these studies, we suggest that current prophylactic antibiotic choices, despite high penetration into the synovial fluid, may need to be reexamined.

Targeted release of tobramycin from a pH-responsive grafted bilayer challenged with S. aureus.

A stimuli-responsive, controlled release bilayer for the prevention of bacterial infection on biomaterials is presented. Drug release is locally controlled by the pH-responsiveness of the bilayer, comprised of an inner poly(acrylic acid) (PAA) monolayer grafted to a biomaterial and cross-linked with an outer chitosan (CH) brush. Tobramycin (TOB) is loaded in the inner PAA in part to minimize bacteria resistance. Because biofilm formation causes a decrease in local pH, TOB is released from PAA and permeates through the CH, which is in contact with the biofilm. Antibiotic capacity is controlled by the PAA thickness, which depends on PAA brush length and the extent of cross-linking between CH and PAA at the bilayer interface. This TOB-loaded, pH-responsive bilayer exhibits significantly enhanced antibacterial activity relative to controls.

Porphyrin-adsorbed Allograft Bone: A Photoactive, Antibiofilm Surface.

BACKGROUND: Allograft bone is commonly used to augment bone stock. Unfortunately, allograft is prone to bacterial contamination and current antimicrobial therapies are inadequate. Photoactivated porphyrins combat bacterial growth by production of reactive oxygen species (ROS); however, to our knowledge, they have not been tested in the setting of allograft bone. QUESTIONS/PURPOSES: We asked: (1) Does 5,10,15,20-tetrakis-(4-aminophenyl)-porphyrin (TAPP) stably adsorb to morselized, mineralized allograft? (2) Does Staphylococcus aureus acquire TAPP from TAPP-allograft? (3) Is TAPP-allograft antibacterial to S. aureus? (4) Is ROS production critical for antimicrobial activity? (5) Does illuminated TAPP-allograft dislodge biofilm? (6) Could other photoactive dyes (TAPP, TMPyP, TSP, THP, and methylene blue) confer antimicrobial properties to allograft? METHODS: TAPP adsorption to allograft (TAPP-allograft), its localization in S. aureus, and TAPP-allograft long-term stability were determined spectrophotometrically. Antimicrobial activity was measured while activated with light or in the dark during incubation with S. aureus or after allograft biofilm formation. Glutathione was added to illuminated TAPP-allograft to quench ROS and antimicrobial activity was determined. Light-dependent antimicrobial activity of other photoactive dyes (TMPyP, TSP, THP, and methylene blue) adsorbed to allograft was also tested. RESULTS: We found (1) porphyrins strongly adhere to bone allograft; and (2) the bacteria are not able to sequester TAPP from the TAPP-allograft; (3) when illuminated, TAPP-allograft is resistant to bacterial adherence; (4) the effects of TAPP are inhibited by the radical scavenger glutathione, indicating ROS-dependent antimicrobial activity; (5) illumination of TAPP-allograft disrupts biofilms; and, (6) other photoactive dyes impede biofilm formation on allograft bone in the presence of light. CONCLUSIONS: Porphyrins stably associate with allograft and are inactive until illuminated. Illuminated TAPP-allograft markedly reduces bacterial colonization, which is restored in the presence of radical scavengers. Finally, illuminated TAPP-allograft disrupts biofilms. CLINICAL RELEVANCE: The findings of this in vitro study suggest that loading bone allograft with biocompatible porphyrins before surgery might allow increased sterility of the allograft during implantation. Future testing in an animal model will determine if these in vitro activities can be used to prevent allograft-based infection in an establishing osteomyelitis.

The 2023 Orthopaedic Research Society International Consensus Meeting on musculoskeletal infection.

The Orthopaedic Research Society's Research Interest Group completed its international consensus meeting (ICM) on musculoskeletal infections (MSKI) following the 2023 Annual Meeting. The work products from this ICM include the 65 questions with recommendation and rationale, and the voting results from the 72 delegates. There are also five Consensus Articles in this issue of the Journal of Orthopaedic Research from the ICM Sections: Host Immunity, Established Infection-Treatment, Clinical Questions not addressed by the prior MSKI ICMs, In Vitro, and Animal Models. This Introduction summarizes the 3-year Delphi process used by the ICM with timelines and critical milestones. It also highlights several challenges that had to be addressed, and a large body of work that remains.

Addressing the need for preclinical study of penile prosthesis infection: a new animal model and narrative review.

Background and Objective: Erectile dysfunction (ED) is a common condition in men, and many patients refractory to conservative treatment may undergo penile prostheses (PPs) placement. The primary concern following PP implantation is device infection. Although antibiotic and hydrophilic coatings have reduced the incidence of inflatable PP (IPP) infections, there remains room for improvement. Optimization of PP outcomes requires a practical in vivo model to better understand mechanisms of infection and to test new infection control strategies. We aimed to describe a new rabbit model which contains a functional IPP and review previously reported animal PP models. Methods: An IPP was placed into rabbit flanks and cycled for functionality testing. Rabbits were evaluated for signs of pain and distress over 14 days. Separately, narrative review methodology was utilized to search the PubMed and Scopus databases for all publications through March 21, 2023, which studied PP within an in vivo setting. Three independent reviewers ultimately selected 12 papers from 1992-2021 for inclusion. Key Content and Findings: Several animal studies highlighted the initial functionality or feasibility of devices for ED before their introduction in the clinical setting. There are several subsequent studies aimed at optimizing the type of antibiotic use or coating material using segments of PP material in an in vivo setting. However, the literature lacks a contemporary animal model containing a functional IPP. Our novel rabbit model offers a safe, practical way to implant a functioning IPP and investigate new perioperative infection prevention and treatment strategies before trials in the clinical setting. Conclusions: Animal models have played a key role in testing medical devices, including PPs, prior to their clinical introduction. Our review uncovered no modern animal studies involving placement of a functional PP. A new animal model can facilitate study of evolving microorganism profiles, novel methods to enhance antibiotic delivery, and proposed treatment options.

The 2023 Orthopedic Research Society's international consensus meeting on musculoskeletal infection: Summary from the in vitro section.

Antimicrobial strategies for musculoskeletal infections are typically first developed with in vitro models. The In Vitro Section of the 2023 Orthopedic Research Society Musculoskeletal Infection international consensus meeting (ICM) probed our state of knowledge of in vitro systems with respect to bacteria and biofilm phenotype, standards, in vitro activity, and the ability to predict in vivo efficacy. A subset of ICM delegates performed systematic reviews on 15 questions and made recommendations and assessment of the level of evidence that were then voted on by 72 ICM delegates. Here, we report recommendations and rationale from the reviews and the results of the internet vote. Only two questions received a ≥90% consensus vote, emphasizing the disparate approaches and lack of established consensus for in vitro modeling and interpretation of results. Comments on knowledge gaps and the need for further research on these critical MSKI questions are included.

Antibacterial Biomimetic Hybrid Films.

In this work, we present a novel method to prepare a hybrid coating based on dextran grafted to a substrate and embedded with silver nanoparticles (Ag NPs). First, the Ag NPs are synthesized in situ in the presence of oxidized dextran in solution. Second, the oxidized dextran is exposed to an amine functionalized surface resulting in the simultaneous grafting of dextran and the trapping of Ag NPs within the layer. The NP loading is controlled by the concentration of silver nitrate, which is 2 mM (DEX-Ag2) and 5 mM (DEX-Ag5). The dried film thickness increases with silver nitrate concentration from 2 nm for dextran to 7 nm and 12 nm for DEX-Ag2 and DEX-Ag5, respectively. The grafted dextran film displays features with a diameter and height of ~ 50 nm and 2 nm, respectively. For the DEX-Ag2 and DEX-Ag5, the dextran features as well as individual Ag NPs (~ 5 nm) and aggregates of Ag NPs are observed. Larger and more irregular aggregates are observed for DEX-Ag5. Overall, the Ag NPs are embedded in the dextran film as suggested by AFM and UVO studies. In terms of its antimicrobial activity, DEX-Ag2 resists bacterial adhesion to a greater extent than DEX-Ag5, which in turn is better than dextran and silicon. Because these antibacterial hybrid coatings can be grafted to a variety of surfaces, many biomedical applications can be envisioned, ranging from coating implants to catheters.

Berberine disrupts staphylococcal proton motive force to cause potent anti-staphylococcal effects in vitro.

The presence of antibiotic resistance has increased the urgency for more effective treatments of bacterial infections. Biofilm formation has complicated this issue as biofilm bacteria become tolerant to antibiotics due to environmental factors such as nutrient deprivation and adhesion. In septic arthritis, a disease with an 11% mortality rate, bacteria in synovial fluid organize into floating, protein-rich, bacterial aggregates (mm-cm) that display depressed metabolism and antibiotic tolerance. In this study, Staphylococcus aureus (S. aureus), which is the most common pathogen in septic arthritis, was tested against different inhibitors that modulate bacterial surface protein availability and that should decrease bacterial aggregation. One of these, berberine, a quaternary ammonium compound, was found to reduce bacterial counts by 3-7 logs in human synovial fluid (aggregating medium) with no effect in tryptic soy broth (TSB, non-aggregating). Unlike traditional antibiotics, the bactericidal activity of berberine appeared to be independent of bacterial metabolism. To elucidate the mechanism, we used synovial fluid fractionation, targeted MRSA transposon insertion mutants, dyes to assess changes in membrane potential (DiSC3(5)) and membrane permeability (propidium iodide (PI)), colony counting, and fluorescence spectroscopy. We showed that berberine's activity was dependent on an alkaline pH and berberine killed both methicillin-sensitive S. aureus and MRSA in alkaline media (pH 8.5-9.0; p < 0.0001 vs. same pH controls). Under these alkaline conditions, berberine localized to S. aureus where berberine was isolated in cytoplasmic (∼95%) and DNA (∼5%) fractions. Importantly, berberine increased bacterial cell membrane permeability, and disrupted the proton motive force, suggesting a mechanism whereby it may be able to synergize with other antibacterial compounds under less harsh conditions. We suggest that berberine, which is cheap and readily available, can be made into an effective treatment.

Multiple Sterile Withdrawals from Iohexol Bottles Does Not Increase Contamination Risk.

BACKGROUND: There is a global shortage of iohexol contrast media, commonly used in epidural injections, as a result of lockdown and decreased production due to COVID-19. Iohexol bottles are designated for single use, which, depending on the vials available, often leads to wasting up to 95% of this limited resource. However, avoiding multiple withdrawals may be unnecessary if withdrawing multiple times using sterile technique does not increase the risk for contamination. OBJECTIVES: The purpose of our study is to determine whether multiple withdrawals from iohexol injection bottles using a sterile technique poses a greater risk of introducing contaminants than a single withdrawal. Furthermore, we wish to determine the extent to which bacteria can survive and grow in the contrast media. STUDY DESIGN: Experimental. SETTING: Outpatient fluoroscopic suite and laboratory. METHODS: Twenty-one 100 mL 300 mg(iodine)/mL iohexol injection bottles, after one clinical use, were tested after the first and last withdrawals (withdrawal one and withdrawal 9 or 10) for bacterial and fungal specimens using culture media and 3M™ Petrifilms™. To determine the ability of methicillin-susceptible Staphylococcus aureus (MSSA) to survive or grow in the media, MSSA was added to different concentrations (0, 25, 50, 75, and 100%) of iohexol contrast media. RESULTS: There was no growth observed in cultures or on Petrifilms among the first and last draws of any of the samples. When bacteria were grown in different dilutions of the media, there was a significant, approximately one log decrease in counts from 0% contrast media to 100% contrast media (8.4 x 108 vs 5.6 x 107, P < 0.01). LIMITATIONS: Our study is limited in the number of samples tested and would benefit from additional investigation before consideration of clinical application. CONCLUSIONS: Our results suggest that single-use 300 iohexol bottles may be reusable and that the contrast media is mildly antimicrobial, but not enough to retard contamination. In setting of shortages, contrast media bottles can safely be reused. This is valuable for conserving resources and limiting unnecessary health care-associated costs.

Evaluation of bacterial attachment on mineralized collagen scaffolds and addition of manuka honey to increase mesenchymal stem cell osteogenesis.

The design of biomaterials to regenerate bone is likely to increasingly require modifications that reduce bacterial attachment and biofilm formation as infection during wound regeneration can significantly impede tissue repair and typically requires surgical intervention to restart the healing process. Further, much research on infection prevention in bone biomaterials has focused on modeling of non-resorbable metal alloy materials, whereas an expanding direction of bone regeneration has focused on development of bioresorbable materials. This represents a need for the prevention and understanding of infection in resorbable biomaterials. Here, we investigate the ability of a mineralized collagen biomaterial to natively resist infection and examine how the addition of manuka honey, previously identified as an antimicrobial agent, affects gram positive and negative bacterial colonization and mesenchymal stem cell osteogenesis and vasculature formation. We incorporate manuka honey into these scaffolds via either direct fabrication into the scaffold microarchitecture or via soaking the scaffold in a solution of manuka honey after fabrication. Direct incorporation results in a change in the surface characteristics and porosity of mineralized collagen scaffolds. Soaking scaffolds in honey concentrations higher than 10% had significant negative effects on mesenchymal stem cell metabolic activity. Soaking or incorporating 5% honey had no impact on endothelial cell tube formation. Although solutions of 5% honey reduced metabolic activity of mesenchymal stem cells, MSC-seeded scaffolds displayed increased calcium and phosphorous mineral formation, osteoprotegerin release, and alkaline phosphatase activity. Bacteria cultured on mineralized collagen scaffolds demonstrated surfaces covered in bacteria and no method of preventing infection, and using 10 times the minimal inhibitory concentration of antibiotics did not completely kill bacteria within the mineralized collagen scaffolds, indicating bioresorbable scaffold materials may act to shield bacteria from antibiotics. The addition of 5% manuka honey to scaffolds was not sufficient to prevent P. aeruginosa attachment or consistently reduce the activity of methicillin resistant staphylococcus aureus, and concentrations above 7% manuka honey are likely necessary to impact MRSA. Together, our results suggest bioresorbable scaffolds may create an environment conducive to bacterial growth, and potential trade-offs exist for the incorporation of low levels of honey in scaffolds to increase osteogenic potential of osteoprogenitors while high-levels of honey may be sufficient to reduce gram positive or negative bacteria activity but at the cost of reduced osteogenesis.

Cold atmospheric pressure plasma-antibiotic synergy in Pseudomonas aeruginosa biofilms is mediated via oxidative stress response.

Cold atmospheric-pressure plasma (CAP) has emerged as a potential alternative or adjuvant to conventional antibiotics for the treatment of bacterial infections, including those caused by antibiotic-resistant pathogens. The potential of sub-lethal CAP exposures to synergise conventional antimicrobials for the eradication of Pseudomonas aeruginosa biofilms is investigated in this study. The efficacy of antimicrobials following or in the absence of sub-lethal CAP pre-treatment in P. aeruginosa biofilms was assessed. CAP pre-treatment resulted in an increase in both planktonic and biofilm antimicrobial sensitivity for all three strains tested (PAO1, PA14, and PA10548), with both minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs) of individual antimicrobials, being significantly reduced following CAP pre-treatment of the biofilm (512-fold reduction with ciprofloxacin/gentamicin; and a 256-fold reduction with tobramycin). At all concentrations of antimicrobial used, the combination of sub-lethal CAP exposure and antimicrobials was effective at increasing time-to-peak metabolism, as measured by isothermal microcalorimetry, again indicating enhanced susceptibility. CAP is known to damage bacterial cell membranes and DNA by causing oxidative stress through the in situ generation of reactive oxygen and nitrogen species (RONS). While the exact mechanism is not clear, oxidative stress on outer membrane proteins is thought to damage/perturb cell membranes, confirmed by ATP and LDH leakage, allowing antimicrobials to penetrate the bacterial cell more effectively, thus increasing bacterial susceptibility. Transcriptomic analysis, reveals that cold-plasma mediated oxidative stress caused upregulation of P. aeruginosa superoxide dismutase, cbb3 oxidases, catalases, and peroxidases, and upregulation in denitrification genes, suggesting that P. aeruginosa uses these enzymes to degrade RONS and mitigate the effects of cold plasma mediated oxidative stress. CAP treatment also led to an increased production of the signalling molecule ppGpp in P. aeruginosa, indicative of a stringent response being established. Although we did not directly measure persister cell formation, this stringent response may potentially be associated with the formation of persister cells in biofilm cultures. The production of ppGpp and polyphosphate may be associated with protein synthesis inhibition and increase efflux pump activity, factors which can result in antimicrobial tolerance. The transcriptomic analysis also showed that by 6 h post-treatment, there was downregulation in ribosome modulation factor, which is involved in the formation of persister cells, suggesting that the cells had begun to resuscitate/recover. In addition, CAP treatment at 4 h post-exposure caused downregulation of the virulence factors pyoverdine and pyocyanin; by 6 h post-exposure, virulence factor production was increasing. Transcriptomic analysis provides valuable insights into the mechanisms by which P. aeruginosa biofilms exhibits enhanced susceptibility to antimicrobials. Overall, these findings suggest, for the first time, that short CAP sub-lethal pre-treatment can be an effective strategy for enhancing the susceptibility of P. aeruginosa biofilms to antimicrobials and provides important mechanistic insights into cold plasma-antimicrobial synergy. Transcriptomic analysis of the response to, and recovery from, sub-lethal cold plasma exposures in P. aeruginosa biofilms improves our current understanding of cold plasma biofilm interactions.

Microbubble cavitation restores Staphylococcus aureus antibiotic susceptibility in vitro and in a septic arthritis model.

Treatment failure in joint infections is associated with fibrinous, antibiotic-resistant, floating and tissue-associated Staphylococcus aureus aggregates formed in synovial fluid (SynF). We explore whether antibiotic activity could be increased against Staphylococcus aureus aggregates using ultrasound-triggered microbubble destruction (UTMD), in vitro and in a porcine model of septic arthritis. In vitro, when bacterially laden SynF is diluted, akin to the dilution achieved clinically with lavage and local injection of antibiotics, amikacin and ultrasound application result in increased bacterial metabolism, aggregate permeabilization, and a 4-5 log decrease in colony forming units, independent of microbubble destruction. Without SynF dilution, amikacin + UTMD does not increase antibiotic activity. Importantly, in the porcine model of septic arthritis, no bacteria are recovered from the SynF after treatment with amikacin and UTMD-ultrasound without UTMD is insufficient. Our data suggest that UTMD + antibiotics may serve as an important adjunct for the treatment of septic arthritis.

Making waves: how ultrasound-targeted drug delivery is changing pharmaceutical approaches.

Administration of drugs through oral and intravenous routes is a mainstay of modern medicine, but this approach suffers from limitations associated with off-target side effects and narrow therapeutic windows. It is often apparent that a controlled delivery of drugs, either localized to a specific site or during a specific time, can increase efficacy and bypass problems with systemic toxicity and insufficient local availability. To overcome some of these issues, local delivery systems have been devised, but most are still restricted in terms of elution kinetics, duration, and temporal control. Ultrasound-targeted drug delivery offers a powerful approach to increase delivery, therapeutic efficacy, and temporal release of drugs ranging from chemotherapeutics to antibiotics. The use of ultrasound can focus on increasing tissue sensitivity to the drug or actually be a critical component of the drug delivery. The high spatial and temporal resolution of ultrasound enables precise location, targeting, and timing of drug delivery and tissue sensitization. Thus, this noninvasive, non-ionizing, and relatively inexpensive modality makes the implementation of ultrasound-mediated drug delivery a powerful method that can be readily translated into the clinical arena. This review covers key concepts and areas applied in the design of different ultrasound-mediated drug delivery systems across a variety of clinical applications.

A Platelet-Rich Plasma-Derived Biologic Clears Staphylococcus aureus Biofilms While Mitigating Cartilage Degeneration and Joint Inflammation in a Clinically Relevant Large Animal Infectious Arthritis Model.

The leading cause of treatment failure in Staphylococcus aureus infections is the development of biofilms. Biofilms are highly tolerant to conventional antibiotics which were developed against planktonic cells. Consequently, there is a lack of antibiofilm agents in the antibiotic development pipeline. To address this problem, we developed a platelet-rich plasma (PRP)-derived biologic, termed BIO-PLY (for the BIOactive fraction of Platelet-rich plasma LYsate) which has potent in vitro bactericidal activity against S. aureus synovial fluid free-floating biofilm aggregates. Additional in vitro studies using equine synoviocytes and chondrocytes showed that BIO-PLY protected these cells of the joint from inflammation. The goal of this study was to test BIO-PLY for in vivo efficacy using an equine model of infectious arthritis. We found that horses experimentally infected with S. aureus and subsequently treated with BIO-PLY combined with the antibiotic amikacin (AMK) had decreased bacterial concentrations within both synovial fluid and synovial tissue and exhibited lower systemic and local inflammatory scores compared to horses treated with AMK alone. Most importantly, AMK+BIO-PLY treatment reduced the loss of infection-associated cartilage proteoglycan content in articular cartilage and decreased synovial tissue fibrosis and inflammation. Our results demonstrate the in vivo efficacy of AMK+BIO-PLY and represents a new approach to restore and potentiate antimicrobial activity against synovial fluid biofilms.