Expanded library of novel 2,3-pyrrolidinedione analogues exhibit anti-biofilm activity.

Herein we report a new library of 2,3-pyrrolidinedione analogues that expands on our previous report on the antimicrobial studies of this heterocyclic scaffold. The novel 2,3-pyrrolidinediones reported herein have been evaluated against S. aureus and methicillin-resistant S. aureus (MRSA) biofilms, and this work constitutes our first report on the antibiofilm properties of this class of compounds. The antibiofilm activity of these 2,3-pyrrolidinediones has been assessed through minimum biofilm eradication concentration (MBEC) and minimum biofilm inhibition concentration (MBIC) assays. The compounds displayed antibiofilm properties and represent intriguing scaffolds for further optimization and development.

Antimicrobial Properties of Equine Stromal Cells and Platelets and Future Directions.

Increasing antimicrobial resistance in veterinary practice has driven the investigation of novel therapeutic strategies including regenerative and biologic therapies to treat bacterial infection. Integration of biological approaches such as platelet lysate and mesenchymal stromal cell (MSC) therapy may represent adjunctive treatment strategies for bacterial infections that minimize systemic side effects and local tissue toxicity associated with traditional antibiotics and that are not subject to antibiotic resistance. In this review, we will discuss mechanisms by which biological therapies exert antimicrobial effects, as well as potential applications and challenges in clinical implementation in equine practice.

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.

Effect of BIO-PLYTM, a Platelet-Rich Plasma Derived Biologic on PRRSV-2-Infected Macrophages.

Porcine Reproductive and Respiratory Syndrome (PRRS) is the one of the most devastating diseases impacting the swine industry worldwide. Control and prevention methods rely on biosafety measures and vaccination. As an RNA virus with a high rate of mutation, vaccines are only partially effective against circulating and newly emerging strains. To reduce the burden of this disease, research on alternative control methods is needed. Here, we assess the in vitro antiviral effect of a novel platelet-rich plasma-derived biologic termed BIO-PLYTM (for the BIOactive fraction of Platelet-rich plasma LYsate) from both swine and equine origin. Our results show that BIO-PLYTM significantly reduces the amount of PRRSV viral load determined by RT-qPCR and the number of infectious viral particles measured by TCID50 in infected porcine alveolar and parenchymal macrophages. This study also showed limited toxicity of BIO-PLYTM in vitro and aspects of its immunomodulatory capacity evaluating the regulation of reactive oxygen species and cytokines production in infected cells. Finally, this study presents promising data on the effect of BIO-PLYTM on other RNA viruses such as human A influenza viruses and coronavirus.

Interleukin-1ß in tendon injury enhances reparative gene and protein expression in mesenchymal stem cells.

Tendon injury in the horse carries a high morbidity and monetary burden. Despite appropriate therapy, reinjury is estimated to occur in 50-65% of cases. Although intralesional mesenchymal stem cell (MSC) therapy has improved tissue architecture and reinjury rates, the mechanisms by which they promote repair are still being investigated. Additionally, reevaluating our application of MSCs in tendon injury is necessary given recent evidence that suggests MSCs exposed to inflammation (deemed MSC licensing) have an enhanced reparative effect. However, applying MSC therapy in this context is limited by the inadequate quantification of the temporal cytokine profile in tendon injury, which hinders our ability to administer MSCs into an environment that could potentiate their effect. Therefore, the objectives of this study were to define the temporal cytokine microenvironment in a surgically induced model of equine tendon injury using ultrafiltration probes and subsequently evaluate changes in MSC gene and protein expression following in vitro inflammatory licensing with cytokines of similar concentration as identified in vivo. In our in vivo surgically induced tendon injury model, IL-1ß and IL-6 were the predominant pro-inflammatory cytokines present in tendon ultrafiltrate where a discrete peak in cytokine concentration occurred within 48 h following injury. Thereafter, MSCs were licensed in vitro with IL-1ß and IL-6 at a concentration identified from the in vivo study; however, only IL-1ß induced upregulation of multiple genes beneficial to tendon healing as identified by RNA-sequencing. Specifically, vascular development, ECM synthesis and remodeling, chemokine and growth factor function alteration, and immunomodulation and tissue reparative genes were significantly upregulated. A significant increase in the protein expression of IL-6, VEGF, and PGE2 was confirmed in IL-1ß-licensed MSCs compared to naïve MSCs. This study improves our knowledge of the temporal tendon cytokine microenvironment following injury, which could be beneficial for the development and determining optimal timing of administration of regenerative therapies. Furthermore, these data support the need to further study the benefit of MSCs administered within the inflamed tendon microenvironment or exogenously licensed with IL-1ß in vitro prior to treatment as licensed MSCs could enhance their therapeutic benefit in the healing tendon.

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.

Host fibrinogen drives antimicrobial function in Staphylococcus aureus peritonitis through bacterial-mediated prothrombin activation.

The blood-clotting protein fibrinogen has been implicated in host defense following Staphylococcus aureus infection, but precise mechanisms of host protection and pathogen clearance remain undefined. Peritonitis caused by staphylococci species is a complication for patients with cirrhosis, indwelling catheters, or undergoing peritoneal dialysis. Here, we sought to characterize possible mechanisms of fibrin(ogen)-mediated antimicrobial responses. Wild-type (WT) (Fib+) mice rapidly cleared S. aureus following intraperitoneal infection with elimination of ∼99% of an initial inoculum within 15 min. In contrast, fibrinogen-deficient (Fib-) mice failed to clear the microbe. The genotype-dependent disparity in early clearance resulted in a significant difference in host mortality whereby Fib+ mice uniformly survived whereas Fib- mice exhibited high mortality rates within 24 h. Fibrin(ogen)-mediated bacterial clearance was dependent on (pro)thrombin procoagulant function, supporting a suspected role for fibrin polymerization in this mechanism. Unexpectedly, the primary host initiator of coagulation, tissue factor, was found to be dispensable for this antimicrobial activity. Rather, the bacteria-derived prothrombin activator vWbp was identified as the source of the thrombin-generating potential underlying fibrin(ogen)-dependent bacterial clearance. Mice failed to eliminate S. aureus deficient in vWbp, but clearance of these same microbes in WT mice was restored if active thrombin was administered to the peritoneal cavity. These studies establish that the thrombin/fibrinogen axis is fundamental to host antimicrobial defense, offer a possible explanation for the clinical observation that coagulase-negative staphylococci are a highly prominent infectious agent in peritonitis, and suggest caution against anticoagulants in individuals susceptible to peritoneal infections.

Comprehensive phenotypic and genotypic characterization and comparison of virulence, biofilm, and antimicrobial resistance in urinary Escherichia coli isolated from canines.

Urinary tract infections (UTIs) affect nearly half of women and an estimated 14 % of the canine companion animal population at least once in their lifetime. As with humans, Escherichia coli is the most commonly isolated bacteria from canine UTIs and infections are dominated by specific phylogenetic groups with notable virulence attributes. In this study, we evaluated uropathogenic E. coli (UPEC) (n = 69) isolated from canine UTIs phenotypically and genotypically for virulence factors, biofilm formation and antimicrobial resistance profiles. Biofilm formation in UPEC strains was positively associated with common virulence factors including papG (p = 0.006), fimH (p < 0.0001), sfaS (p = 0.004), focA (p = 0.004), cnf-1 (p = 0.009) and hlyA (p = 0.006). There was a negative association between biofilm formation and phenotypic antimicrobial resistance for ampicillin (p < 0.0004), ciprofloxacin (p < 0.0001), and trimethoprim-sulfamethoxazole (p < 0.02), as well as multidrug resistance (isolates resistant to ≥ 3 classes of antimicrobials) (p < 0.0002), and the presence of extended spectrum beta-lactamase (ESBL)-producing genes (p < 0.05). In conclusion, UPECs isolated from clinical cases of canine UTIs show a broad negative association between antimicrobial resistance and biofilm formation, and this observation is supported both by phenotypic and genotypic endpoints. As the biofilm formation may result in antimicrobial tolerance, this could be a secondary evasive tactic of UPEC lacking traditional antimicrobial resistance traits. This observation is important for veterinary practitioners to consider when treating puzzling chronic intractable and/or recurrent cases of UTI that appear to be susceptible to antimicrobial therapy via traditional antimicrobial susceptibility testing (AST) methods.

Platelet-rich plasma lysate displays antibiofilm properties and restores antimicrobial activity against synovial fluid biofilms in vitro.

Infectious arthritis is difficult to treat in both human and veterinary clinical practice. Recent literature reports Staphylococcus aureus as well as other gram-positive and gram-negative isolates forming free-floating biofilms in both human and equine synovial fluid that are tolerant to traditional antimicrobial therapy. Using an in vitro equine model, we investigated the ability of platelet-rich plasma (PRP) formulations to combat synovial fluid biofilm aggregates. Synovial fluid was infected, and biofilm aggregates allowed to form over a 2-hour period. PRP was collected and processed into different formulations by platelet concentration, leukocyte presence, and activation or lysis. Infected synovial fluid was treated with different PRP formulations with or without aminoglycoside cotreatment. Bacterial load (colony-forming unit/mL) was determined by serial dilutions and plate counting at 8 hours posttreatment. All PRP formulations displayed antimicrobial properties; however, formulations containing higher concentrations of platelets without leukocytes had increased antimicrobial activity. Lysis of PRP and pooling of the PRP lysate (PRP-L) from multiple horses as compared to individual horses further increased antimicrobial activity. This activity was lost with the removal of the plasma component or inhibition of the proteolytic activity within the plasma. Fractionation of pooled PRP-L identified the bioactive components to be cationic and low-molecular weight (<10 kDa). Overall, PRP-L exhibited synergism with amikacin against aminoglycoside tolerant biofilm aggregates with greater activity against gram-positive bacteria. In conclusion, the use of PRP-L has the potential to augment current antimicrobial treatment regimens which could lead to a decrease in morbidity and mortality associated with infectious arthritis.

5-Benzylidene-4-Oxazolidinones Are Synergistic with Antibiotics for the Treatment of Staphylococcus aureus Biofilms.

The failure of frontline antibiotics in the clinic is one of the most serious threats to human health and requires a multitude of novel therapeutics and innovative approaches to treatment so as to curtail the growing crisis. In addition to traditional resistance mechanisms resulting in the lack of efficacy of many antibiotics, most chronic and recurring infections are further made tolerant to antibiotic action by the presence of biofilms. Herein, we report an expanded set of 5-benzylidene-4-oxazolidinones that are able to inhibit the formation of Staphylococcus aureus biofilms, disperse preformed biofilms, and, in combination with common antibiotics, are able to significantly reduce the bacterial load in a robust collagen-matrix model of biofilm infection.

Effects of acellular equine amniotic allografts on the healing of experimentally induced full-thickness distal limb wounds in horses.

OBJECTIVE: To characterize the growth factors contained in equine amniotic membrane allograft (eAM; StemWrap scaffold and StemWrap+ injection) and to evaluate the effect of eAM on equine distal limb wound healing. STUDY DESIGN: Prospective experimental controlled study. SAMPLE POPULATION: Eight adult horses. METHODS: Transforming growth factor (TGF)-ß1, vascular endothelial growth factor (VEGF), epidermal growth factor, platelet-derived growth factor-BB, and prostaglandin E2 (PGE2 ) concentrations in StemWrap+ were assessed with enzyme-linked immunosorbent assay. Two full-thickness 6.25-cm2 skin wounds were created on each metacarpus. On one forelimb, one wound was treated with eAM, and the other was left untreated (eAM control). On the contralateral limb, one wound was treated with a silicone dressing, and the other served as negative control. Three-dimensional images were obtained to determine wound circumference and surface area analyses at each bandage change until healed. Excessive granulation tissue was debrided once weekly for 4 weeks. Biopsy samples were taken to evaluate quality of wound healing via histologic and immunohistochemistry assays. RESULTS: StemWrap+ contained moderate concentrations of TGF-ß1 (494.10 pg/mL), VEGF (212.52 pg/mL), and PGE2 (1811.61 pg/mL). Treatment of wounds with eAM did not affect time to healing or histologic quality of the healing compared with other groups but was associated with increased granulation tissue production early in the study, particularly on day 7. CONCLUSION: Application of eAM resulted in increased granulation tissue production while maintaining appropriate healing of experimental wounds. CLINICAL SIGNIFICANCE: Use of eAM is likely most beneficial for substantial wounds in which expedient production of large amounts of granulation tissue is desirable.

Equine or porcine synovial fluid as a novel ex vivo model for the study of bacterial free-floating biofilms that form in human joint infections.

Bacterial invasion of synovial joints, as in infectious or septic arthritis, can be difficult to treat in both veterinary and human clinical practice. Biofilms, in the form of free-floating clumps or aggregates, are involved with the pathogenesis of infectious arthritis and periprosthetic joint infection (PJI). Infection of a joint containing an orthopedic implant can additionally complicate these infections due to the presence of adherent biofilms. Because of these biofilm phenotypes, bacteria within these infected joints show increased antimicrobial tolerance even at high antibiotic concentrations. To date, animal models of PJI or infectious arthritis have been limited to small animals such as rodents or rabbits. Small animal models, however, yield limited quantities of synovial fluid making them impractical for in vitro research. Herein, we describe the use of ex vivo equine and porcine models for the study of synovial fluid induced biofilm aggregate formation and antimicrobial tolerance. We observed Staphylococcus aureus and other bacterial pathogens adapt the same biofilm aggregate phenotype with significant antimicrobial tolerance in both equine and porcine synovial fluid, analogous to human synovial fluid. We also demonstrate that enzymatic dispersal of synovial fluid aggregates restores the activity of antimicrobials. Future studies investigating the interaction of bacterial cell surface proteins with host synovial fluid proteins can be readily carried out in equine or porcine ex vivo models to identify novel drug targets for treatment of prevention of these difficult to treat infectious diseases.

Gram-negative multi-drug resistant bacteria influence survival to discharge for horses with septic synovial structures: 206 Cases (2010-2015).

Bacterial colonization of synovial structures can cause infections that are difficult to treat. Systemic and local antimicrobials and repeated joint lavages are the mainstays of therapy. However, despite aggressive treatments, infection may persist, leading to significant tissue damage or death of the patient. In order to investigate the impact of bacterial culture and antimicrobial resistance on survival to discharge, we reviewed medical records of horses admitted to the University of Pennsylvania's large animal teaching hospital from 2010-2015. Two-hundred and six cases with a definitive diagnosis of septic synovitis and a synovial fluid sample submitted for microbiological culture were included in the study. Of these horses, 48% were culture negative and 52% were positive for any bacterial growth, of which 66% were gram-positive and 28% were gram-negative aerobic organisms with 4% anaerobic and 2% fungal organisms. Overall survival to discharge from hospital was 86%. Horses that had negative growth on culture were more likely to survive until discharge (p < 0.02). Multivariable analyses revealed that the likelihood of euthanasia was significantly associated with identification of coagulase positive Staphylococcus spp. (OR 7.66, 5.46-10.74, p < 0.0001), ß-hemolytic Streptococcus spp. (OR 5.18, 3.56-7.55, p < 0.0001), Enterococcus spp. (OR 18.38, 11.45-29.52, p = 0.002), Enterobacteriaceae (OR 31.37, 22.28-44.17, p < 0.0001), Pseudomonas aeruginosa (OR 9.31, 5.30-16.34, p = 0.0004) or other gram-negative species (OR 3.51, 2.07-5.94, p = 0.001). Multi-drug resistance and gram-negative bacteria species were associated with significantly decreased survival rates (OR 119.24, 70.57-201.46, p < 0.0001). In conclusion, prognosis for survival to discharge was poor for horses that were infected with gram-negative organisms, particularly those with MDR phenotypes.

Characteristics of Dogs with Biofilm-Forming Escherichia Coli Urinary Tract Infections.

BACKGROUND: Bacterial urinary tract infections (UTIs) are common in companion animals. Increasing awareness of biofilm-forming bacteria raises concern regarding the appropriate diagnosis, treatment, and prognosis of UTIs associated with these organisms. HYPOTHESIS/OBJECTIVES: To (1) describe the population of dogs with UTIs associated with biofilm-forming Escherichia coli and (2) determine whether or not clinical differences exist between dogs with biofilm-forming E. coli UTIs and dogs with nonbiofilm-forming E. coli UTIs. We hypothesized that there would be no difference in the population characteristics, but that biofilm-formation would be more prevalent in dogs with chronic, complicated, and asymptomatic UTIs. ANIMALS: Seventy-six client-owned dogs with E. coli UTIs, divided into 2 groups based on the biofilm-forming capability of stored bacterial isolates as assessed by the crystal violet assay. METHODS: Retrospective cross-sectional study. Medical records of the affected dogs were reviewed and their population and infection characteristics were compared. RESULTS: Most (52.6%) E. coli isolates were capable of forming biofilms. Biofilm-forming E. coli had a lower likelihood (P < .001) of multidrug resistance than did nonbiofilm-forming E. coli. No statistically significant differences were identified between the population or infection characteristics of the 2 groups of dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Escherichia coli isolated from canine urinary tracts are frequently capable of forming biofilms. Because no reliable clinical features allowed exclusion of biofilm formation, the potential for biofilm formation should be considered whenever E. coli UTI is diagnosed. The association of antibiotic resistance and biofilm potential may affect treatment of UTIs, but additional investigation is warranted.

Pooled Platelet-Rich Plasma Lysate Therapy Increases Synoviocyte Proliferation and Hyaluronic Acid Production While Protecting Chondrocytes From Synoviocyte-Derived Inflammatory Mediators.

Platelet-rich plasma (PRP) preparations are being used with moderate success to treat osteoarthritis (OA) in humans and in veterinary species. Such preparations are hindered, however, by being autologous in nature and subject to tremendous patient and processing variability. For this reason, there has been increasing interest in the use of platelet lysate preparations instead of traditional PRP. Platelet lysate preparations are acellular, thereby reducing concerns over immunogenicity, and contain high concentrations of growth factors and cytokines. In addition, platelet lysate preparations can be stored frozen for readily available use. The purpose of this study was to evaluate the effects of a pooled allogeneic platelet-rich plasma lysate (PRP-L) preparation on equine synoviocytes and chondrocytes challenged with inflammatory mediators in-vitro to mimic the OA joint environment. Our hypothesis was that PRP-L treatment of inflamed synoviocytes would protect chondrocytes challenged with synoviocyte conditioned media by reducing synoviocyte pro-inflammatory cytokine production while increasing synoviocyte anti-inflammatory cytokine production. Synoviocytes were stimulated with either interleukin-1ß (IL-1ß) or lipopolysaccharide (LPS) for 24 h followed by no treatment or treatment with platelet-poor plasma lysate (PPP-L) or PRP-L for 48 h. Synoviocyte growth was evaluated at the end of the treatment period and synoviocyte conditioned media was assessed for concentrations of hyaluronic acid (HA), IL-1ß, tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6). Chondrocytes were then challenged for 48 h with synoviocyte conditioned media from each stimulation and treatment group and examined for gene expression of collagen types I (COL1A1), II (COL2A1), and III (COL3A1), aggrecan (ACAN), lubricin (PRG4), and matrix metallopeptidase 3 (MMP-3) and 13 (MMP-13). Treatment of inflamed synoviocytes with PRP-L resulted in increased synoviocyte growth and increased synoviocyte HA and IL-6 production. Challenge of chondrocytes with conditioned media from PRP-L treated synoviocytes resulted in increased collagen type II and aggrecan gene expression as well as decreased MMP-13 gene expression. The results of this study support continued investigation into the use of pooled PRP-L for the treatment of osteoarthritis and warrant further in-vitro studies to discern the mechanisms of action of PRP-L.

Age-related variation in the cellular composition of equine bronchoalveolar lavage fluid.

BACKGROUND: Previous reports reveal variation in the cellular composition of equine bronchoalveolar lavage fluid (BALF). OBJECTIVES: The purpose of this study was to compare the profiles of BALF from horses to assess age-related differences. Serial BALF samples were collected from the same individuals over a one-year period to identify changes in individual animals as they aged. METHODS: Collection of BALF was performed on horses aged one week and one, 2, 6, and 12 months. Total nucleated cell count (TNCC), protein concentration, and cytology were assessed. Longitudinal analysis was performed and compared to healthy adults. RESULTS: Foals at one week and 6 months of age had significantly higher TNCC than adults (medians: 320/µL, 285/µL, and 90/µL, respectively); no differences in total protein were found. Foals at one month had the highest proportion of macrophages (median: 87.3%), differing significantly from both yearlings and adults (medians: 45.5% and 48.7%, respectively). Foals aged one week and one month had significantly lower proportions of lymphocytes than yearlings and adults (medians: 3.2% and 4.7% vs 43.2% and 45.8%, respectively). Eosinophil percentage was lowest in foals aged one week, one month, and 2 months (median: 0.0%) and highest in foals aged 6 months (median: 2.2%). Mast cell percentages were highest in yearlings and adults (medians: 2.2% and 3.3%, respectively) and neutrophil percentage was highest in foals aged one week (13.7%). CONCLUSIONS: Cytologic profiles of BALF from foals and adult horses differed considerably. Significant changes in TNCC and percentages of lymphocytes, macrophages, and eosinophils occurred with age.