Suppression of Ornithine Decarboxylase Gene Expression by Retinoids in Cultured Human Keratinocytes

Modulation of ornithine decarboxylase (ODC) gene expression by retinoids was analyzed in human keratinocyte cultures maintained in serum-free medium containing 0.15mM Ca++. Cells were incubated with all-trans-retinoic acid, 13-cis-retinoic acid or arotinoid Ro15-0778 (10−10 to 10−5 M), total RNA was isolated, and mRNA transcripts for ODC were analyzed by Northern and slot blot hybridizations with a human ODC cDNA. Treatment of cells for 24h resulted in a dose-dependent decrease in ODC mRNA levels, with an estimated IC50 of ∼1 × 10−8 M for all-trans- and 13-cis-retinoic acid, while Ro15-0778 was somewhat less effective (IC50 ∼1-5 × 10−7 M). The suppression of ODC mRNA levels by retinoids was detectable at ∼3h of incubation, with essentially a maximal inhibition at 12h. Reduced ODC mRNA levels noted after 24h of incubation with 5 × 10−7 M all-trans-retinoic acid were accompanied by a reduction in ODC enzyme activity. To determine if all-trans-retinoic acid was regulating ODC gene expression directly, or if protein synthesis was required, ODC expression was analyzed in cultures treated with protein synthesis inhibitors. In the presence of cycloheximide or puromycin, all-trans-retinoic acid did not suppress ODC mRNA levels. These findings suggest that suppression of ODC gene expression is not a direct effect of all-trans-retinoic acid, but depends on ongoing protein synthesis

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

Ultrasound-triggered antibiotic release from PEEK clips to prevent spinal fusion infection: Initial evaluations.

Despite aggressive peri-operative antibiotic treatments, up to 10% of patients undergoing instrumented spinal surgery develop an infection. Like most implant-associated infections, spinal infections persist through colonization and biofilm formation on spinal instrumentation, which can include metal screws and rods for fixation and an intervertebral cage commonly comprised of polyether ether ketone (PEEK). We have designed a PEEK antibiotic reservoir that would clip to the metal fixation rod and that would achieve slow antibiotic release over several days, followed by a bolus release of antibiotics triggered by ultrasound (US) rupture of a reservoir membrane. We have found using human physiological fluid (synovial fluid), that higher levels (100–500 μg) of vancomycin are required to achieve a marked reduction in adherent bacteria vs. that seen in the common bacterial medium, trypticase soy broth. To achieve these levels of release, we applied a polylactic acid coating to a porous PEEK puck, which exhibited both slow and US-triggered release. This design was further refined to a one-hole or two-hole cylindrical PEEK reservoir that can clip onto a spinal rod for clinical use. Short-term release of high levels of antibiotic (340 ± 168 μg), followed by US-triggered release was measured (7420 ± 2992 μg at 48 h). These levels are sufficient to prevent adhesion of Staphylococcus aureus to implant materials. This study demonstrates the feasibility of an US-mediated antibiotic delivery device, which could be a potent weapon against spinal surgical site infection. Statement of Significance: Spinal surgical sites are prone to bacterial colonization, due to presence of instrumentation, long surgical times, and the surgical creation of a dead space (≥5 cm 3 ) that is filled with wound exudate. Accordingly, it is critical that new approaches are developed to prevent bacterial colonization of spinal implants, especially as neither bulk release systems nor controlled release systems are available for the spine. This new device uses non-invasive ultrasound (US) to trigger bulk release of supra-therapeutic doses of antibiotics from materials commonly used in existing surgical implants. Thus, our new delivery system satisfies this critical need to eradicate surviving bacteria, prevent resistance, and markedly lower spinal infection rates

Berberine Disrupts Staphylococcal Proton Motive Force to Cause Potent Anti-Staphylococcal Effects

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\u27s 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 \u3c 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

Biomolecules as Model Indicators of In Vitro and In Vivo Cold Plasma Safety

The potential applications for cold plasma in medicine are extensive, from microbial inactivation and induction of apoptosis in cancer cells to stimulating wound healing and enhancing the blood coagulation cascade. The safe bio-medical application of cold plasma and subsequent effect on complex biological pathways requires precision and a distinct understanding of how physiological redox chemistry is manipulated. Chemical modification of biomolecules such as carbohydrates, proteins, and lipids treated with cold plasma have been characterized, however, the context of how alterations of these molecules affect cell behavior or in vivo functionality has not been determined. Thus, this study examines the cytotoxic and mutagenic effects of plasmatreated molecules in vitro using CHO-K1 cells and in vivo in Galleria mellonella larvae. Specifically, albumin, glucose, cholesterol, and arachidonic acid were chosen as representative biomolecules, with established involvement in diverse bioprocesses including; cellular respiration, intracellular transport, cell signaling or membrane structure. Long- and short-term effects depended strongly on the molecule type and the treatment milieu indicating the impact of chemical and physical modifications on downstream biological pathways. Importantly, absence of short-term toxicity did not always correlate with absence of longer-term effects, indicating the need to comprehensively assess ongoing effects for diverse biological applications

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 \u3c 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

Minimizing Penile Prosthesis Implant Infection: What Can We Learn From Orthopedic Surgery?

The implantation of penile protheses for the surgical treatment of erectile dysfunction has risen in popularity over the past several decades. Considerable advances have been made in surgical protocol and device design, specifically targeting infection prevention. Despite these efforts, device infection remains a critical problem, which causes significant physical and emotional burden to the patient. The aim of this review is to broaden the discussion of best practices by not only examining practices in urology, but additionally delving into the field of orthopedic surgery to identify techniques and approaches that may be applied to penile prothesis surgery

The inhibition of \u3cem\u3eStaphylococcus epidermidis\u3c/em\u3e biofilm formation by vancomycinmodified titanium alloy and implications for the treatment of periprosthetic infection

Peri-prosthetic infections are notoriously difficult to treat as the biomaterial implant is ideal for bacterial adhesion and biofilm formation, resulting in decreased antibiotic sensitivity. Previously, we reported that vancomycin covalently attached to a Ti alloy surface (Vanc-Ti) could prevent bacterial colonization. Herein we examine the effect of this Vanc-Ti surface on Staphylococci epidermidis, a Gram-positive organism prevalent in orthopaedic infections. By direct colony counting and fluorescent visualization of live bacteria, S. epidermidis colonization was significantly inhibited on Vanc-Ti implants. In contrast, the gram-negative organism Escherichia coli readily colonized the Vanc-Ti rod, suggesting retention of antibiotic specificity. By histochemical and SEM analysis, Vanc-Ti prevented S. epidermidis biofilm formation, even in the presence of serum. Furthermore, when challenged multiple times with S. epidermidis, Vanc-Ti rods resisted bacterial colonization. Finally, when S. epidermidis was continuously cultured in the presence of Vanc-Ti, the bacteria maintained a Vanc sensitivity equivalent to the parent strain. These findings indicate that antibiotic derivatization of implants can result in a surface that can resist bacterial colonization. This technology holds great promise for the prevention and treatment of periprosthetic infections