Investigation of Antibiotic Release from Bone Allograft in an Experiment on Rabbits

BACKGROUND: The treatment of chronic osteomyelitis, despite the use of new methods, is still an urgent problem. Local use of antibacterial drugs in combination with systemic antibiotic therapy has become popular in recent decades. Autologous bone grafts are considered ideal for bone defects filling. Different methods of allograft preparation may have differences in the rate and duration of antibiotic release. Moreover, it can affect the effectiveness of microbial agent eradication. The study analyzed the differences in the release of gentamicin from different types of allografts in dynamics and methods of preparation: «PerOssal» medium, whole bone allograft soaked in antibiotic, whole bone allograft, welded with an antibiotic, and perforated bone allograft soaked in an antibiotic solution. AIM: The objective of the study was to study the stability of antibiotic release and to determine the effectiveness of local transport systems. Evaluation of the difference in gentamicin release from different types of allografts in dynamics and methods of preparation had been realized: “PerOssal” medium, whole bone allograft soaked in antibiotic, whole bone allograft welded with an antibiotic, and perforated bone allograft, soaked in antibiotic solution. MATERIALS AND METHODS: The research was conducted between September 2020 and March 2021. The experiments were performed on 120 laboratory rabbits (weight – 3000–3500 g, age – 6–8 months), which were divided into four groups (30 animals in each group). Group 1 consisted of animals treated with “PerOssal.” The whole bone allograft soaked in an antibiotic was used in the treatment of animals of Group 2. The whole bone allograft, welded with an antibiotic, was used in the treatment of animals of Group 3. Perforated bone allograft soaked in an antibiotic was used in Group 4. Osteomyelitis of the proximal femur was formed in experimental animals. RESULTS AND DISCUSSION: Statistically insignificant decrease in the concentration of gentamicin was observed by the 7th day in all experimental groups. In rabbits whose bone defect was filled with a whole bone allograft welded with antibiotic and perforated bone allograft impregnated with an antibiotic (Groups 3 and 4), the most stable concentration of gentamicin was noted throughout the study period. Statistically significant differences were revealed between the experimental groups in relation to the dynamics of changes in the concentration of gentamicin in blood plasma. It was found that the group using the biodegradable material “PerOssal” on the 1st day showed a high concentration of the antibiotic in the blood plasma. However, by the 2nd day, a lower concentration of the antibiotic was recorded compared to all comparison groups of the bone allograft. CONCLUSIONS: The results of the analysis of the dynamics of gentamicin concentration may indicate significant differences between the methods of graft preparation, especially in the relationship with antibiotic release into the blood plasma. The most stable antibiotic concentration was registered in the groups of animals that underwent the filling of bone defect using a whole bone allograft welded with an antibiotic and a perforated bone allograft impregnated with antibiotic. A significant decrease of gentamicin concentration in the femur homogenate by the 7th day after transplantation was observed when using a whole bone allograft impregnated with an antibiotic. At the same time, a stable concentration of the antibiotic in the blood plasma was registered. The highest initial antibiotic concentration in the homogenate with a gradual decrease over 7 days was observed when using the antibiotic-impregnated biodegradable material “PerOssal.”

Influence of Tranexamic Acid on Elution Characteristics and Compressive Strength of Antibiotic-Loaded PMMA-Bone Cement with Gentamicin

Purpose: The topical application of tranexamic acid (TXA) into the joint space during total joint arthroplasty (TJA) with no increase of complications, has been widely reported. We investigated the influence of TXA on antibiotic release, activity of the released antibiotic against a clinical isolate of S. aureus, and compressive strength of a widely used commercially prepared gentamicin-loaded cement brand (PALACOS R + G). Method: 12 bone cement cylinders (diameter and height = 6 and 12 mm, respectively) were molded. After curing in air for at least 1 h, six of the cylinders were completely immersed in 5 mL of fetal calf serum (FCS) and the other six were completely immersed in a solution consisting of 4.9 mL of FCS and 0.1 mL (10 mg) of TXA. Gentamicin elution tests were performed over 7 d. Four hundred µL of the gentamicin eluate were taken every 24 h for the first 7 d without renewing the immersion fluid. The gentamicin concentration was determined in a clinical analyzer using a homogeny enzyme immuno-assay. The antimicrobial activity of the eluate, obtained after day 7, was tested. An agar diffusion test regime was used with Staphylococcus aureus. Bacteria were grown in a LB medium and plated on LB agar plates to get a bacterial lawn. Fifty µL of each eluate were pipetted on 12-mm diameter filter discs, which were placed in the middle of the agar gel. After 24 h of cultivation at 37 °C, the zone of inhibition (ZOI) for each specimen was measured. The compressive strength of the cements was determined per ISO 5833. Results: At each time point in the gentamicin release test, the difference in gentamicin concentration, obtained from specimens immersed in the FCS solution only and those immersed in the FCS + TXA solution was not significant (p = 0.055–0.522). The same trend was seen in each of the following parameters, after 7 d of immersion: (1) Cumulative gentamicin concentration (p < 0.297); (2) gentamicin activity against S. aureus (strongly visible); (3) ZOI size (mostly > 20 mm) (p = 0.631); and (4) compressive strength (p = 0.262). Conclusions: For the PALACOS R + G specimens, the addition of TXA to FCS does not produce significant decreases in gentamicin concentration, in the activity of the gentamicin eluate against a clinical isolate of S. aureus, the zone of inhibition of S. aureus, and in the compressive strength of the cement, after 7 d of immersion in the test solution.

Optimizing gentamicin dosing in different pediatric age groups using population pharmacokinetics and Monte Carlo simulation

Introduction The use of once daily dosing of aminoglycosides in pediatrics is increasing but studies on dose optimization targeting the pediatric population are limited. This study aimed to derive a population pharmacokinetic model of gentamicin and apply it to design optimal dosing regimens in pediatrics. Methods Population pharmacokinetics of gentamicin in pediatrics was described from a retrospective chart review of plasma gentamicin concentration data (peak/ trough levels) of pediatric patients (1 month − 12 years), admitted to non-critically ill pediatrics. Monte Carlo simulations were performed on the resulting pharmacokinetic model to assess the probability of achieving a Cmax/MIC target of 10 mg/L over a range of gentamicin MICs of 0.5–2 mg/L and once daily gentamicin dosing regimens. Results: A two-compartment model with additive residual error best described the model with weight incorporated as a significant covariate for both clearance and volume of distribution. Monte Carlo simulations demonstrated a good probability of target attainment even at a MIC of 2 mg/L, where neonates required doses of 6-7 mg/kg/day and older pediatrics required lower daily doses of 4–5 mg/kg/day while maintaining trough gentamicin concentration below the toxicity limit of 1 mg/L. Conclusion: Once daily dosing is a reasonable option in pediatrics that allows target attainment while maintaining trough gentamicin level below the limits of toxicity.

Degradation of Internal Fixation Materials Based on Antibacterial and Absorbable Silk Containing Different Gentamicin Concentrations

Adding gentamicin to silk fibroin enhances both the antibacterial performance and degradation rate of silk-based materials. The increased material degradation rate can affect the strength of early internal fixation, resulting in internal fixation failure. This study sought to adjust the gentamicin concentration to control the material degradation rate, thereby better meeting clinical application requirements. The in vitro degradation, water absorption rate, and expansion rate of silk-based materials containing different gentamicin concentrations were studied. A gentamicin-loaded silk-based screw (GSS) was implanted into the femurs of New Zealand rabbits. Micro-computed tomography (Micro-CT) was used to measure the screw diameter, which was then used to calculate the degradation rate. The in vitro results revealed increasing material degradation rates with increasing gentamicin concentration but no significant differences in water absorption rates with different gentamicin concentrations. The degradation rates of gentamicin-loaded (4 mg/g) silk-based rod-like materials were approximately 11.08% at three months in vitro and 9.4% in the animal experiment. The time for complete degradation was predicted from the fitting curve to be approximately 16 months. The degradation of material containing 4 mg/g gentamicin meets clinical application requirements, and previous experimental results demonstrate good antibacterial performance is retained by materials containing this gentamicin concentration.

Microsampling for monitoring gentamicin in neonates.

Gentamicin is recommended as first-line treatment of neonatal sepsis. The use of gentamicin is associated with toxicity which complicates neonatal dosing and necessitates therapeutic drug monitoring (TDM). In a proof-of-concept investigation, we sought to compare (1) gentamicin concentrations obtained using volumetric absorptive microsampling (VAMS) to standard TDM plasma samples, and (2) the time taken to report results obtained using VAMS compared to standard TDM by the local hospital chemical pathology service. The difference between gentamicin concentrations obtained from plasma collected for routine clinical care and calculated plasma concentrations, based on samples collected in whole blood using VAMS, was -18.0% and -0.4% for two patients. The research laboratory reported results within the time taken for the routine chemical pathology laboratory to report results. This proof-of-concept study demonstrates that the use of microsampling for TDM by pathology services can fulfil the requirements of providing an accurate gentamicin concentration in a timely manner.

Estimated glomerular filtration rate in patients overdosed with gentamicin

Background The aim of this study was to compare the estimated glomerular filtration rate (eGFR) from serum creatinine (eGFRcrea) and cystatin C (eGFRcys) in patients with elevated serum trough levels of gentamicin before the next planned dose during treatment in the intensive care unit (ICU). Methods This was a retrospective observational study. Patients who stayed in an ICU, received a once-daily dose of gentamicin, and who had results from all serum gentamicin trough levels, eGFRcrea and eGFRcys analyses were included in the study. Overdosed patients were defined as those with serum gentamicin trough levels above 1 mg/L before the next dose. Gentamicin was measured by a particle-enhanced turbidimetric immunoassay (PETIA). Creatinine and cystatin C were measured by standardized methods. Results The median age (range) was lower in all patients with gentamicin concentration measurements than in overdosed patients (67 [19–96] vs. 75 [48–99] years, respectively; p < 0.0001). The median interquartile range (IQR) of the eGFRcrea was higher than that of the eGFRcys in overdosed patients (60 [44–79] mL/min/1.73 m2 vs. 41 [29–58] mL/min/1.73 m2, respectively; p < 0.0001). The median IQR of the eGFRcrea was higher than that of the eGFRcys in controls (87 [78–98] mL/min/1.73 m2 vs. 66 [54–93] mL/min/1.73 m2, respectively; p < 0.0001). Conclusions Overdosed patients had both a lower eGFRcrea and eGFRcys than controls. Elderly patients are the most commonly overdosed patients. We recommend measuring cystatin C and calculating the eGFRcys and combined equation (eGFRcrea + cys) in ICU patients over 65 years of age, which would enable improved gentamicin dosage adjustments.

Gentamicin Alone Is Inadequate to Eradicate Neisseria Gonorrhoeae From the Pharynx

Background Centers for Disease Control and Prevention (CDC) guidelines recommend 240 mg gentamicin plus 2 g azithromycin for the treatment of gonorrhea in cephalosporin-allergic patients. The efficacy of gentamicin alone in the treatment of pharyngeal gonorrhea is uncertain. Methods Between September 2018 and March 2019, we enrolled men who have sex with men with nucleic acid amplification test–diagnosed pharyngeal gonorrhea in a single-arm, unblinded clinical trial. Men received a single 360-mg intramuscular dose of gentamicin and underwent test of cure by culture 4–7 days later. The study measured creatinine at enrollment and test of cure, serum gentamicin concentration postdose to establish peak concentration (Cmax), and standard antimicrobial minimum inhibitory concentrations (MICs) by agar dilution. The trial was designed to establish a point estimate for gentamicin’s efficacy for pharyngeal gonorrhea. We planned to enroll 50 evaluable participants; assuming gentamicin was 80% efficacious, the trial would establish a 95% confidence interval (CI) of 66%–90%. We planned interim analyses at n = 10 and n = 25. Results The study was stopped early due to poor efficacy. Of 13 enrolled men, 10 were evaluable, and only 2 (20% [95% CI, 2.5%–55.6%]) were cured. Efficacy was not associated with gentamicin Cmax or MIC. No participants experienced renal insufficiency. The mean creatinine percentage change was +5.2% (range, −6.7% to 21.3%). Six (46%) participants experienced headache, all deemed unrelated to treatment. Conclusions Gentamicin alone failed to eradicate Neisseria gonorrhoeae from the pharynx. Clinicians should use caution when treating gonorrhea with the CDC’s current alternative regimen (gentamicin 240 mg plus azithromycin 2 g) given increases in azithromycin resistance and gentamicin’s poor efficacy at the pharynx. Clinical Trials Registration NCT03632109.

Synergistic Action of Phage and Antibiotics: Parameters to Enhance the Killing Efficacy Against Mono and Dual-Species Biofilms

Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens and are commonly found in polymicrobial biofilm-associated diseases, namely chronic wounds. Their co-existence in a biofilm contributes to an increased tolerance of the biofilm to antibiotics. Combined treatments of bacteriophages and antibiotics have shown a promising antibiofilm activity, due to the profound differences in their mechanisms of action. In this study, 48 h old mono and dual-species biofilms were treated with a newly isolated P. aeruginosa infecting phage (EPA1) and seven different antibiotics (gentamicin, kanamycin, tetracycline, chloramphenicol, erythromycin, ciprofloxacin, and meropenem), alone and in simultaneous or sequential combinations. The therapeutic efficacy of the tested antimicrobials was determined. Phage or antibiotics alone had a modest effect in reducing biofilm bacteria. However, when applied simultaneously, a profound improvement in the killing effect was observed. Moreover, an impressive biofilm reduction (below the detection limit) was observed when gentamicin or ciprofloxacin were added sequentially after 6 h of phage treatment. The effect observed does not depend on the type of antibiotic but is influenced by its concentration. Moreover, in dual-species biofilms it was necessary to increase gentamicin concentration to obtain a similar killing effect as occurs in mono-species. Overall, combining phages with antibiotics can be synergistic in reducing the bacterial density in biofilms. However, the concentration of antibiotic and the time of antibiotic application are essential factors that need to be considered in the combined treatments.

In vitro validation of a method for neonatal urine collection and analysis

ObjectiveUrine collection and analysis is important for diagnosis, monitoring of clinical progress, and research in neonates. This study aims to validate a novel methodology for neonatal urine collection, which combines the convenience of cotton ball collection with accurate timing via a urine continence monitor.DesignLaboratory model using a combined cotton ball and urinary incontinence monitor method with and without the presence of an impermeable membrane to prevent desiccation.Main outcome measuresAccuracy, bias and precision in measurement of urine volume, electrolytes (sodium, potassium, chloride), creatinine and gentamicin. Changes in analyte concentration over time, and evaporative loss of water, were tested using analysis of variance. The effects of time, temperature and humidity were explored using multivariate analysis of variance.ResultsWith the use of an impermeable membrane, sodium concentration increased from a mean (SD) of 3.57% (0.68) at 1 min to 5.03% (0.74) at 120 min. There was no significant change in potassium, chloride or creatinine concentrations. Gentamicin concentration decreased by a mean (SD) of 9.05% (1.37) by 30 min. Multivariate analysis found that absolute change in weight, sodium and chloride were only dependent on duration. Gentamicin concentration was affected by duration, humidity and temperature. Relative evaporative loss was minimal at −0.58% (0.31), and the urinary continence monitor was 100% successful at detecting urination for all time points.ConclusionsThis novel methodology provides a standardisable and practical method to collect small volumes of neonatal urine for accurate measurement of both urine output and analyte concentrations.