Ingenuity pathways analysis of urine metabonomics phenotypes toxicity of gentamicin in multiple organs

We introduce the use of Ingenuity Pathway Analysis to analyzing global metabonomics in order to characterize phenotypically biochemical perturbations and the potential mechanisms of the gentamicin-induced toxicity in multiple organs. A single dose of gentamicin was administered to Sprague Dawley rats (200 mg/kg, n = 6) and urine samples were collected at -24-0 h pre-dosage, 0-24, 24-48, 48-72 and 72-96 h post-dosage of gentamicin. The urine metabonomics analysis was performed by UPLC/MS, and the mass spectra signals of the detected metabolites were systematically deconvoluted and analyzed by pattern recognition analyses (Heatmap, PCA and PLS-DA), revealing a time-dependency of the biochemical perturbations induced by gentamicin toxicity. As result, the holistic metabolome change induced by gentamicin toxicity in the animal's organisms was characterized. Several metabolites involved in amino acid metabolism were identified in urine, and it was confirmed that gentamicin biochemical perturbations can be foreseen from these biomarkers. Notoriously, it was found that gentamicin induced toxicity in multiple organs system in the laboratory rats. The proof-of-knowledge based Ingenuity Pathway Analysis revealed gentamicin induced liver and heart toxicity, along with the previously known toxicity in kidney. The metabolites creatine, nicotinic acid, prostaglandin E2, and cholic acid were identified and validated as phenotypic biomarkers of gentamicin induced toxicity. Altogether, the significance of the use of metabonomics analyses in the assessment of drug toxicity is highlighted once more; furthermore, this work demonstrated the powerful predictive potential of the Ingenuity Pathway Analysis to study of drug toxicity and its valuable complementation for metabonomics based assessment of the drug toxicity.

Eradication of endotracheal tube biofilm by nebulised gentamicin.

Objective: To compare the efficacy of gentamicin, nebulised via the endotracheal tube (ET), with that of parenteral cefotaxime or parenteral cefuroxime in preventing the formation of ET biofilm.

Setting: General intensive care units in two university teaching hospitals.

Design: The microbiology of ET biofilm from 36 ICU patients eligible to receive antibiotic prophylaxis was examined. Peak and trough tracheal concentrations of gentamicin, cefotaxime or cefuroxime were measured in each patient group, on the 2nd day of intubation.

Patients: Twelve patients received gentamicin (80 mg) nebulised in 4 ml normal saline every 8 h, 12 cefotaxime (1 g, 12 hourly) and 12 cefuroxime (750 mg, 8 hourly). Prophylaxis was continued for the duration of intubation.

Measurements and results: Samples of tracheal secretions were taken on the 2nd day of ventilation for determination of antibiotic concentrations. Following extubation, ETs were examined for the presence of biofilm. Pathogens considered to be common aetiological agents for VAP included Staphylococcus aureus, enterococci, Enterobacteriaceae and pseudomonads. While microbial biofilm was found on all ETs from the cephalosporin group, microbial biofilm of these micro-organisms was found on 7 of the 12 ET tubes from patients receiving cefotaxime [S. aureus (4), pseudomonads (1), Enterobacteriaceae (1), enterococcus (1)] and 8 of the 12 ET tubes from patients receiving cefuroxime [Enterobacteriaceae (6), P. aeruginosa (1) and enterococcus (1)]. While microbial biofilm was observed on five ETs from patients receiving nebulised gentamicin, none of these were from pathogens for ventilator-associated pneumonia (VAP). Tracheal concentrations of both cephalosporins were lower than those needed to inhibit the growth of pathogens recovered from ET tube biofilm. The median (and range) concentrations for cefotaxime were 0.90 (<0.23–1.31) mg/l and 0.28 (<0.23–0.58) mg/l for 2 h post-dose and trough samples, respectively. Two hours post-dose concentrations of cefuroxime (median and range) were 0.40 (0.34–0.83) mg/l, with trough concentrations of 0.35 (<0.22–0.47) mg/l. Tracheal concentrations (median and range) of gentamicin measured 1 h post-nebulisation were 790 (352–>1250) mg/l and then, before the next dose, were 436 (250–1000) mg/l.

Conclusion: Nebulised gentamicin attained high concentrations in the ET lumen and was more effective in preventing the formation of biofilm than either parenterally administered cephalosporin and therefore may be effective in preventing this complication of mechanical ventilation.

In vitro study of the efficacy of acrylic bone cement loaded with supplementary amounts of gentamicin: Effect on mechanical properties, antibiotic release, and biofilm formation

Background: Infection remains a severe complication following a total hip replacement. If infection is suspected when revision surgery is being performed, additional gentamicin is often added to the cement on an ad hoc basis in an attempt to reduce the risk of recurrent infection.

Methods and results: In this in vitro study, we determined the effect of incorporating additional gentamicin on the mechanical properties of cement. We also determined the degree of gentamicin release from cement, and also the extent to which biofilms of clinical Staphylococcus spp. isolates form on cement in vitro. When gentamicin was added to unloaded cement (1–4 g), there was a significant reduction in the mechanical performance of the loaded cements compared to unloaded cement. A significant increase in gentamicin release from the cement over 72 h was apparent, with the amount of gentamicin released increasing significantly with each additional 1 g of gentamicin added. When overt infection was modeled, the incorporation of additional gentamicin did result in an initial reduction in bacterial colonization, but this beneficial effect was no longer apparent by 72 h, with the clinical strains forming biofilms on the cements despite the release of high levels of gentamicin.

Interpretation: Our findings indicate that the addition of large amounts of gentamicin to cement is unlikely to eradicate bacteria present as a result of an overt infection of an existing implant, and could result in failure of the prosthetic joint because of a reduction in mechanical performance of the bone cement.

In vitro testing of chitosan in gentamicin-loaded bone cement No antimicrobial effect and reduced mechanical performance

Background and purpose Efforts to prevent infection of arthroplasties, including the use of antibiotic-loaded bone cement, are not always successful. We investigated whether the incorporation of chitosan in gentamicin-loaded bone cement increases antibiotic release, and prevents bacterial adherence and biofilm formation by clinical isolates of Staphylococcus spp. In addition, we performed mechanical and degradation tests.

Gentamicin-loaded nanoparticles show improved antimicrobial effects towards Pseudomonas aeruginosa infection

Gentamicin is an aminoglycoside antibiotic commonly used for treating Pseudomonas infections, but its use is limited by a relatively short half-life. In this investigation, developed a controlled-release gentamicin formulation using poly(lactide-co-glycolide) (PLGA) nanoparticles. We demonstrate that entrapment of the hydrophilic drug into a hydrophobic PLGA polymer can be improved by increasing the pH of the formulation, reducing the hydrophilicity of the drug and thus enhancing entrapment, achieving levels of up to 22.4 µg/mg PLGA. Under standard incubation conditions, these particles exhibited controlled release of gentamicin for up to 16 days. These particles were tested against both planktonic and biofilm cultures of P. aeruginosa PA01 in vitro, as well as in a 96-hour peritoneal murine infection model. In this model, the particles elicited significantly improved antimicrobial effects as determined by lower plasma and peritoneal lavage colony-forming units and corresponding reductions of the surrogate inflammatory indicators interleukin-6 and myeloperoxidase compared to free drug administration by 96 hours. These data highlight that the controlled release of gentamicin may be applicable for treating Pseudomonas infections.

Construction of aminoglycoside-sensitive Burkholderia cenocepacia strains for use in studies of intracellular bacteria with the gentamicin protection assay

Burkholderia cenocepacia is a multidrug-resistant opportunistic pathogen that infects the airways of patients with cystic fibrosis (CF) and can survive intracellularly in macrophages and epithelial cells. The gentamicin protection assay, which relies on the poor ability of gentamicin or other aminoglycosides to permeate eukaryotic cell membranes, is traditionally employed to quantify intracellular bacteria. However, the high resistance of these bacteria to aminoglycosides hampers the use of the gentamicin protection assay to investigate intracellular infection by B. cenocepacia. Here, we report the construction of gentamicin-sensitive strains of B. cenocepacia carrying a deletion of the BCAL1674, BCAL1675, and BCAL1676 genes that form an operon encoding an AmrAB-OprA-like efflux pump. We show that bacteria carrying this deletion are hypersensitive to gentamicin and also delay phagolysosomal fusion upon infection of RAW 264.7 murine macrophages, as previously demonstrated for the parental strain. We also demonstrate for the first time that low concentrations of gentamicin can be used to effectively kill extracellular bacteria and reliably quantify the intracellular infection by B. cenocepacia, which can replicate in RAW 264.7 macrophages.

Controlled delivery of gentamicin using poly(3-hydroxybutyrate) microspheres

Poly(3-hydroxybutyrate), P(3HB), produced from Bacillus cereus SPV using a simple glucose feeding strategy was used to fabricate P(3HB) microspheres using a solid-in-oil-water (s/o/w) technique. For this study, several parameters such as polymer concentration, surfactant and stirring rates were varied in order to determine their effect on microsphere characteristics. The average size of the microspheres was in the range of 2 μm to 1.54 μm with specific surface areas varying between 9.60 m(2)/g and 6.05 m(2)/g. Low stirring speed of 300 rpm produced slightly larger microspheres when compared to the smaller microspheres produced when the stirring velocity was increased to 800 rpm. The surface morphology of the microspheres after solvent evaporation appeared smooth when observed under SEM. Gentamicin was encapsulated within these P(3HB) microspheres and the release kinetics from the microspheres exhibiting the highest encapsulation efficiency, which was 48%, was investigated. The in vitro release of gentamicin was bimodal, an initial burst release was observed followed by a diffusion mediated sustained release. Biodegradable P(3HB) microspheres developed in this research has shown high potential to be used in various biomedical applications.

Overexpression of catalase prevents gentamicin – induced apoptosis of renal proximal tubular cells in transgenic mice.

Introduction : La néphrotoxicité est une complication majeure de la gentamicine, qui est largement utilisée dans le traitement des infections bactériennes, en particulier celles provoquées par des bactéries à Gram-négatif. La gentamicine induit l'apoptose tubulaire, mais les mécanismes moléculaires impliqués demeurent mal compris. Dans l’étude présente, nous avons examiné le rôle des espèces réactives de l'oxygène (ROS) , des proteins Bax, Bmf et Caspase-12 (Csp-12) dans le mécanisme d’action de la gentamicine sur l'apoptose des tubules proximaux rénaux (RPT) et les dommages rénaux induits par ce médicament chez la souris. Méthode: Des souris adultes (âgées 18-19 semaines) mâles non-Tg et des souris transgéniques (CAT-Tg) qui surexpriment la catalase spécifiquement dans leurs cellules des RPT ont été traitées par injections intra-péritonéales de gentamicine (20 mg/kg/jour) pour 5 jours consécutifs, puis euthanasiés. Les reins ont été examinés et analysés par histologie, immunohistochimie pour presansance de la stress oxidative, expression des proteins Bax, Bmf et Csp-12 et essai TUNEL pour étudier de l’apoptose . Nous avons aussi examiné l'effet de la gentamicine sur génération des ROS et l’apoptose dans les cellules RPTC immortalisées de rat (IRPTC) in vitro. Résultats: In vivo, chez les souris non-Tg, la gentamicine induit une tubulopathie et l'apoptose des RPT , stimule la production de ROS et induit une augmentation de Bax et Bmf détectée par immunohistochimie et augmont activité du caspase-12. Ces changements sont atténués chez les souris Cat-Tg. In vitro, la gentamicine induit l’apoptos des cellulles. Le co-traitement avec la catalase normalise ces effets dans les IRPTC. Conclusion : Ces données démontrent que l'apoptose des RPTC induite par la gentamicine s’effectue, au moins en partie, par l'intermédiaire de la génération des ROS.

Successful prevention of tunneled central catheter infection by antibiotic lock therapy using cefazolin and gentamicin

Catheter-related bacteremia (CRB) is one of the various complications related to hemodialysis (HD). As a result of this high rate of infection, the antibiotic lock technique (ALT) has been recommended to prevent CRB. However, adverse effects of ALT such as increased emergence of strains resistant to antibiotics and increased mechanical dysfunction catheter were poorly evaluated. We prospectively evaluated the efficacy of catheter-restricted filling using an antibiotic lock solution in preventing CRB. A total of 233 HD patients requiring 325 new tunneled catheters while waiting for placement and maturation of an arteriovenous fistula or graft were enrolled in this study. Patients with a tunneled catheter were assigned to receive either an antibiotic-heparin lock solution (antibiotic group: cefazolin 10 mg/ml, gentamicin 5 mg/ml, heparin 1,000 U/ml) or a heparin lock solution (no-antibiotic group: heparin 1,000 U/ml) as a catheter lock solution during the interdialytic period. The present study aimed to assess the efficacy of ALT using cefazolin and gentamicin in reducing CRB in patients undergoing HD with tunneled central catheter and to identify its adverse effects. CRB developed in 32.4 % of patients in the no-antibiotic group and in 13.1 % of patients in the antibiotic group. CRB rates per 1,000 catheter-days were 0.57 in the antibiotic group versus 1.74 in the no-antibiotic group (p < 0.0001). Kaplan-Meier analysis also showed that mean CRB-free catheter survival was significantly higher in the antibiotic group than in the no-antibiotic group (log-rank statistic 17.62, p < 0.0001). There was statistically significant difference between the two groups in causative organisms of CRB, with predominance of negative culture in both groups, but this prevalence was higher in ALT group (57.9 vs 90.1 %, p < 0.0001), and the two groups also were different in prevalence of gram-positive bacteria as causing organisms (ALT group 21.05 vs = 0 % in control group, p < 0.0001). There was no statistically significant difference between the two groups in drug-resistant germs. There were statistically significant differences between the two groups in the catheter removal causes, with higher rate of infectious cause in control group (12.32 vs 2.22 %, p < 0.0001) and mechanical cause in ALT group (28.26 vs 37.78 %, p < 0.0001). The results suggest that ALT may be a beneficial means of reducing the CRB rate in HD patients with tunneled catheter, without association between ALT and emergence of strains resistant. However, mechanical complications were more prevalent in antibiotic group. Further studies are required to determine the optimal drug regimen, concentrations for ALT, and its adverse effects. © 2012 Springer Science+Business Media Dordrecht.

Effect of endogenous hydrogen sulfide inhibition on structural and functional renal disturbances induced by gentamicin

Animal models of gentamicin nephrotoxicity present acute tubular necrosis associated with inflammation, which can contribute to intensify the renal damage. Hydrogen sulfide (H2S) is a signaling molecule involved in inflammation. We evaluated the effect of DL-propargylglycine (PAG), an inhibitor of endogenous H2S formation, on the renal damage induced by gentamicin. Male Wistar rats (N = 8) were injected with 40 mg/kg gentamicin (im) twice a day for 9 days, some of them also received PAG (N = 8, 10 mg·kg-1·day-1, ip). Control rats (N = 6) were treated with saline or PAG only (N = 4). Twenty-four-hour urine samples were collected one day after the end of these treatments, blood samples were collected, the animals were sacrificed, and the kidneys were removed for quantification of H2S formation and histological and immunohistochemical studies. Gentamicin-treated rats presented higher sodium and potassium fractional excretion, increased plasma creatinine [4.06 (3.00; 5.87) mg%] and urea levels, a greater number of macrophages/monocytes, and a higher score for tubular interstitial lesions [3.50 (3.00; 4.00)] in the renal cortex. These changes were associated with increased H2S formation in the kidneys from gentamicin-treated rats (230.60 ± 38.62 µg·mg protein-1·h-1) compared to control (21.12 ± 1.63) and PAG (11.44 ± 3.08). Treatment with PAG reduced this increase (171.60 ± 18.34), the disturbances in plasma creatinine levels [2.20 (1.92; 4.60) mg%], macrophage infiltration, and score for tubular interstitial lesions [2.00 (2.00; 3.00)]. However, PAG did not interfere with the increase in fractional sodium excretion provoked by gentamicin. The protective effect of PAG on gentamicin nephrotoxicity was related, at least in part, to decreased H2S formation.

Lokalisation und Quantifizierung von 4-Hydroxynonenal in der Kochlea des Meerschweinchens nach Applikation von Gentamicin

Einleitung und Literaturdiskussion: Gentamicin ist ein aus Bakterien gewonnenes Aminoglykosid-Antibiotikum, das seit vielen Jahren im klinischen Alltag zur Therapie von bakteriellen Infektionen und zur Behandlung des Morbus Ménière eingesetzt wird. Ein bedeutender, jedoch noch nicht vollständig verstandener, Pathomechanismus ist dabei die Entstehung von 4-HNE durch Lipid Peroxidation und die konsekutive Schädigung durch das gebildete Aldehyd. Ziel dieser Arbeit war es, die Beeinflussung der Expression von 4-HNE in sieben verschiedenen Regionen der Kochlea (SV, SL, CO, NF, LF, IDZ und SGZ) durch Gentamicin zu beschreiben und quantitativ zu ermitteln.rnMaterial und Methoden: Die Meerschweinchen wurden in vier Gruppen unterteilt: eine unbehandelte Kontrollgruppe und je eine Gruppe 1, 2 und 7 Tage nach Gentamicin-Applikation. Nach Ablauf der Inkubationszeit wurden die Kochleae den Tieren entnommen, das Gewebe fixiert, geschnitten und auf Objektträgern aufgebracht. Die Schnitte wurden mit 4-HNE-Antikörpern behandelt und die Immunreaktion mikroskopisch lokalisiert und zelluläre quantitative Unterschiede am Computer berechnet.rnErgebnisse: Die Auswertung der Daten ergab signifikante Anstiege der Immunreaktion auf 4-HNE von der Kontrollgruppe zu allen drei Behandlungsgruppen in vier der sieben untersuchten Regionen (Stria vascularis, Spirales Ligament, Cortisches Organ und Nervenfasern). In zwei Bereichen (Fibrozyten im Limbus und Interdentalzellen) kam es zwischen Kontrollgruppe und nur einer Behandlungsgruppe D (7d) zu einer signifikanten Erhöhung. Lediglich die Spiralganglionzellen erbrachten keine signifikanten Differenzen. Der Vergleich der Einzelwindungen erbrachte für die Stria vascularis, das Spirale Ligament, das Cortische Organ und die Nervenfasern signifikante Anstiege innerhalb der drei Windungen von der Kontrollgruppe zu den drei Behandlungsgruppen. Bei der Stria vascularis zeigte sich als einzige Region eine signifikant erhöhte Immunfärbung in allen drei Einzelwindungen von der Kontrollgruppe zu allen Behandlungsgruppen. Beim Vergleich der Gesamtwindungen ließ sich ausschließlich für die Region der Stria vascularis von der ersten zur dritten Windung ein Anstieg der Braunfärbung feststellen. Zudem konnten Korrelationen der Färbeintensitäten einerseits zwischen den beiden Regionen der Lateralen Wand und andererseits zwischen zwei Zelltypen im Limbus aufgezeigt werden.rnDiskussion: Die durch Gentamicin-gesteigerte 4-HNE-Expression kann durch genomische und nicht-genomische Prozesse hervorgerufen werden.

Vancomycin acts synergistically with gentamicin against penicillin-resistant pneumococci by increasing the intracellular penetration of gentamicin

Vancomycin and gentamicin act synergistically against penicillin-resistant pneumococci in vitro and in experimental rabbit meningitis. The aim of the present study was to investigate the underlying mechanism of this synergism. The intracellular concentration of gentamicin was measured by using the following experimental setting. Bacterial cultures were incubated with either gentamicin alone or gentamicin plus vancomycin for a short period (15 min). The gentamicin concentration was determined before and after grinding of the cultures by using the COBAS INTEGRA fluorescence polarization system (Roche). The grinding efficacies ranged between 44 and 54%, as determined by viable cell counts. In the combination regimen the intracellular concentration of gentamicin increased to 186% compared to that achieved with gentamicin monotherapy. These data suggest that the synergy observed in vivo and in vitro is based on an increased intracellular penetration of the aminoglycoside, probably due to the effect of vancomycin on the permeability of the cell wall.