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Colistin, used as a last resort drug, has a narrow therapeutic range that justifies therapeutic drug monitoring. Few data are available in the literature concerning the in vivo unbound fraction of colistin. The objectives of this study were to develop a method to isolate unbound colistin in clinical samples by ultrafiltration and to quantify it. The association between unbound colistin and biological parameters (total protein, albumin, alpha-1-acid glycoprotein, and creatinine) was investigated. The measured ranges were 0.036-7.160 mg/L for colistin A and 0.064-9.630 mg/L for colistin B. The process of isolation and determination of unbound colistin was applied to clinical samples (n=30) within 40 minutes and no non-specific binding was observed during the ultracentrifugation step. The median unbound fractions of colistin measured were 34.3% (12.8%-51.0%) and 53.4% (27.0%-77.8%) for colistin A and colistin B, respectively. High inter-individual biological variation of binding was observed for colistin A and B which was not explained by the biochemical parameters studied. The method developed could be useful to improve outcomes for patients.

is a highly prevalent pathogen in the respiratory tract of young patients with cystic fibrosis (CF) and causes biofilm-related infections. Here, we set up an model of a biofilm grown in Trypticase soy broth supplemented with glucose and NaCl (TGN) or in artificial sputum medium (ASM) and used it to evaluate on a pharmacodynamic basis the activity of antibiotics used in CF patients and active on staphylococci (meropenem, vancomycin, azithromycin, linezolid, rifampin, ciprofloxacin, tobramycin). Rheological studies showed that ASM was more elastic than viscous, as was also observed for sputa from CF patients, with elastic and viscous moduli being, respectively, similar to and slightly lower than those of CF sputa. Biofilms formed by methicillin-sensitive strain ATCC 25923 and methicillin-resistant strain ATCC 33591 reached maturity after 24 h, with biomass (measured by crystal violet staining) and metabolic activity (assessed by following resazurin metabolization) being lower in ASM than in TGN and viability (assessed by bacterial counts) being similar in both media. Full concentration-response curves of antibiotics obtained after 24 h of incubation of biofilms showed that all antibiotics were drastically less potent and less efficient in ASM than in TGN toward viability, metabolic activity, and biomass. Tobramycin selected for small-colony variants, specifically in biofilms grown in ASM; the auxotrophism of these variants could not be established. These data highlight the major influence exerted by the culture medium on responsiveness to antibiotics in biofilms. The use of ASM may help to determine effective drug concentrations or to evaluate new therapeutic options against biofilms in CF patients.

Background Pulsed washing (PW) is a major step during debridement surgery in the context of orthopaedic device-related infections (ODRI), but its effects on biofilms are poorly understood. Previous studies showed a weak effect of PW alone or combined with antiseptics on MRSA biofilms in-vitro. The combined effect of PW and antibiotics has never been published in the orthopaedic literature. Objectives The present study aims at evaluating the effect of PW alone or followed by vancomycin treatment on MRSA biofilms grown on titanium Ti6Al4V coupons in-vitro. Study Design & Methods MRSA strains: ATCC33591 and clinical isolates 25755749 and 27353676 (isolates from ODRI). Biofilm culture: Ti6Al4V coupons were incubated during 24h at 37°C with a bacterial suspension (starting inoculum ~6.6Log10CFU/mL in TGN [TSB + 1% glucose + 2% NaCl], under shaking at 50rpm. Treatment: Half of the coupons received PW with 50mL of physiological serum using a Stryker Interpulse from a distance of 5cm; the coupons were then either analysed or reincubated for 24h in TGN or TGN + vancomycin at two concentrations (MIC and a therapeutic concentration of 20µg/mL). Analysis: Coupons were rinsed twice with PBS. Total biomass was determined by crystal violet (CV) staining. CFUs were counted after recovering bacteria from coupons (alternating vortexing and sonication) and TSA plating. Results For control biofilms, 24h reincubation without or with vancomycin had no influence on CFU counts or total biomass, except for a minor biomass decrease in isolate 27353676 at 20µg/mL of vancomycin. For biofilms exposed to PW, a significant reduction in both CFU (2.58-3.12log10) and biomass (77.81-96.15%) was observed, but full regrowth was observed after 24 h reincubation in control conditions. Reincubation with vancomycin prevented regrowth when used at its MIC but further reduced CFUs at 20 µg/mL in a synergistic way. Conclusions PW alone only has a transient effect on coupon colonisation by MRSA biofilms and v

Background: Pulsed washing (PW) is a major step during debridement surgery in the context of orthopaedic device-related infections (ODRI), but its effects on biofilms are poorly characterized. The present study aims at evaluating the effect of PW alone or followed by antibiotics on MSSA and MRSA biofilms grown on titanium Ti6Al4V in-vitro. Materials/methods: Strains: 1 reference (MSSA: ATCC25923; MRSA: ATCC33591) and 2 clinical MSSA and MRSA isolated from ODRI. Biofilm culture: Incubation for 24h at 37°C with bacteria (starting inoculum ~6.6Log10CFU/mL in TGN [TSB+1%glucose+2%NaCl]), under continuous agitation. Treatment: Half the coupons received PW (50mL physiological serum, Stryker Interpulse); the coupons were then either analysed or reincubated for 24h in TGN or TGN + flucloxacillin (MSSA) or vancomycin (MRSA) at two concentrations (MIC and 20mg/L). Analysis: Biomass was determined by crystal violet (CV) staining. CFUs were counted after recovering bacteria from coupons using sonication and TSA plating. Results: See Figure. Antibiotics alone: Flucloxacillin significantly reduced both CFU and biomass for ATCC25923 but not for clinical isolates of MSSA. Vancomycin had no statistically significant effects on CFUs for MRSAs and a weak effect on biomass for one clinical isolate. PW alone markedly reduced CFUs and biomass for all strains but had no persistent effect. PW + antibiotics: Antibiotics prevented bacterial regrowth after PW when used at their MIC and further decreased CFUs when used at 20 mg/L. Conclusions: PW alone only has a transient effect on S. aureus biofilms. Vancomycin alone at therapeutic concentrations is ineffective and flucloxacillin has a strain-dependent effect. Yet, these treatments show synergistic effects, indicating the importance of PW followed by high antibiotic doses. However, PW in-vivo is limited by poor access to parts of the implants, providing arguments for the development of alternative biofilm-disrupting treatments.

Oxazolidinones cause rare but severe adverse effects in patients receiving prolonged treatment with these antibiotics. One of them is thrombocytopenia. The aim of the thesis was to better understand the cellular and biochemical mechanisms of this toxicity. In this context, we compared linezolid, the first drug marketed in this class, with a new, more active derivative, tedizolid. Focusing our work on mitochondria, we demonstrated that both antibiotics inhibited the synthesis of proteins encoded by the mitochondrial genome and studied the functional consequences of this inhibition on mitochondrial metabolism and ultrastructure in human cultured cells exposed to clinically relevant concentrations of both drugs. We also investigated the effects of oxazolidinones on the differentiation process of platelets precursors., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Aim: Irrigation is a major step during debridement surgery in the context of Prosthetic Joint Infections (PJI), but its effects on biofilms are poorly described. The present study aims at evaluating the effect of PW alone or followed by antibiotics on MSSA and MRSA biofilms grown on Ti6Al4V coupons in-vitro. Method: Strains: 1 reference (MSSA: ATCC25923; MRSA: ATCC33591) and 2 clinical MSSA and MRSA isolated from PJI. Biofilm culture: Coupons were incubated for 24h at 37°C with bacteria (starting inoculum ~6.6Log10CFU/mL in TGN [TSB + 1% glucose + 2% NaCl]), under shaking at 50rpm. Treatment: Half of the coupons were irrigated with 50mL physiological serum from 5cm using a Stryker Interpulse; the coupons were then either analysed (ControlT0 and PWT0) or reincubated for 24h in TGN or TGN containing flucloxacillin (MSSA) or vancomycin (MRSA) at MIC or 20mg/L. Analysis: Coupons were rinsed twice with PBS. Biomass was measured by crystal violet (CV) assay. CFUs were counted after recovering bacteria from coupons using sonication and TSA plating. Results: Antibiotics alone: Flucloxacillin reduced CFU and biomass for ATCC25923 and 611 but not 578 (Fig b and d). Vancomycin had no statistically significant effects on CFUs for all MRSA and only a weak effect on biomass for 676. Irrigation alone markedly reduced CFUs and biomass for all strains but had no persistent effect after 24h reincubation in TGN. PW + antibiotics: Antibiotics prevented bacterial regrowth after PW when used at their MIC and further decreased CFUs when used at 20 mg/L. Conclusions: PW alone has a transient effect on coupon colonisation by S. aureus biofilms. Vancomycin at therapeutic concentrations is ineffective and flucloxacillin has a strain-dependent effect. In combination, these treatments show synergistic effects, indicating the importance of irrigation followed by high antibiotic doses for ODRI.

Thrombocytopenia is commonly seen in patients receiving linezolid for >14 days. Linezolid is also a reversible inhibitor of mitochondrial functions in various cell types. We have studied the inhibitory effects of linezolid and tedizolid and their potential recovery on (i) CYTox I expression (subunit I of cytochrome c-oxidase; encoded by the mitochondrial genome), (ii) cytochrome c-oxidase activity, and (iii) mitochondrial respiration (Seahorse bioanalysis) in two megakaryocytic cell lines (UT-7 WT [human acute megakaryoblastic leukemia cells] and UT-7 MPL [transduced to stably express the thrombopoietin (TPO) receptor]). Cells were exposed to linezolid (0.5-25 mg/L) or tedizolid (0.1-5 mg/L) for up to 5 days and recovery followed after drug removal. Both oxazolidinones caused a concentration- and time-dependent inhibition of CYTox I expression, cytochrome c-oxidase activity and mitochondrial spare capacity. In the electron microscope, mitochondria appeared dilated with a loss of cristae. Globally, tedizolid exerted stronger effects than linezolid. While the expression of CYTox I was completely recovered after 6 days of drug washout, we observed only a partial (linezolid) or no (tedizolid) recovery of cytochrome c-oxidase activity, and no rescue of mitochondrial spare capacity (after 3 days). Thus, and in contrast with previous studies using a variety of cell lines unrelated to megakaryocytic lineages, the inhibitory effects exerted by oxazolidinones on mitochondrial functions of megakaryoblastic cells appear to be particularly protracted. Given the dynamics of platelet production and destruction, these results may explain why oxazolidinone-induced thrombocytopenia emerges as one of the most frequent untoward effects in patients exposed to these antibiotics.

Category: Basic - Poster 9

Fluoroquinolones have been in clinical use for over 50 years with significant efficacy. However, increasing resistance and emergence of some marked adverse events have limited their usage. The most recently approved class member, delafloxacin, is the only available anionic (non-zwitterionic) fluoroquinolone. Its unique molecular structure provides improved in vitro activity against most Gram-positive pathogens, including quinolone-resistant strains, which is further enhanced at acid pH. Delafloxacin shows favorable pharmacological properties, with about 60% bioavailability after oral administration, only mild inhibition of cytochrome P450 3A, and no evidence of cardiac- or phototoxicity in healthy volunteers (tested against positive controls). Its twice daily dosing, suitability for intravenous, oral, or switch dosing, the lack of many clinically significant drug-drug interactions, and acceptable adverse event profile in registration clinical trials supports its use in the treatment of acute bacterial skin and skin structure infections, and potentially in other infections, where resistance to other agents, safety, and/or the need for early discharge is of concern.

Infections are the second leading cause of death for patients with end-stage renal disease (ESRD). Temocillin, a beta-lactam antimicrobial agent, is an interesting therapeutic option for Gram-negative infections. However, temocillin pharmacokinetic (PK) data in the haemodialysis population is limited, and data on its dialysability with contemporary haemodialysers is inexistent. This thesis is in three parts. The first focuses on the development and validation of an analytical method for the quantification of temocillin concentrations in human serum. In the second, temocillin PK is characterised in the ESRD population based on data from a study conducted in a Belgian hospital. The licensed dosage regimens were found to be far below target, leading to an update of the Summary of Product Characteristics. Finally, population PK methodologies were used to investigate the performance of clinically practical dosing regimens and their ability to achieve set PK/PD targets. Model-based Monte Carlo simulations lead to an improved temocillin dosage regimen applicable in routine clinical practice and likely to meet contemporary treatment targets for this patient population. More generally, this thesis addresses the current need, acknowledged by regulatory bodies, to study PK/PD relationships for 'old-revived' antibiotics such as temocillin, including in specific patient populations for whom data is lacking. This thesis provides such data for ESRD patients and makes concrete propositions on how to optimise treatment in this population., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Colistin, used as a last-resort drug, has a narrow therapeutic range that justifies therapeutic drug monitoring. Few data are available in the literature regarding the in vivo unbound fraction of colistin. The objectives of this study were to develop a method to isolate unbound colistin in clinical samples by ultrafiltration and to quantify it. The association between unbound colistin and biological parameters (total protein, albumin, alpha-1-acid glycoprotein and creatinine) was investigated. The measured ranges were 0.036–7.160 mg/L for colistin A and 0.064–9.630 mg/L for colistin B. The process of isolation and determination of unbound colistin was applied to clinical samples (n = 30) within 40 min and no non-specific binding was observed during the ultracentrifugation step. The median unbound fractions of colistin measured were 34.3% (12.8–51.0%) and 53.4% (27.0–77.8%) for colistin A and B, respectively. High interindividual biological variation of binding was observed for colistin A and B that was not explained by the biochemical parameters studied. The method developed could be useful to improve outcomes for patients., SCOPUS: ar.j, info:eu-repo/semantics/published