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Introduction: Linezolid is a last-resort antibiotic for infections caused by multidrug-resistant microorganisms. It is widely used for off-label indications and for longer than recommended treatment durations, exposing patients at higher risk of adverse drug reactions (ADRs), notably thrombocytopenia. This study aimed to investigate ADR incidence and risk factors, identify thrombocytopenia-related trough levels based on treatment duration, and evaluate the performance of predictive scores for ADR development. Methods: Adult in- and outpatients undergoing linezolid therapy were enrolled in three hospitals and ADRs and linezolid trough levels prospectively monitored over time. A population pharmacokinetic (pop-PK model) was used to estimate trough levels for blood samples collected at varying times. Results: A multivariate analysis based on 63 treatments identified treatment duration ≥10 days and trough levels >8 mg/L as independent risk factors of developing thrombocytopenia, with high trough values correlated with impaired renal function. Five patients treated for >28 days did not develop thrombocytopenia but maintained trough values in the target range (<8 mg/L). The Buzelé predictive score, which combines an age-adjusted Charlson comorbidity index with treatment duration, demonstrated 77% specificity and 67% sensitivity to predict the risk of ADR. Conclusion: Our work supports the necessity of establishing guidelines for dose adjustment in patients with renal insufficiency and the systematic use of TDM in patients at-risk in order to keep trough values ≤8 mg/L. The Buzelé predictive score (if ≥7) may help to detect these at-risk patients, and pop-PK models can estimate trough levels based on plasma samples collected at varying times, reducing the logistical burden of TDM in clinical practice., SCOPUS: ar.j, info:eu-repo/semantics/published

Background : In a retrospective study in 4 Belgian hospitals, we showed that the use of linezolid (LZD) was larger and the rate of adverse drug reactions (ADRs) higher (especially anemia and thrombocytopenia) than described in the summary of product characteristics (1). Associated risk factors for ADRs were long treatment duration, renal impairment at the treatment initiation, and presence of comorbidities (evaluated by the Charlson index (2)). Moreover, literature suggests that trough levels (Cmin) > 9 mg/L increase the risk of hematological toxicity (HT) [...].

The emergence of antibiotic-resistant Staphylococcus aureus has become a major public health concern, necessitating the discovery of new antimicrobial compounds. Given that the skin microbiome plays a critical role in the host defense against pathogens, the development of therapies that target the interactions between commensal bacteria and pathogens in the skin microbiome offers a promising approach. Here, we report the discovery of two bacteriocins, cerein 7B and cerein B4080, that selectively inhibit S. aureus without affecting Staphylococcus epidermidis, a commensal bacterium on the skin.In this study, structural and mutational characterization of cerein 7B allowed us to propose a hypothesis of the mechanism of action for this bacteriocin, where it is believed to accumulate at the cell membrane, inducing the formation of pores. Furthermore, the study revealed that exposure of S. aureus to both discovered bacteriocins resulted in mutations in the walK/R two-component system, leading to a thickening of the cell wall visible by transmission electron microscopy and subsequent decreased sensitivity to vancomycin. Following RNA sequencing results indicate an upregulation of genes known to enhance the abundance of positively charged components at the cell membrane, thereby resulting in reduced sensitivity to other cationic bacteriocins.Our findings prompt a nuanced discussion of the potential of those bacteriocins for selective targeting of S. aureus on the skin, given the emergence of resistance and co-resistance with vancomycin. The idea put forward implies that by preserving commensal bacteria, selective compounds could limit the emergence of resistance in pathogenic cells by promoting competition with remaining commensal bacteria, ultimately reducing chronic infections and limiting the spread of antibiotic resistance., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Gram-negative ESKAPE pathogens are responsible for increases in multidrug-resistant infections worldwide. Poor permeability and active efflux cause intrinsic resistance to many antibiotics. Besides, these pathogens can also adopt specific lifestyles like intracellular survival and biofilm formation where bacteria can switch to dormant phenotypes poorly responsive to antibiotics. This thesis investigated the capacity of the polyaminoisoprenyl potentiator NV716 to revive disused antibiotics against Gram-negative bacteria using planktonic, intracellular and biofilm models. We showed that NV716 re-sensitizes Gram-negative bacteria to disused antibiotics by binding to the lipopolysaccharides (LPS) of the outer membrane, permeabilizing this membrane as well as downregulating quorum sensing regulated processes. Therefore, our work highlights a strong potential for this compound as an adjuvant therapy against difficult-to-treat Gram-negative organisms, and suggests that NV716 deserves further investigations, notably in in-vivo models of infections., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

Persisters are transiently nongrowing and antibiotic-tolerant phenotypic variants identified in major human pathogens, including intracellular Staphylococcus aureus. Due to their capacity to regrow once the environmental stress is relieved and to promote resistance, persisters possibly contribute to therapeutic failures. While persistence and its related quiescence have been mostly studied under starvation, little is known within host cell environments. Here, we examined how the level of reactive oxygen species (ROS) in different host cells affects dormancy depth of intracellular S. aureus. Using single-cell approaches, we found that host ROS induce variable dormant states in S. aureus persisters, displaying heterogeneous and increased lag times for resuscitation in liquid medium. Dormant persisters displayed decreased translation and energy metabolism, but remained infectious, exiting from dormancy and resuming growth when reinoculated in low-oxidative-stress cells. In high-oxidative-stress cells, ROS-induced ATP depletion was associated with the formation of visible dark foci similar to those induced by the protein aggregation inducer CCCP (carbonyl cyanide m-chlorophenylhydrazone) and with the recruitment of the DnaK-ClpB chaperone system involved in the clearance of protein aggregates. ATP depletion led to higher fractions of dormant persisters than ROS, due to a counterbalancing effect of ROS-induced translational repression, suggesting a pivotal role of translation in the dormant phenotype. Consistently, protein synthesis inhibition limited dormancy to levels similar to those observed in low-oxidative-stress cells. This study supports the hypothesis that intracellular S. aureus persisters can reach heterogeneous dormancy depths and highlights the link between ROS, ATP depletion, dark focus formation, and subsequent dormancy state.

Alpha-1 antitrypsin (AAT) is a serine protease inhibitor. A low level of serum AAT (<11 µM) is defined as AAT deficiency. The limitations of the current treatments for AAT deficiency-related lung disease are the low AAT lung penetration rate following intravenous infusion and the rapid AAT clearance from the lung following inhalation. Here, we present a PEGylated version of AAT, which shows improved resistance to proteolysis in vitro. In vivo, PEGylated AAT exhibits prolonged body residency after delivery by the intravenous and intratracheal routes. In a murine COPD model, a low intranasal dose of PEGylated AAT achieves better efficacy than a 45-fold higher intravenous dose of AAT. PEGylated AAT represents a promising alternative for AAT augmentation therapy., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

Background Orthopedic device-related infection (ODRI) is a leading cause of failure following arthroplasty or osteosynthesis. In this setting, antibiotic activity against micro-organisms is reduced by biofilm formation. The use of matrix-targeting enzymes is a promising option to restore this activity. However, the ideal drug form for this application is undetermined. We propose the use of a thermoresponsive hydrogel containing a previously described enzyme combination (TEC; Poilvache et al. 2021) associated with vancomycin and describe the release kinetics of TEC and the activity of the TEC and vancomycin combination against S.aureus biofilms. Methods Release kinetics: A solution of 20%(w/v) Pluronic F-127 in a TEC solution (Poilvache et al. 2021) containing 10mg/ml of vancomycin, was prepared at 4°C. 1mL was placed in 15mL tubes and gelled at room temperature. 9ml of Tris-HCl buffer was added and tubes were incubated at 37°C. 0.5mL samples were regularly collected over 7 days and replaced with fresh buffer to maintain total volume. Samples proteins contents were determined using the BCA assay. Antibiofilm activity: ATCC33591 S.aureus biofilms were grown for 24h at 37°C with a continuous 50rpm agitation on Ti-6Al-4V coupons by adding 3ml of a 6log10 suspension in TGN (TSB+1%glucose+2%NaCl) to 12wells plates containing the coupons. Samples were placed in 6wells plates containing 1ml of the hydrogel containing buffer (controls), TEC, vancomycin, or both. 9ml of TGN was added and samples were incubated in identical conditions for 24h. CFU counts were obtained after samples sonication, dilutions, and TSA plating. Results (a)Figure 1. A reproducible release of proteins from the hydrogels was observed following an exponential plateau model, with a rate constant of 0.08h-1, a plateau reached after 72h, and 80% proteins release after 17h30. (b)Figure 2. Exposure of mature S. aureus biofilms to hydrogels containing either TEC or vancomycin reduced CFU counts by respectively

The intense use and misuse of antibiotics is undoubtedly the main factor associated with the high numbers of antibiotic-resistant pathogenic and commensal bacteria worldwide. In low-income countries, this misuse and overuse is widespread, with great consequences at the personal and global levels. In the context of user fee exemptions in caesarean sections, we performed a descriptive study in women to assess the use of antibiotics on three levels—antenatal, during caesarean section, and postpartum—in four Beninese hospitals. Out of the 141 women included, 56.7% were using antibiotics. More than the half (71.3%) were taking more than one antibiotic, either for a long time or in acute treatment. In prophylaxis, the timing, dose, and duration of administration were not correctly achieved. Only 31.2% of women received optimal antibiotic prophylaxis. Various antibiotics including broad-spectrum molecules were used in the patients after caesarean section. The use of antibiotics was improper on the three levels studied. The high rate of self-administered antibiotics, the poor achievement of antibiotic prophylaxis, and the postpartum overuse of antibiotics showed a poor quality of care provided in pregnancy. A national policy is essential to improve the use of antibiotics by the general public as well as by professionals.

Most clinical guidelines are developed by high income countries institutions with little consideration given to either the evidence base for interventions in low and middle income countries, or the specific challenges low and middle income countries’ health systems may face in implementing recommendations In our previous studies, we noticed a misuse and overuse of antibiotics, and a poor match with the bacteria in concern The aim of this study was to develop, based on the results of antimicrobial susceptibility of bacteria isolated in surgical site infections, and antibiotics available, a guideline for the rational use of antibiotics in surgery in order to improve the quality of antibiotic prophylaxis, antibiotherapy and reduce the rate of antimicrobial resistance in Benin The study is a part of the project named “MUltidisciplinary STrategy for Prevention and Infection Control(MUST PIC)

The setting up of the fee exemption policy in cesarean-sections is spread in Sub-Saharan Africa. In our country, since 2009, a free kit as a guideline, containing the required materials and antibiotics has been available. An agency was set up to manage the policy. Unfortunately, some contradictions were noted in antibiotic prophylaxis practices.

A low adherence to recommendations on antibiotic prophylaxis has been reported worldwide. Since 2009, cesarean sections have been performed under user fee exemption in Benin with a free kit containing the required supplies and antibiotics for prophylaxis. Despite the kit, the level of antibiotic prophylaxis achievement remains low. We conducted a convergent parallel design study in 2017 using a self-administered questionnaire and interviews to assess the knowledge and explore the beliefs of healthcare professionals regarding antibiotic prophylaxis in three hospitals. Of the 35 participants, 33 filled out the questionnaire. Based on the five conventional criteria of antibiotic prophylaxis, the mean level of knowledge was 3.3 out of 5, and only 15.2% scored 5 out of 5. From the verbatim of 19 interviewees, determinants such as suboptimal patient status health, low confidence in antibiotics, some disagreement with the policy, inappropriate infrastructures and limited financial resources in hospitals, poor management of the policy in the central level, and patient refusal to buy antibiotics can explain poor practices. Because of the dysfunction at these levels, the patient becomes the major determinant of adequate antibiotic prophylaxis. Policymakers have to consider these determinants for improving antibiotic prophylaxis in a way that ensures patient safety and reduces the incidence of antimicrobial resistance.

Pseudomonas aeruginosa is intrinsically resistant to many antibiotics due to the impermeability of its outer membrane and to the constitutive expression of efflux pumps. Here, we show that the polyaminoisoprenyl compound NV716 at sub-MIC concentrations re-sensitizes P. aeruginosa to abandoned antibiotics by binding to the lipopolysaccharides (LPS) of the outer membrane, permeabilizing this membrane and increasing antibiotic accumulation inside the bacteria. It also prevents selection of resistance to antibiotics and increases their activity against biofilms. No stable resistance could be selected to NV716-itself after serial passages with subinhibitory concentrations, but the transcriptome of the resulting daughter cells shows an upregulation of genes involved in the synthesis of lipid A and LPS, and a downregulation of quorum sensing-related genes. Accordingly, NV716 also reduces motility, virulence factors production, and biofilm formation. NV716 shows a unique and highly promising profile of activity when used alone or in combination with antibiotics against P. aeruginosa, combining in a single molecule anti-virulence and potentiator effects. Additional work is required to more thoroughly understand the various functions of NV716.

Potentiators can improve antibiotic activity against difficult-to-treat Gram-negative bacteria like Escherichia coli, Klebsiella pneumoniae or Acinetobacter baumannii. They represent an appealing strategy in view of the paucity of therapeutic alternatives in case of multidrug resistance. Here, we examine the ability of the polyamino-isoprenyl compound NV716 to restore the activity of a series of disused antibiotics (rifampicin, azithromycin, linezolid, fusidic acid, novobiocin, chloramphenicol, and doxycycline, plus ciprofloxacin as an active drug) against these three species in planktonic cultures, but also in infected human monocytes and biofilms and we study its underlying mechanism of action. NV716 considerably reduced the MICs of these antibiotics (2-11 doubling dilutions), the highest synergy being observed with the more lipophilic drugs. This potentiation was related to a strong interaction of NV716 with LPS, ensuing permeabilization of the outer membrane, and leading to an increased accumulation of the antibiotics inside bacteria. Moreover, NV716 increased the relative potency of all drugs against intracellular infection by the same bacteria as well as their maximal efficacy, probably related to an improvement of antibiotic activity against persisters. Lastly, NV716 also enhanced rifampicin activity against biofilms from these three species. All these effects were observed at sub-MIC concentrations of NV716 (and thus unrelated to a bactericidal effect), and in conditions for which no toxicity was evidenced towards eukaryotic cells. Altogether, these data highlight for the first time the potential interest of NV716 as an adjuvant against these Gram-negative pathogens placed in the priority list of WHO for search of new therapies.

INTRODUCTION: Prosthetic Joint Infection (PJI) are catastrophic complications of joint replacement. Debridement, implant retention, and antibiotic therapy (DAIR) is the usual strategy in acute infections but fails in 45% of MRSA infections. We describe the development of a model of infected arthroplasty in rabbits, treated with debridement and a course of vancomycin with clinically relevant dosage. MATERIALS AND METHODS: A total of 15 rabbits were assigned to three groups: vancomycin pharmacokinetics (A), infection (B), and DAIR (C). All groups received a tibial arthroplasty using a Ti-6Al-4V implant. Groups B and C were infected per-operatively with a 5.5 log10 MRSA inoculum. After 1 week, groups C infected knees were surgically debrided. Groups A and C received 1 week of vancomycin. Pharmacokinetic profiles were obtained in group A following 1st and 5th injections. Animals were euthanized 2 weeks after the arthroplasty. Implants and tissue samples were processed for bacterial counts and histology. RESULTS: Average vancomycin AUC0-12 h were 213.0 mg*h/L (1st injection) and 207.8 mg*h/L (5th injection), reaching clinical targets. All inoculated animals were infected. CFUs were reproducible in groups B. A sharp decrease in CFU was observed in groups C. Serum markers and leukocytes counts increased significantly in infected groups. CONCLUSION: We developed a reproducible rabbit model of PJI treated with DAIR, using vancomycin at clinically relevant concentrations.

Surgical site infections are one of the most common healthcare associated infections and contribute significantly to increase the patient's morbidity, healthcare costs and antibiotic resistance. In Benin, due mainly to the irrational prescription of antibiotics, and poor infection prevention and control programs, we observed a scarcity of local data regarding the rates of SSIs and the frequency of multi drug bacteria isolated (MDR). Almost all Gram negative bacteria isolated on pus samples were resistant to at least five classes of antibiotics. Moreover, healthcare workers missed two opportunities out of three to apply hand hygiene and when HH was applied, technique and duration were not appropriate. Our interventions focused on implementation of world health organization multimodal strategy with high availability of alcohol-based hand rub solution in hospitals. However, future work is needed to largely describe the molecular epidemiology of MDR., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

The inappropriate utilization of antibiotics drove to a global public health threat: the antimicrobial resistance. Several strategies were set up to fight the problem such as Infection Prevention and Control which aims to improve the use of antibiotic. Our work identified the bottlenecks of the rational use of antibiotics in Benin in the context of the user fees exemption policy in caesarean section with a national free kit. The findings of our five studies showed: - a misuse, overuse, and underuse of antibiotics before, during and after caesarean section, - a low level of knowledge of antibiotic prophylaxis in healthcare professionals, a lack of confidence in the kit, and some general disagreement with the policy, - some cases of overdosage of active ingredient, - a mixed knowledge of antibiotics and their utilization by patients, - a low involvement of end-implementers during the policy formulation, a mixed-consensual context without scientific evidence-based considerations in the choice of antibiotics in the kit. The analysis of these findings revealed that the bottlenecks of the rational use of antibiotics were in all levels of the Benin healthcare system (healthcare professionals, patients and policymakers). Considering the burden the inappropriate use of antibiotics induces for patient and public health, it is imperative to implement actions to improve the use of antibiotics. Based on the literature, a program named “antimicrobial stewardship” permits to coordinate a set of actions for promoting the prudent use of antimicrobials, with the ultimate goal of optimizing clinical outcomes while minimizing unfavorable consequences including resistance selection as well as adverse drug reactions. This program includes various actions for all actors of the healthcare system such as patient education, healthcare professional training and involvement in Infection Prevention and Control strategies, and policymakers’ commitment. The implementation of this kind of program, (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

This work investigated the blind spots of the mechanisms of action of fluoroquinolones (F-QNL) on the Gram-negative bacterium Escherichia coli from two perspectives (1) how cells die and (2) how certain cells manage to survive: the ‘persister cells’. F-QNLs are frequently prescribed against bacterial infections, though the lethal mode of action is still elusive. As a frequent treatment failure is associated with antibiotic resistance, it is critical to better understand the antibiotic action. In this light, the principal aim was to find out more about the putative role of Reactive Oxygen Species (ROS) in F-QNL-dependent killing. Using a single-cell approach, this work enabled to pinpoint the source, timing and level of ROS contribution during F-QNL treatment. Characteristic physiological aspects of persister cells were uncovered. Finally, new aspects of the more-kills-less phenomenon in F-QNLs were revealed., Cette étude porte sur les éléments manquants des connaissances des mécanismes d'action des fluoroquinolones (F-QNL) sur la bactérie Gram-négative Escherichia coli sous deux angles (1) comment les bactéries meurent et (2) comment certaines parviennent à survivre, connu sous le terme de « Persistance ». Les F-QNLs sont fréquemment prescrits contre les infections bactériennes, bien que l'activité létale soit méconnue. Étant donné que le fréquent échec thérapeutique est associé à la résistance aux antibiotiques, il est essentiel de mieux comprendre leur mode d’action. Dans cette optique, l'objectif principal était d'en savoir plus sur l'implication présumée des espèces réactives de l'oxygène (ROS). Avec une approche au niveau de la cellule unique, ces travaux ont permis d'identifier la source, le moment et le degré de contribution des ROS lors du traitement par les F-QNLs. Des aspects physiologiques des cellules persistantes ont été découverts. Enfin, l'énigme du phénomène ‘more-kills-less’ des F-QNLs a été éclairé., Option Biologie moléculaire du Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Biofilms colonize medical devices and are often recalcitrant to antibiotics. Inter-kingdom biofilms, when at least a bacterium and a fungus are co-isolated, increase the likelihood of therapeutic failures. In this work, a three-species in vitro biofilm model including S. aureus, E. coli and C. albicans was used to study the activity of the antibiotics moxifloxacin and meropenem, the antifungal caspofungin, and combinations of them against inter-kingdom biofilms. The culturable cells and total biomass were evaluated to determine the pharmacodynamic parameters of the drug response for the incubation with the drugs alone. The synergic or antagonistic effects (increased/decreased effects) of the combination of drugs were analysed with the highest single agent method. Biofilms were imaged in confocal microscopy after live/dead staining. The drugs had limited activity when used alone against single-, dual- or three-species biofilms. When used in combination, additive effects were observed against single- or dual-species biofilms, and increased effects (synergy) against biomass of three-species biofilms. In addition, the two antibiotics showed different patterns, moxifloxacin being more active when targeting S. aureus and meropenem when targeting E. coli. All these observations were confirmed by confocal microscopy images. Our findings highlight the interest in combining caspofungin with antibiotics against inter-kingdom biofilms.

Medical devices-associated infections are characterised by the formation of a biofilm that encapsulates the microorganisms in an extracellular matrix. The thesis focused on inter-kingdom biofilms, when at least a bacterium and a fungus are present. They are hard to treat infections with high failure rates. Our aim was to determine if the disruption of the biofilm matrix with hydrolytic enzymes could potentiate the activity of the antimicrobials against inter-kingdom biofilms. An in-vitro three-species biofilm model was set up co-culturing the bacteria S. aureus and E. coli, and the fungus C. albicans. The results confirmed that the antimicrobials on their own had a limited activity, but the enzymes permitted the antimicrobials to reduce further the biofilm biomass and C. albicans culturable cells. The inclusion of the strategy proposed in this thesis to current clinical practices could lead to a better outcome of the treatment of inter-kingdom medical device-associated infections., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2021

Sickle cell disease is the most common genetic disease affecting more than five million people worldwide. For its treatment, compounds such as senicapoc and voxelotor have recently proven their efficacy in clinical studies with sickle cell patients. However, their low aqueous solubility and sensitivity to metabolism make them poorly bioavailable by the oral route. This project aims to develop self-emulsifying systems to increase the aqueous solubility and oral bioavailability of senicapoc and voxelotor. Self-emulsifying systems loaded with senicapoc and voxelotor were developed and optimized. These formulations increased both the aqueous solubility and cellular transport of senicapoc and voxelotor through enterocyte monolayers (Caco-2). The lipid formulation loaded with voxelotor significantly increased its oral bioavailability compared to a suspension., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2021

Background: Carbapenem-resistant Acinetobacter baumannii is considered a top priority pathogen by the World Health Organization for combatting increasing antibiotic resistance and development of new drugs. Since it was originally reported in Klebsiella pneumoniae in 2009, the quick spread of the blaNDM-1 gene encoding a New-Delhi metallo-beta-lactamase-1 (NDM-1) is increasingly recognized as a serious threat. This gene is usually carried by large plasmids and has already been documented in diverse bacterial species, including A. baumannii. Here, we report the first detection of a NDM-1-producing A. baumannii strain isolated in Benin. Case presentation: A 31-year-old woman was admitted to a surgical unit with a diagnosis of post-cesarean hematoma. An extensively-drug resistant A. baumannii strain solely susceptible to amikacin, colistin and ciprofloxacin, and resistant to several other antibiotics including ceftazidime, imipenem, meropenem, gentamicin, tobramycin, ceftazidime/ avibactam, and sulfamethoxazole-trimethoprim, was isolated from the wound. Production of NDM-1 was demonstrated by immunochromatographic testing. Whole genome sequencing of the isolate confirmed the presence of blaNDM-1, but also antibiotic resistance genes against multiple beta-lactamases and other classes of antibiotics, in addition to several virulence genes. Moreover, the blaNDM-1 gene was found to be present in a Tn125 transposon integrated on a plasmid. Conclusions: The discovery of this extensively-drug resistant A. baumannii strain carrying blaNDM-1 in Benin is worrying, especially because of its high potential risk of horizontal gene transfer due to being integrated into a transposon located on a plasmid. Strict control and prevention measures should be taken, once NDM-1 positive A. baumannii has been identified to prevent transfer of this resistance gene to other Enterobacterales. Capacity building is required by governmental agencies to provide suitable antibiotic treatment options a

During the last decade, the advent of multi-drug resistant pathogens responsible for skin infections tends to make conventional treatments obsolete. Even though many studies have reported the antimicrobial properties of essential oils (EOs), the inconsistent use of various susceptibility testing methods has made information on antimicrobial potential of many EO varieties fragmentary. Using a single method approach, the objective of this work was to assess and to compare the antibacterial and antifungal properties, against skin pathogens, of EOs extracted from West African aromatic plants. Twenty-three plant samples collected in Benin and Burkina Faso were screened against 20 bacterial and fungal isolates obtained from skin lesions. Activity was evaluated by the determination of minimal inhibitory concentrations (MICs), with readings facilitated by the use of resazurin, a blue dye metabolized into pink resorufin by viable cells. Following this screening, nine EOs were found particularly active with MICs lower than 0.35% v/v. Gas Chromatography-Mass Spectrometry (GC/MS) analysis was used to determine the phytochemical profile of these active EOs which were found exceptionally rich in oxygenated monoterpenes, especially aldehydes, alcohols or phenols and their derivatives. Through this study, we demonstrated that several West African EOs have a significant antimicrobial potential which could, however, be considerably impacted by plant growing or harvesting place due to phytochemical composition variation. These EOs, even if their antimicrobial effects appeared lower than those of conventional antibiotics, constitute easily available mixtures of active compounds and could nevertheless be considered, in the context of increasing multidrug resistance, as complementary or alternative therapies in common skin infections management.

Staphylococcus aureus is an etiological agent involved in a multitude of infectious diseases, ranging from minor skin infections to more serious diseases. Bacterial persisters are subpopulations that adopt a transient phenotype characterized by a non-growing state and a tolerance to lethal concentrations of antibiotics. When the antibiotic treatment is discontinued, persisters give rise to a population that is, again, mostly drug susceptible. Their rare and transient nature has long hampered their experimental study, but there is now rising evidence on their implication in relapses of chronic infections. A switch to a persister phenotype has been suggested to occur inside eukaryotic cells for only very few intracellular bacteria, and the mechanisms leading to their formation are still poorly understood. Given the capacity of S. aureus to promote persistent or recurrent infections, we demonstrated that antibiotics could induce persisters within host cells and thus explain the impossibility of eradicating intracellular reservoirs. Our data further indicate that permissive host cells can host alternatively persistent bacterial forms upon antibiotic exposure, and replicative forms upon antibiotic removal and could therefore constitute a viable reservoir and a source of dissemination. Further transcriptomic analysis contributed to a better understanding of the mechanism of persistence formation. Persisters remained metabolically active and redirect energy-consuming processes to the benefit of multiple stress responses that contribute to the maintenance of vital processes, notably cell wall, DNA and transcription. In non-permissive cells, host cell oxidative stress modulates dormancy of S. aureus persisters, which may then reach different levels of dormancy but remain infectious, unveiling complex strategies developed by S. aureus to cope with the host and antibiotic treatments., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2021

The infection by the human immunodeficiency virus (HIV) is typically treated using a combination of pharmacological agents targeting different stages of the viral cycle. Darunavir is a second-generation protease inhibitor that occupies a place of choice in this therapeutic arsenal and for which an important inter-individual pharmacokinetic variability has been described in the literature. First, we characterized darunavir pharmacokinetics by developing a population model based on data collected in a large cohort of HIV patients. Alpha-1 acid glycoprotein level, sex, and genetic polymorphisms in the CYP3A5 and SLCO3A1 genes were found to be significant predictors of darunavir pharmacokinetics. The model was thoroughly evaluated using internal and external validation techniques. The model also allowed us to simulate the effect of alternative dose regimens in populations representative of clinical practice. A reduction of the standard 800 mg once-daily dosage to 600 or 400 mg once-daily was found to be safe in a large proportion of patients, based on commonly defined pharmacokinetic targets for this molecule. On the other hand, intermittent therapy (five out of seven days) constituted an unsafe option in most subjects. Whether individual patients could benefit from these alternative regimens could be predicted by our model. Additionally, optimal sampling strategies for darunavir were derived, showing how to best design future studies or how to optimize therapeutic monitoring for this drug. Finally, in vitro experiments helped determine the contribution of polymorphisms in the P-glycoprotein gene to the aforementioned variability. These experiments showed that neither the c.1199G>A polymorphism nor the haplotype defined by c.1236C>T, c.2677G>T and c.3435C>T influence intracellular accumulation of darunavir., L'infection par le virus de l'immunodéficience humaine (VIH) est typiquement traitée par une combinaison d'agents pharmacologiques ciblant différentes étapes du cycle viral. Le darunavir est un inhibiteur de protéase de seconde génération qui occupe une place de choix dans cet arsenal thérapeutique et pour lequel une importante variabilité pharmacocinétique inter-individuelle a été décrite dans la littérature. Premièrement, nous avons caractérisé la pharmacocinétique du darunavir en développant un modèle de population sur base de données collectées dans une large cohorte de patients VIH-positifs. Il a été démontré que le taux d’alpha-1 glycoprotéine acide, le sexe, et des polymorphismes dans les gènes CYP3A5 et SLCO3A1 étaient des prédicteurs significatifs de la pharmacocinétique du darunavir. Le modèle a été évalué en profondeur par le biais de techniques de validation interne et externe. Le modèle a également permis de simuler l’effet de régimes posologiques alternatifs dans des populations représentatives de la pratique clinique. Une réduction de la dose journalière standard de 800 mg à 600 ou 400 mg a été démontrée sure pour une large proportion de patients, sur base de cibles pharmacocinétiques couramment définies pour cette molécule. En revanche, la thérapie intermittente (cinq jours sur sept) ne constituait pas une option sure pour la plupart des sujets. L’existence d’un bénéfice individuel de ces stratégies alternatives pourrait être prédite par notre modèle. Par ailleurs, des stratégies de prélèvement optimales pour le darunavir ont été définies, montrant comment concevoir de futures études de manière optimale ou comment optimiser le monitoring thérapeutique pour ce médicament. Enfin, des expériences in vitro ont permis d’établir la contribution de polymorphismes dans le gène de la P-glycoprotéine à la variabilité précédemment mentionnée. Ces expériences ont montré que ni le polymorphisme c.1199G>A ni l’haplotype défini par c.1236C>T, c.2677G>T et c.3435C>T, (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2021

Background: Ceftazidime and imipenem have been increasingly used to treat Acute Exacerbations of Chronic Obstructive Pulmonary Disease (AECOPD) due to their extended-spectrum covering Pseudomonas aeruginosa. This study aims to describe the population pharmacokinetic (PK) and pharmacodynamic (PD) target attainment for ceftazidime and imipenem in patients with AECOPD. Methods: We conducted a prospective PK study at Bach Mai Hospital (Viet Nam). A total of 50 (ceftazidime) and 44 (imipenem) patients with AECOPD were enrolled. Population PK analysis was performed using Monolix 2019R1 and Monte Carlo simulations were conducted to determine the optimal dose regimen with respect to the attainment of 60% and 40% fT>MIC for ceftazidime and imipenem, respectively. A dosing algorithm was developed to identify optimal treatment doses. Results: Ceftazidime and imipenem PK was best described by a one-compartment population model with a volume of distribution and clearance of 23.7 L and 8.74 L/h for ceftazidime and 15.1 L and 7.88 L/h for imipenem, respectively. Cockcroft–Gault creatinine clearance represented a significant covariate affecting the clearance of both drugs. Increased doses with prolonged infusion were found to cover pathogens with reduced susceptibility. Conclusions: This study describes a novel and versatile three-level dosing algorithm based on patients’ renal function and characteristic of the infective pathogen to explore ceftazidime and imipenem optimal regimen for AECOPD

In Belgium, linezolid is indicated for pneumonia and skin and soft tissue infections, but is more broadly used, due to its oral bioavailability and activity against multiresistant organisms. This could increase the risk of adverse drug reactions (ADR), notably hematological disorders (anemia, thrombocytopenia), neuropathy, or lactic acidosis. We analyzed linezolid clinical use in relationship with occurrence of ADR in Belgian hospitals and highlighted risk factors associated with the development of thrombocytopenia. A retrospective analysis of electronic medical records and laboratory tests of adult patients treated with linezolid in four Belgian hospitals in 2016 allowed the collection of ADR for 248 linezolid treatments. Only 19.7% of indications were in-label. ADR included 43 thrombocytopenia, 17 anemia, 4 neuropathies, and 4 increases in lactatemia. In a multi-variate analysis, risk factors of thrombocytopenia were a treatment duration > 10 days, a glomerular filtration rate < 60 mL/min, and a Charlson index ≥ 4. Off-label use of linezolid is frequent in Belgium, and ADR more frequent than reported in the summary of product characteristics, but not statistically associated with any indication. This high prevalence of ADR could be related to a high proportion of patients presenting risk factors in our population, highlighting the importance of detecting them prospectively

Background: Periprosthetic joint infections (PJI) are catastrophic complications of joint replacement surgeries. Current treatment options are unsatisfactory due to poor functional results following staged-replacements and high failure rates after debridement, implant retention and antibiotic therapy (DAIR). New treatment options have been explored in-vitro, and require the development of specific in-vivo models mimicking PJI. Here we describe a novel model of infected knee arthroplasty in New-Zealand White (NZW) rabbits and examined it suitability to evaluate antibiotic pharmacokinetics or treatment efficacy, focusing on a standard DAIR procedure. Materials: 15 NZW rabbits were assigned to three groups: vancomycin pharmacokinetics (A), infection (B), and DAIR (C). All rabbits received a unicompartimental tibial arthroplasty using a custom 3D-printed TI-6Al-4V implant. The joint of groups B and C rabbits were inoculated with 5 log10 CFU of ATCC33591 MRSA injected in the knee at the end of the surgery. All rabbits benefitted from a post-operative analgesia with transcutaneous fentanyl. One week after the initial surgery, the joint space of group C rabbits were surgically debrided. Rabbits of groups A and C received i.v. injections of vancomycin 50mg/kg b.i.d. for one week. Serum concentrations of vancomycin were measured in group A rabbits after the first and fifth doses. Rabbits were euthanized two weeks after the initial surgery. Bacterial counts from joint tissues and the implant were obtained in groups B and C rabbits. Results: Vancomycin pharmacokinetics: The average AUC0-12 was of 213.0mg*h/L for the first dose FIG.I) and 207.8mg*h/L for the fifth dose (FIG.II), with complete elimination of the drug in 12h. As the MIC for vancomycin of ATCC33591 is 1mg/L, the AUC0-24/MIC ratio is above 400, i.e. the usual pharmacokinetic/pharmacodynamic target against staphylococci. Infection and DAIR (FIG.III): All animals of the B and C groups were infected. CFU counts were h

Biofilms are an important medical burden, notably for patients with orthopaedic device-related infections. When polymicrobial, these infections are more lethal and recalcitrant. Inter-kingdom biofilm infections are poorly understood and challenging to treat. Here, an in vitro three-species model including Staphylococcus aureus, Escherichia coli and Candida albicans was developed, to represent part of the diversity observed in orthopaedic infections or other clinical contexts. The importance of fungal hyphae for biofilm formation and virulence factor expression was explored. Two protocols were set up, allowing, or not, for hyphal formation. Culturable cells and biomass were characterised in both models, and biofilms were imaged in bright-field, confocal and electron microscopes. The expression of genes related to virulence, adhesion, exopolysaccharide synthesis and stress response was analysed in early-stage and mature biofilms. It was found that biofilms enriched in hyphae had larger biomass and showed higher expression levels of genes related to bacterial virulence or exopolysaccharides synthesis.

Prosthetic joint infection (PJI) is a devastating complication of joint replacement surgeries, affecting 0.5 to 2% of patients following a hip or a knee arthroplasty. Its treatment is complicated by the rapid formation of bacterial biofilms on the implants’ surfaces. Biofilms are bacterial communities enveloped in a complex self-produced matrix that isolates bacteria from their environment, leading to an increased tolerance to antimicrobials. Therefore, new therapeutic options targeting biofilms are in development to improve the outcome of PJI. The objectives of this work were to investigate biofilm disruption using a standard irrigation technique (pulsed-lavage) or an enzyme combination targeting the matrix of the biofilms, and its impact on the susceptibility of bacteria to antibiotics. Pulsed-lavage and the enzymatic combination were studied in two successive in-vitro studies, using a model of biofilms grown on titanium alloy substrates. Our experiments showed that the successive application of biofilm disrupting treatments and antibiotics, used at clinically relevant concentrations, was synergistic. Considering these encouraging results, an in-vivo model of PJI was developed to study the use of the enzymatic combination as an adjuvant to a treatment with debridement, systemic antibiotics, and implant retention. Regrettably, the addition of the enzymatic combination did not appear to improve the outcome of the treatment. However, these experiments open new perspectives to optimize the use of enzymes in this setting, and the tools developed for this work could prove useful to investigate the application of other strategies for the treatment of PJI., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2021

Intracellular bacteria are poorly responsive to antibiotic treatment. Pharmacological studies are thus needed to determine the antibiotics which are the most potent or effective against intracellular bacteria as well as to explore the reasons for poor bacterial responsiveness. An in vitro pharmacodynamic model is described, consisting of (1) phagocytosis of preopsonized bacteria by eukaryotic cells, (2) elimination of noninternalized bacteria with gentamicin, (3) incubation of infected cells with antibiotics, and (4) determination of surviving bacteria by viable cell counting and normalization of the counts based on sample protein content. The use of strains expressing fluorescent proteins under the control of an inducible promoter allows to follow intracellular bacterial division at the individual level and therefore to monitor bacterial persisters that do not multiply anymore.

Staphylococcus aureus biofilms are poorly responsive to antibiotics. Underlying reasons include a matrix effect preventing drug access to embedded bacteria, or the presence of dormant bacteria with reduced growth rate. Using 18 clinical isolates previously characterized for their moxifloxacin-resistant and moxifloxacin-persister character in stationary-phase culture, we studied their biofilm production and matrix composition and the anti-biofilm activity of moxifloxacin. Biofilms were grown in microtiter plates and their abundance quantified by crystal violet staining and colony counting; their content in polysaccharides, extracellular DNA and proteins was measured. Moxifloxacin activity was assessed after 24 h of incubation with a broad range of concentrations to establish full concentration-response curves. All clinical isolates produced more biofilm biomass than the reference strain ATCC 25923, the difference being more important for those with high relative persister fractions to moxifloxacin, most of which being also resistant. High biofilm producers expressed icaA to higher levels, enriching the matrix in polysaccharides. Moxifloxacin was less potent against biofilms from clinical isolates than from ATCC 25923, especially against moxifloxacin-resistant isolates with high persister fractions, which was ascribed to a lower concentration of moxifloxacin in these biofilms. Time-kill curves in biofilms revealed the presence of a moxifloxacin-tolerant subpopulation, with low multiplication capacity, whatever the persister character of the isolate. Thus, moxifloxacin activity depends on its local concentration in biofilm, which is reduced in most isolates with high-relative persister fractions due to matrix effects, and insufficient to kill resistant isolates due to their high MIC.

In Belgium, linezolid is indicated for pneumonia and skin and soft tissue infections, but is more broadly used, due to its oral bioavailability and activity against multiresistant organisms. This could increase the risk of adverse drug reactions (ADR), notably hematological disorders (anemia, thrombocytopenia), neuropathy, or lactic acidosis. We analyzed linezolid clinical use in relationship with occurrence of ADR in Belgian hospitals and highlighted risk factors associated with the development of thrombocytopenia. A retrospective analysis of electronic medical records and laboratory tests of adult patients treated with linezolid in four Belgian hospitals in 2016 allowed the collection of ADR for 248 linezolid treatments. Only 19.7% of indications were in-label. ADR included 43 thrombocytopenia, 17 anemia, 4 neuropathies, and 4 increases in lactatemia. In a multivariate analysis, risk factors of thrombocytopenia were a treatment duration > 10 days, a glomerular filtration rate < 60 mL/min, and a Charlson index ≥ 4. Off-label use of linezolid is frequent in Belgium, and ADR more frequent than reported in the summary of product characteristics, but not statistically associated with any indication. This high prevalence of ADR could be related to a high proportion of patients presenting risk factors in our population, highlighting the importance of detecting them prospectively., SCOPUS: ar.j, info:eu-repo/semantics/published

Staphylococcus aureus may cause relapsing infections. We previously showed that S. aureus SH1000 surviving intracellularly to bactericidal antibiotics are persisters. Here, we used 54 non-duplicate clinical isolates to assess links between persistence, resistance evolution, and intracellular survival, using moxifloxacin throughout as test bactericidal antibiotic. The relative persister fraction (RPF: percentage of inoculum surviving to 100× MIC moxifloxacin in stationary phase culture for each isolate relative to ATCC 25923) was determined to categorize isolates with low (≤10) or high (>10) RPF. Evolution to resistance (moxifloxacin MIC ≥ 0.5 mg/L) was triggered by serial passages at 0.5× MIC (with daily concentration readjustments). Intracellular moxifloxacin maximal efficacy (Emax) was determined by 24 h concentration-response experiments [pharmacodynamic model (Hill-Langmuir)] with infected THP-1 monocytes exposed to moxifloxacin (0.01 to 100× MIC) after phagocytosis. Division of intracellular survivors was followed by green fluorescence protein dilution (FACS). Most (30/36) moxifloxacin-susceptible isolates showed low RPF but all moxifloxacin-resistant (n = 18) isolates harbored high RPF. Evolution to resistance of susceptible isolates was faster for those with high vs. low RPF (with SOS response and topoisomerase-encoding genes overexpression). Intracellularly, moxifloxacin Emax was decreased (less negative) for isolates with high vs. low RPF, independently from resistance. Moxifloxacin intracellular survivors were non-dividing. The data demonstrate and quantitate persisters in clinical isolates of S. aureus, and show that this phenotype accelerates resistance evolution and is associated with intracellular survival in spite of high antibiotic concentrations. Isolates with high RPF may represent a possible cause of treatment failure not directly related to resistance in patients receiving active antibiotics.

BACKGROUND: Increasing multidrug resistance rates in Neisseria gonorrhoeae have raised concerns and an urgent call for new antibiotics for treatment of gonorrhoea. Several decades of subdued drug development in this field and the recent failures of two new antibiotics to show non-inferiority compared with the current first-line antibiotics ceftriaxone plus azithromycin highlight the need for improved preclinical tools to predict clinical outcome of new drugs in the development process. OBJECTIVES: To summarize current pharmacokinetic/pharmacodynamic (PK/PD) knowledge and dose-finding strategies for antibiotics against gonorrhoea. SOURCES: Literature review of published papers and discussions by global experts at a special workshop on this topic. CONTENT: We review current knowledge of gonococcal specific PK/PD principles and provide an update on new in vitro and in vivo models to correlate drug exposure with clinical outcome, and identify challenges and gaps in gonococcal therapeutic research. IMPLICATIONS: Identifying the ideal antimicrobial agent and dose for treating uncomplicated urogenital and pharyngeal gonococcal disease requires appropriate validated non-clinical PK/PD models. Recent advances in adapting in vitro and in vivo models for use in gonorrhoea are an important step for enabling the development of new drugs with reduced risk of failure in Phase 3 clinical development and diminish the risk of emergence of resistance.

Burkholderia pseudomallei, Yersinia pestis and Francisella tularensis are facultative intracellular bacteria causing life-threatening infections. We have (a) compared the activity of finafloxacin (a fluoroquinolone in development showing improved activity at acidic pH) with that of ciprofloxacin, levofloxacin and imipenem against the extracellular and intracellular (THP-1 monocytes) forms of infection by attenuated surrogates of these species (B. thailandensis, Y. pseudotuberculosis, F. philomiragia) and (b) assessed finafloxacin cellular pharmacokinetics (accumulation, distribution, efflux). Bacteria in broth or in infected monocytes were exposed to antibiotics at pH 7.4 or 5.5 for 24 hr. Maximal relative efficacies (E) and static concentrations (C) were calculated using the Hill equation (concentration-response curves). Finafloxacin pharmacokinetics in cells at pH 7.4 or 5.5 was investigated using C-labelled drug. Extracellularly, all drugs sterilized the cultures, with finafloxacin being two to six times more potent at acidic pH. Intracellularly, E reached the limit of detection (4-5 log cfu decrease) for finafloxacin against all species, but only against B. thailandensis and F. philomiragia for ciprofloxacin and levofloxacin, while imipenem caused less than 2 log cfu decrease for all species. At acid pH, C shifted to two to five times lower values for finafloxacin and to one to four times higher values for the other drugs. Finafloxacin accumulated in THP-1 cells by approximately fivefold at pH 7.4 but up to 20-fold at pH 5.5, and distributed in the cytosol. Fluoroquinolones have proven to be effective in reducing the intracellular reservoirs of B. thailandensis, Y. pseudotuberculosis and F. philomiragia, with finafloxacin demonstrating an additional advantage in acidic environments.

Resistance is notoriously high in Asia but may not entirely explain therapeutic failures. Specific modes of bacterial life, such as biofilm or intracellular survival, may also contribute to the persistent and/or recurrent character of infections. Most isolates form biofilm and many survive and even thrive intracellularly. We collected 36 nonduplicate isolates (including 18 methicillin-resistant ) from patients with clinical evidence of persistent or recurrent infections in a large tertiary Vietnamese hospital. We examined their antibiotic resistance profile (minimal inhibitory concentration determination) and clonal relatedness ( and typing, pulsed field gel electrophoresis profiles). We then assessed the activity of moxifloxacin in both biofilms and infected phagocytes (moxifloxacin previously proved to be one of the most active antibiotics against reference strains in these models). types t189 and t437 and group I were the most frequent. Among the 36 isolates, 30 were multidrug resistant but 30 were recovered from patients having received an active drug. All tested isolates produced biofilm and survived inside phagocytes. At its human , moxifloxacin was inactive on biofilms made by moxifloxacin-susceptible as well as moxifloxacin-resistant isolates. It caused only a modest intracellular colony-forming unit decrease against moxifloxacin-susceptible isolates and was inactive against those resistant to moxifloxacin. While our data confirm for this collection the high resistance levels and prevalence of endemic or types in Asia, they show that tolerance in both biofilm and phagocytes are correlated and markedly limit moxifloxacin activity, which goes in line with the suggested role of these modes of life in persistence or recurrence of infections.

BACKGROUND: Surgical site infections are related to high morbidity, mortality and healthcare costs. Because the emergence of multidrug-resistant bacteria in hospitals is becoming a worldwide challenge for surgeons who treat healthcare-associated infections, we wished to identify the causative agents involved in these infections and the rate of multidrug-resistant bacteria in six public hospitals in Benin. METHODS: Using standard microbiological procedures, we processed pus specimens collected from obstetrics and gastrointestinal surgery wards. Mass spectrometry (MALDI-TOF) was used for confirmation. For the antibiotic susceptibility test, we first used the Kirby-Bauer disk diffusion method. The secondary test (by microdilution) used the Beckton Dickinson Phoenix automated system (Becton Dickinson Diagnostic, USA). RESULTS: We included 304 patients, whose median length of stay was 9 days. A total of 259 wound swabs (85.2%) had positive aerobic bacterial growth. In obstetrics, S. aureus (28.5%, n = 42) was the most common isolate. In contrast, Gram-negative bacteria (GNB) were predominant in gastrointestinal surgery, the most dominant being E.coli (38.4%, n = 31). Overall, 90.8% (n = 208) of aerobic bacteria were multidrug resistant. Two-thirds of S. aureus (65.3%, n = 32) were methicillin-resistant Staphylococcus aureus (MRSA), three of which carried both MRSA and induced clindamycin resistance (ICR). GNB showed high resistance to ceftazidime, ceftriaxone and cefepime. Extended-spectrum beta-lactamases were presented by 69.4% of E.coli (n = 43/62) and 83.3% of K. pneumoniae (n = 25/30). Overall, twelve Gram-negative bacteria (5.24%) showed resistance to at least one carbapenem. No isolates showed a wild-type susceptible phenotype. CONCLUSION: This study shows the alarming prevalence of multidrug-resistant organisms from surgical site infections in Benin hospitals. To reduce the spread of such bacteria in Benin, periodic surveillance of surgical site infections and st