Biology subjects

Buyck, J. M.

Publications and source records attributed to Buyck, J. M..

6 recordsLinked to original sources

bioRxiv2026

Replacing In Vivo Experiments for PK/PD Target Determination Through In Vitro Time-Kill Experiments and PK/PD Modelling Incorporating Inter-strain Variability: Application to Meropenem Against Pseudomonas aeruginosa

Background. Optimal antibiotic dosing regimens depend on the pharmacokinetic/pharmacodynamic (PK/PD) index that best predicts antibacterial efficacy. PK/PD targets are traditionally determined using murine infection models based on a limited number of bacterial isolates. Objective. This study aimed to investigate whether animal experiments could be replaced by in vitro time-kill experiments performed on a large collection of clinical isolates and analyzed using a modelling approach accounting for inter-strain variability. The proposed framework was evaluated using meropenem against Pseudomonas aeruginosa. Materials and Methods. In vitro time-kill experiments were performed on 66 clinical isolates of P. aeruginosa. A population pharmacodynamic model was developed from experimental data. A murine pharmacokinetic model was reproduced from literature and combined with the pharmacodynamic model to simulate in vivo bacterial burden over time. The relationships between simulated bacterial counts at 24 h and the three main PK/PD indices (fCmax/MIC, fAUC/MIC and %fT>MIC) were characterized using nonlinear mixed-effects Imax models. Results. The PK/PD index showing the strongest correlation with meropenem efficacy at 24 h was %fT>MIC (R2 = 0.989), compared with fAUC/MIC (R2 = 0.373) and fCmax/MIC (R2 = 0.284). These findings are consistent with previous studies using murine thigh infection models. The %fT>MIC target required to achieve a 2-log CFU reduction was estimated at 44%, with substantial inter-strain variability (10th and 90th percentiles: 27% and 71%, respectively). Conclusions. Using meropenem against P. aeruginosa as a proof of concept, we demonstrate that in vitro time-kill experiments combined with pharmacometric modelling can identify the same PK/PD efficacy targets as animal infection models. Moreover, performing experiments on a large panel of clinical isolates enables the quantification of inter-strain variability in PK/PD targets, providing information that may improve their translation to clinical dosing optimization.

pharmacology and toxicology↗

bioRxiv2026

Model-based evaluation of Targeted-Antibacterial-Plasmids (TAPs) transfer kinetics and resensitization of pOXA-48 carbapenem-resistant Escherichia coli

Background Targeted-Antibacterial Plasmids (TAPs) are engineered mobile genetic elements that use bacterial conjugation to deliver selective CRISPR/Cas9 antibacterial activity against a specific target strain. Yet, the efficiency of TAPs is typically evaluated at a single time point, whereas the success of TAP-mediated resensitization critically depends on the dynamics of plasmid transfer and the complex interactions between bacterial subpopulations. This is the first study to evaluate the efficiency of a conjugation-based antibacterial approach at the subpopulation level, using an analytical framework analogous to that used for conventional antibiotics. Here, we investigate which process limits resensitization by TAPF-dCas9-OXA48: plasmid delivery, dCas9 activity, or the emergence of refractory and escape populations. Methods We fitted a mechanistic model of five interacting subpopulations (donors, recipients, transconjugants, escapers, and recusants) to 44 longitudinal conjugation experiments and used the fitted model to explore a range of biologically relevant scenarios. Results Using longitudinal conjugation data spanning 24 h, we show that up to 24% of recipients become recusants within 24h, refractory to further conjugation via entry exclusion, while secondary transconjugant emergence stays below 0.01%. Overall resensitization efficiency reaches up to 80%. Conclusion Plasmid transfer, rather than dCas9 repression, therefore appears to be the main bottleneck limiting the efficiency of TAPF-dCas9-OXA48 efficiency. These results identify plasmid delivery as a key engineering target for improving the performance of future TAPs.

bioinformatics↗

bioRxiv2026

Zeta potential measurements of Escherichia coli to evaluate colistin susceptibility and gain insight in resistance mechanisms

Colistin resistance in Escherichia coli arises from outer membrane (OM) remodeling that reduces surface charge and thereby lowers drug binding affinity. In this study, we investigated the zeta potential of E. coli strains with colistin minimum inhibitory concentrations (MICs) ranging from 0.125 to 16 mg/L, following EDTA treatment to chelate divalent cations and unmask intrinsic surface charge. Zeta potential was measured across pH values from 3 to 7, revealing consistent pH-dependent trends and an average 18.5 mV reduction in surface charge with increasing MIC. At pH 7, zeta potential strongly correlated with colistin resistance (R2 = 0.919), and this correlation was further strengthened following exposure to sub-inhibitory colistin (1/8 MIC; R2 = 0.9975). Notably, resistant strains carrying mcr-1 or mcr-4 exhibited significant shifts toward less negative surface charges after sub-MIC exposure, whereas the susceptible parental strain remained unchanged. However, mcr-1 mRNA expression did not consistently increase under these conditions, highlighting a disconnect between transcriptional responses and phenotypic charge alterations. These findings suggest that regulatory pathways beyond mcr-1 transcription, including stress-induced lipid A remodeling, contribute to OM charge modulation. Overall, zeta potential profiling provides a rapid and sensitive readout of resistance-associated membrane alterations and complements molecular assays by capturing functional phenotypes. This approach offers a valuable research tool for dissecting colistin resistance mechanisms and may inform future strategies for monitoring bacterial adaptation to last-resort antibiotics.

microbiology↗

bioRxiv2025

Optimizing Antimicrobial Susceptibility Testing: Cost and Environmental Benefits of MIC Volume Reduction

The determination of Minimum Inhibitory Concentrations (MICs) is essential for evaluating antimicrobial efficacy, guiding both clinical treatment decisions and drug development. The standard broth microdilution method is widely used but requires significant reagent volumes, which can be limiting when working with novel or expensive antimicrobials. This study assesses the feasibility of reducing assay volumes without compromising MIC accuracy. We compared the MIC values obtained in standard 96-well plates (200 {micro}L) to those in 384-well plates with reduced volumes (30-50 {micro}L) for a range of Gram-negative and Gram-positive bacteria, as well as yeast species. Our results demonstrate that except for micafungin, MIC values obtained with reduced volumes remained within the acceptable variability ranges defined by EUCAST and CLSI. Evaporation, a potential source of bias in smaller volumes, was mitigated by conducting experiments in a water-saturated atmosphere. Furthermore, reduced assay volumes significantly lowered material costs and antimicrobial consumption, particularly for expensive drugs such as cefiderocol. This miniaturization approach offers a cost-effective, high-throughput alternative for antimicrobial susceptibility testing while maintaining accuracy and reproducibility.

microbiology↗

bioRxiv2023

EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption upon Pseudomonas infection

The intracellular inflammasome complex have been implicated in the maladaptive tissue damage and inflammation observed in chronic Pseudomonas aeruginosa infection. Human airway and corneal epithelial cells, which are critically altered during chronic infections mediated by P. aeruginosa, specifically express the inflammasome sensor NLRP1. Here, together with a companion study, we report that the NLRP1 inflammasome detects Exotoxin A (EXOA), a ribotoxin released by P. aeruginosa Type 2 Secretion System (T2SS) during chronic infection. Mechanistically, EXOA-driven Eukaryotic Elongation Factor 2 (EEF2) ribosylation and covalent inactivation promotes ribotoxic stress and subsequent NLRP1 inflammasome activation, a process shared with other EEF2-inactivating toxins, Diphtheria Toxin and Cholix Toxin. Biochemically, irreversible EEF2 inactivation triggers ribosome stress-associated kinases ZAK- and P38-dependent NLRP1 phosphorylation and subsequent proteasome-driven functional degradation. Finally, Cystic Fibrosis cells from patients exhibit exacerbated P38 activity and hypersensitivity to EXOA-induced ribotoxic stress-dependent NLRP1 inflammasome activation, a process inhibited by the use of ZAK inhibitors. Altogether, our results show the importance of P. aeruginosa virulence factor EXOA at promoting NLRP1-dependent epithelial damage and identify ZAK as a critical sensor of virulence-inactivated EEF2. KEY POINTSO_LIP. aeruginosa induces NLRP1-dependent pyroptosis in human corneal and nasal epithelial cells C_LIO_LIP. aeruginosa Exotoxin A (EXOA) and other EEF2-inactivating bacterial exotoxins activate the human NLRP1 inflammasome C_LIO_LIEEF2 inactivation promotes ribotoxic stress response and ZAK kinase-dependent NLRP1 inflammasome activation. C_LIO_LIBronchial epithelial cells from Cystic Fibrosis patients show extreme sensitivity to ribotoxic stress-dependent NLRP1 inflammasome activation in response to Exotoxin A C_LIO_LIP38 and ZAK inhibition protects Cystic Fibrosis epithelial cell from EXOA-induced pyroptosis C_LI

immunology↗

bioRxiv2019

Type-3 Secretion System-induced pyroptosis protects Pseudomonas against cell-autonomous immunity

Inflammasome-induced pyroptosis comprises a key cell-autonomous immune process against intracellular bacteria, namely the generation of dying cell structures. These so-called pore-induced intracellular traps (PITs) entrap and weaken intracellular microbes. However, the immune importance of pyroptosis against extracellular pathogens remains unclear. Here, we report that Type-3 secretion system (T3SS)-expressing Pseudomonas aeruginosa (P. aeruginosa) escaped PIT immunity by inducing a NLRC4 inflammasome-dependent macrophage pyroptosis response in the extracellular environment. To the contrary, phagocytosis of Salmonella Typhimurium promoted NLRC4-dependent PIT formation and the subsequent bacterial caging. Remarkably, T3SS-deficient Pseudomonas were efficiently sequestered within PIT-dependent caging, which favored exposure to neutrophils. Conversely, both NLRC4 and caspase-11 deficient mice presented increased susceptibility to T3SS-deficient P. aeruginosa challenge, but not to T3SS-expressing P. aeruginosa. Overall, our results uncovered that P. aeruginosa uses its T3SS to overcome inflammasome-triggered pyroptosis, which is primarily effective against intracellular invaders.\n\nImportanceAlthough innate immune components confer host protection against infections, the opportunistic bacterial pathogen Pseudomonas aeruginosa (P. aeruginosa) exploits the inflammatory reaction to thrive. Specifically the NLRC4 inflammasome, a crucial immune complex, triggers an Interleukin (IL)-1{beta} and -18 deleterious host response to P. aeruginosa. Here, we provide evidence that, in addition to IL-1 cytokines, P. aeruginosa also exploits the NLRC4 inflammasome-induced pro-inflammatory cell death, namely pyroptosis, to avoid efficient uptake and killing by macrophages. Therefore, our study reveals that pyroptosis-driven immune effectiveness mainly depends on P. aeruginosa localization. This paves the way toward our comprehension of the mechanistic requirements for pyroptosis effectiveness upon microbial infections and may initiate targeted approaches in order to ameliorate the innate immune functions to infections.\n\nGraphical abstractMacrophages infected with T3SS-expressing P. aeruginosa die in a NLRC4-dependent manner, which allows bacterial escape from PIT-mediated cell-autonomous immunity and neutrophil efferocytosis. However, T3SS-deficient P. aeruginosa is detected by both NLRC4 and caspase-11 inflammasomes, which promotes bacterial trapping and subsequent efferocytosis of P. aeruginosa-containing-PITs by neutrophils.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC=\"FIGDIR/small/650333v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (15K):\norg.highwire.dtl.DTLVardef@17e05c4org.highwire.dtl.DTLVardef@aca55forg.highwire.dtl.DTLVardef@9d2853org.highwire.dtl.DTLVardef@1c275f9_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗