Spotting method as a high throughput alternative to the conventional spread plating method conventional spread plating method for bacterial time killexperiments

International audienc

PKPD Modeling of the Inoculum Effect of Acinetobacter baumannii on Polymyxin B in vivo

International audienceThe reduction in antimicrobial activity at high bacterial counts is a microbiological phenomenon known as the inoculum effect (IE). In a previous in vitro study, a significant IE was observed for polymyxin B (PMB) against a clinical isolate of Acinetobacter baumannii , and well described by a new pharmacokinetic-pharmacodynamic model. Few in vivo studies have investigated the impact of inoculum size on survival or antibiotic efficacy. Therefore, our objective was to confirm the influence of inoculum size of this A. baumannii clinical isolate on PMB in vivo effect over time. Pharmacokinetics and pharmacodynamics of PMB after a single subcutaneous administration (1, 15 and 40 mg/kg) were studied in a neutropenic murine thigh infection model. The impact of A. baumannii inoculum size (10 5 , 10 6 and 10 7 CFU/thigh) on PMB efficacy was also evaluated. In vivo PMB PK was well described by a two-compartment model including saturable absorption from the subcutaneous injection site and linear elimination. The previous in vitro PD model was modified to adequately describe the decrease of PMB efficacy with increased inoculum size in infected mice. The IE was modeled as a decrease of 32% in the in vivo PMB bactericidal effect when the starting inoculum increases from 10 5 to 10 7 CFU/thigh. Although not as important as previously characterized in vitro an IE was confirmed in vivo

Characterization of Pseudomonas aeruginosa resistance to ceftolozane-tazobactam due to ampC and/or ampD mutations observed during treatment using semi-mechanistic PKPD modeling

A double ampC (AmpC G183D ) and ampD (AmpD H157Y ) genes mutations have been identified by whole genome sequencing in a Pseudomonas aeruginosa (PaS) that became resistant (PaR) in a patient treated by ceftolozane/tazobactam (C/T). To precisely characterize the respective contributions of these mutations on the decreased susceptibility to C/T and on the parallel increased susceptibility to imipenem (IMI), mutants were generated by homologous recombination in PAO1 reference strain (PAO1- AmpC G183D , PAO1-AmpD H157Y , PAO1-AmpC G183D /AmpD H157Y ) and in PaR (PaR-AmpC PaS /AmpD PaS ). Sequential time-kill curve experiments were conducted on all strains and analyzed by semi-mechanistic PKPD modeling. A PKPD model with adaptation successfully described the data, allowing discrimination between initial and time-related (adaptive resistance) effects of mutations. With PAO1 and mutant-derived strains, initial EC 50 values increased by 1.4, 4.1, and 29-fold after AmpC G183D , AmpD H157Y and AmpC G183D /AmpD H157Y mutations, respectively. EC 50 values were increased by 320, 12.4, and 55-fold at the end of the 2 nd experiment. EC 50 of PAO1-AmpC G183D /AmpD H157Y was higher than that of single mutants at any time of the experiments. Within the PaR clinical background, reversal of AmpC G183D , and AmpD H157Y mutations led to an important decrease of EC 50 value, from 80.5 mg/L to 6.77 mg/L for PaR and PaR-AmpC PaS /AmpD PaS , respectively. The effect of mutations on IMI susceptibility mainly showed that the AmpC G183D mutation prevented the emergence of adaptive resistance. The model successfully described the separate and combined effect of AmpC G183D and AmpD H157Y mutations against C/T and IMI, allowing discrimination and quantification of the initial and time-related effects of mutations. This method could be reproduced in clinical strains to decipher complex resistance mechanisms

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

Human airway and corneal epithelial cells, which are critically altered during chronic infections mediated by Pseudomonas 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 promote 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 ZAKα- 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 ZAKα inhibitors. Altogether, our results show the importance of P. aeruginosa virulence factor EXOA at promoting NLRP1-dependent epithelial damage and identify ZAKα as a critical sensor of virulence-inactivated EEF2.Published versionThis project was supported by the ATIP-Avenir program (to E. Meunier), Fondation pour la Recherche Médicale “Amorçage Jeunes Equipes” (AJE20151034460 to E. Meunier), the Agence Nationale de la Recherche (ANR Psicopak to E. Meunier), the Agence nationale de recherche sur le sida et les hépatites-Maladies infectieuses émergentes (to E. Meunier), the European Research Council (StG INFLAME 804249 to E. Meunier), the European Society of Clinical Microbiology and Infectious Diseases (to R. Planès), Invivogen-Conventions industrielles de formation par la recherche PhD grant (to M. Pinilla), Vaincre La Mucoviscidose, and Region Occitanie (Groupement de Recherche pour des Applications INnovantes avec les Entreprises) grants to C. Cougoule

Caspase-1-driven neutrophil pyroptosis and its role in host susceptibility to Pseudomonas aeruginosa

International audienceMultiple regulated neutrophil cell death programs contribute to host defense against infections. However, despite expressing all necessary inflammasome components, neutrophils are thought to be generally defective in Caspase-1-dependent pyroptosis. By screening different bacterial species, we found that several Pseudomonas aeruginosa ( P . aeruginosa ) strains trigger Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P . aeruginosa enhanced neutrophil death to Caspase-1-dependent pyroptosis, suggesting that these exotoxins interfere with this pathway. Mechanistically, P . aeruginosa Flagellin activates the NLRC4 inflammasome, which supports Caspase-1-driven interleukin (IL)-1β secretion and Gasdermin D (GSDMD)-dependent neutrophil pyroptosis. Furthermore, P . aeruginosa -induced GSDMD activation triggers Calcium-dependent and Peptidyl Arginine Deaminase-4-driven histone citrullination and translocation of neutrophil DNA into the cell cytosol without inducing extracellular Neutrophil Extracellular Traps. Finally, we show that neutrophil Caspase-1 contributes to IL-1β production and susceptibility to pyroptosis-inducing P . aeruginosa strains in vivo . Overall, we demonstrate that neutrophils are not universally resistant for Caspase-1-dependent pyroptosis

From Triclosan toward the Clinic: Discovery of Nonbiocidal, Potent FabI Inhibitors for the Treatment of Resistant Bacteria

In this paper, we present some elements of our optimization program to decouple triclosan’s specific FabI effect from its nonspecific cytotoxic component. The implementation of this strategy delivered highly specific, potent, and nonbiocidal new FabI inhibitors. We also disclose some preclinical data of one of their representatives, <b>83</b>, a novel antibacterial compound active against resistant staphylococci and some clinically relevant Gram negative bacteria that is currently undergoing clinical trials