Biology subjects

Royet, K.

Publications and source records attributed to Royet, K..

3 recordsLinked to original sources

bioRxiv2025

Exploring Phage-Staphylococcus aureus Host Dynamics Through Innovative In Vitro Experiment and Pharmacokinetic/pharmacodynamic Modeling

BackgroundPhage therapy is a promising approach against multidrug-resistant Staphylococcus aureus, but its clinical development is limited by gaps in pharmacokinetic/pharmacodynamic (PK/PD) understanding, especially concerning phage self-replication and resistance dynamics. MethodsWe combined in vitro time-kill assays with PK/PD modeling to study three anti-staphylococcal phages (V1SA019, V1SA020, V1SA022) against two S. aureus strains (SH1000 and USA300). A system of differential equations captured the co-dynamics of susceptible, resistant and infected bacteria and free phages. Nonlinear mixed-effect modeling quantified parameter variability. Resistance emergence was monitored through phenotyping and whole-genome sequencing of resistant clones. ResultsPhage-bacteria interactions followed a predator-prey pattern, with early bacterial growth, rapid phage amplification, and subsequent bacterial collapse. However, resistant subpopulations emerged, regrew over time, in a multiplicity of infection (MOI) dependent way. The model accurately described bacterial and phage dynamics and estimated kinetic parameters including adsorption and burst size. Proliferation and inundation thresholds varied by strain and phage. All resistant clones harbored mutations in genes involved in teichoic acid biosynthesis, with associated growth defects. Simulations demonstrated that only phage doses exceeding both susceptible and resistant bacterial inundation thresholds fully suppressed regrowth. ConclusionThis study provides a quantitative framework for understanding phage-S. aureus co-dynamics and resistance emergence. It emphasizes the importance of considering both proliferation and inundation thresholds when designing phage dosing regimens. These findings inform the rational development of phage therapy and support translation toward in vivo and clinical applications.

pharmacology and toxicology↗

bioRxiv2024

High-throughput Tn-seq screens identify both known and novel Pseudomonas putida KT2440 genes involved in metal resistance

Chemical waste with toxic effects is released into the environment by industrial and urban activities. Pseudomonas putida, a rhizosphere bacterium, harbors a wide variety of genes capable of degrading hydrocarbons and xenobiotic compounds in its natural environment. This bacterium harbors also a large set of metal resistance genes. Most studies that identify genes involved in metal resistance in P. putida focus on over/underexpressed genes and may miss other genes important for metal resistance whose expression does not change. In this study, we used a Tn-seq approach to determine the essential genome of P. putida required for growth in the presence of an excess of metals in a culture medium. Tn-seq enables the detection of mutants with reduced or increased fitness in the presence of metal excess. We validated our screen by identifying known metal resistance gene such as czcA-1 (PP_0043), cadA-3 (PP_5139), cadR (PP_5140) and pcoA2 (PP_5380). Their mutants were underrepresented in the presence of zinc, cadmium (for cadA-3 and cadR) or copper respectively. In this study, we demonstrate by targeted mutagenesis and complementation assay that PP_5337 and PP_0887 are putative transcriptional regulators involved in copper and cadmium resistance, respectively, in P. putida. The study revealed the role of two genes, PP_1663 and PP_5002, in cadmium and cobalt resistance respectively. This is the first evidence linking these genes to metal resistance and highlights the incomplete understanding of metal resistance mechanisms in P. putida.

microbiology↗

bioRxiv2017

Genome Wide Identification Of Genes Required For Bacterial Plant Infection By Tn-Seq

Soft rot enterobacteria (Dickeya and Pectobacterium) are major pathogens that cause diseases on plants of agricultural importance such as potato and ornamentals. Long term studies to identify virulence factors of these bacteria focused mostly on plant cell wall degrading enzymes secreted by the type II secretion system and the regulation of their expression. To identify new virulence factors we performed a Tn-seq genome-wide screen of a transposon mutant library during chicory infection followed by high-throughput sequencing. This allowed the detection of mutants with reduced but also increased fitness in the plant. Virulence factors identified differed from those previously known since diffusible ones (secreted enzymes, siderophores or metabolites) were not detected by this screen. In addition to genes encoding proteins of unknown function that could be new virulence factors, others could be assigned to known biological functions. The central role of the FlhDC regulatory cascade in the control of virulence was highlighted with the identification of new members of this pathway. Scarcity of the plant in certain amino acids and nucleic acids required presence of the corresponding biosynthetic genes in the bacteria. Their products could be targets for the development of antibacterial compounds. Among the genes required for full development in chicory we also identified six genes involved in the glycosylation of the flagellin FliC, glycosylation, which in other plant pathogenic bacteria contributes to virulence.\n\nAuthor summaryIdentification of virulence factors of plant pathogenic bacteria has relied on the test of individual mutants on plants, a time-consuming method. New methods like transcriptomic or proteomic can now be used but they only allow the identification of genes induced during the infection process and non-induced genes may be missed. Tn-seq is a very powerful method to identify genes required for bacterial growth in their host. We used for the first time this method in a plant pathogenic bacteria to identify genes required for the multiplication of Dickeya dadantii in chicory. We identified about 100 genes with decreased or increased fitness in the plant. Most of them had no previously described role in bacterial virulence. We unveiled important metabolic genes and regulators of motility and virulence. We showed that D. dadantii flagellin is glycosylated and that this modification confers fitness to the bacteria during plant infection. Our work opens the way to the use of Tn-seq with bacterial phytopathogens. Assay by this method of large collections of environmental pathogenic strains now available will allow an easy and rapid identification of new virulence factors.

microbiology↗