Insight into the outer membrane asymmetry of P. aeruginosa and the role of MlaA in modulating the lipidic composition, mechanical, biophysical, and functional membrane properties of the cell envelope.

In Gram-negative bacteria, the outer membrane (OM) is asymmetric, with lipopolysaccharides (LPS) in the outer leaflet and glycerophospholipids (GPLs) in the inner leaflet. The asymmetry is maintained by the Mla system (MlaA-MlaBCDEF), which contributes to lipid homeostasis by removing mislocalized GPLs from the outer leaflet of the OM. Here, we ascribed how Pseudomonas aeruginosa ATCC 27853 coordinately regulates pathways to provide defense against the threats posed by the deletion of mlaA. Especially, we explored (i) the effects on membrane lipid composition including LPS, GPLs, and lysophospholipids, (ii) the biophysical properties of the OM such as stiffness and fluidity, and (iii) the impact of these changes on permeability, antibiotic susceptibility, and membrane vesicles (MVs) generation. Deletion of mlaA induced an increase in total GPLs and a decrease in LPS level while also triggering alterations in lipid A structures (arabinosylation and palmitoylation), likely to be induced by a two-component system (PhoPQ-PmrAB). Altered lipid composition may serve a physiological purpose in regulating the mechanobiological and functional properties of P. aeruginosa. We demonstrated an increase in cell stiffness without alteration of turgor pressure and inner membrane (IM) fluidity in ∆mlaA. In addition, membrane vesiculation increased without any change in OM/IM permeability. An amphiphilic aminoglycoside derivative (3',6-dinonyl neamine) that targets P. aeruginosa membranes induced an opposite effect on ∆mlaA strain with a trend toward a return to the situation observed for the WT strain. Efforts dedicated to understanding the crosstalk between the OM lipid composition, and the mechanical behavior of bacterial envelope, is one needed step for designing new targets or new drugs to fight P. aeruginosa infections.IMPORTANCEPseudomonas aeruginosa is a Gram-negative bacterium responsible for severe hospital-acquired infections. The outer membrane (OM) of Gram-negative bacteria acts as an effective barrier against toxic compounds, and therefore, compromising this structure could increase sensitivity to antibiotics. The OM is asymmetric with the highly packed lipopolysaccharide monolayer at the outer leaflet and glycerophospholipids at the inner leaflet. OM asymmetry is maintained by the Mla pathway resulting in the retrograde transport of glycerophospholipids from the OM to the inner membrane. In this study, we show that deleting mlaA, the membrane component of Mla system located at the OM, affects the mechanical and functional properties of P. aeruginosa cell envelope. Our results provide insights into the role of MlaA, involved in the Mla transport pathway in P. aeruginosa.

Long-term intensive care unit outbreak of carbapenemase-producing organisms associated with contaminated sink drains.

BACKGROUND: Between 2018 and 2022, a Belgian tertiary care hospital faced a growing issue with acquiring carbapenemase-producing organisms (CPO), mainly VIM-producing P. aeruginosa (PA-VIM) and NDM-producing Enterobacterales (CPE-NDM) among hospitalized patients in the adult intensive care unit (ICU). AIM: To investigate this ICU long-term CPO outbreak involving multiple species and a persistent environmental reservoir. METHODS: Active case finding, environmental sampling, whole-genome sequencing (WGS) analysis of patient and environmental strains, and implemented control strategies were described in this study. FINDINGS: From 2018 to 2022, 37 patients became colonized or infected with PA-VIM and/or CPE-NDM during their ICU stay. WGS confirmed the epidemiological link between clinical and environmental strains collected from the sink drains with clonal strain dissemination and horizontal gene transfer mediated by plasmid conjugation and/or transposon jumps. Environmental disinfection by quaternary ammonium-based disinfectant and replacement of contaminated equipment failed to eradicate environmental sources. Interestingly, efflux pump genes conferring resistance to quaternary ammonium compounds were widespread in the isolates. As removing sinks was not feasible, a combination of a foaming product degrading the biofilm and foaming disinfectant based on peracetic acid and hydrogen peroxide has been evaluated and has so far prevented recolonization of the proximal sink drain by CPO. CONCLUSION: The persistence in the hospital environment of antibiotic- and disinfectant-resistant bacteria with the ability to transfer mobile genetic elements poses a serious threat to ICU patients with a risk of shifting towards an endemicity scenario. Innovative strategies are needed to address persistent environmental reservoirs and prevent CPO transmission.

Deficient Pseudomonas aeruginosa in MlaA/VacJ outer membrane lipoprotein shows decrease in rhamnolipids secretion, motility, and biofilm formation, and increase in fluoroquinolones susceptibility and innate immune response.

Pseudomonas aeruginosa, a Gram-negative bacterium that causes severe hospital acquired infections poses threat by its ability for adaptation to various growth modes and environmental conditions and by its intrinsic resistance to antibiotics. The latter is mainly due to the outer membrane (OM) asymmetry which is maintained by the Mla pathway resulting in the retrograde transport of glycerophospholipids from the OM to the inner membrane. It comprises six Mla proteins, including MlaA, an OM lipoprotein involved in the removal of glycerophospholipids mislocalized at the outer leaflet of OM. To investigate the role of P. aeruginosa OM asymmetry especially MlaA, this study investigated the effect of mlaA deletion on (i) the susceptibility to antibiotics, (ii) the secretion of virulence factors, the motility, biofilm formation, and (iii) the inflammatory response. mlaA deletion in P. aeruginosa ATCC27853 results in phenotypic changes including, an increase in fluoroquinolones susceptibility and in PQS (Pseudomonas Quinolone Signal) and TNF-α release and a decrease in rhamnolipids secretion, motility and biofilm formation. Investigating how the mlaA knockout impacts on antibiotic susceptibility, bacterial virulence and innate immune response will help to elucidate the biological significance of the Mla system and contribute to the understanding of MlaA in P. aeruginosa OM asymmetry.

Optimized Method for Measuring Persistence in Escherichia coli with Improved Reproducibility.

Monitoring persister cells can be extremely difficult due to their transient and stochastic nature, their low abundance, and their resemblance to Viable But Non-Culturable Cells (VBNCs). To date, the predominant method consists of determining the survival rate of a bacterial population after antibiotic treatment as a function of time or antibiotic concentration. Unfortunately, this method is limited, as it shows high levels of dispersion of the data around the mean, making interpretation difficult. Furthermore, additional reproducibility problems arise from the lack of a standard method, different research groups using different protocols. Here, we describe a standard and optimized method for monitoring E. coli persister cells at the population level allowing for maximal reproducibility.