Fluopsin C for Treating Multidrug-Resistant Infections: In vitro Activity Against Clinically Important Strains and in vivo Efficacy Against Carbapenemase-Producing Klebsiella pneumoniae

Abstract

The increasing emergence of multidrug-resistant (MDR) organisms in hospital infections is causing a global public health crisis. The development of drugs with effective antibiotic action against such agents is of the highest priority. In the present study, the action of Fluopsin C against MDR clinical isolates was evaluated under in vitro and in vivo conditions. Fluopsin C was produced in cell suspension culture of Pseudomonas aeruginosa LV strain, purified by liquid adsorption chromatography and identified by mass spectrometric analysis. Bioactivity, bacterial resistance development risk against clinically important pathogenic strains and toxicity in mammalian cell were initially determined by in vitro models. In vivo toxicity was evaluated in Tenebrio molitor larvae and mice. The therapeutic efficacy of intravenous Fluopsin C administration was evaluated in a murine model of Klebsiella pneumoniae (KPC) acute sepsis, using six different treatments. The in vitro results indicated MIC and MBC below 2 μg/mL and low bacterial resistance development frequency. Electron microscopy showed that Fluopsin C may have altered the exopolysaccharide matrix and caused disruption of the cell wall of MDR bacteria. Best therapeutic results were achieved in mice treated with a single dose of 2 mg/kg and in mice treated with two doses of 1 mg/kg, 8 h apart. Furthermore, acute and chronic histopathological studies demonstrated absent nephrotoxicity and moderate hepatotoxicity. The results demonstrated the efficacy of Fluopsin C against MDR organisms in in vitro and in vivo models, and hence it can be a novel therapeutic agent for the control of severe MDR infections.

Determining the Targets of Fluopsin C Action on Gram-Negative and Gram-Positive Bacteria

“…On the other hand, treated MRSA N315 strain bacteria presented the same changes on cell membrane and wall that was observed in Gram-negative bacteria, with cell disruption and changes in the cytoplasm. Similar activity was reported previously (Navarro et al, 2019).…”

“…F4A and Fluopsin C generated the same effect on Xcc 306 that was observed earlier by de Oliveira et al (2011), where the cell membrane and wall were completely disrupted. The same action was observed in Kpn-19 strain treated with Fluopsin C (Navarro et al, 2019), suggesting that the compound acts in the same way in different Gram-negative genera.…”

“…The F4A fraction and Fluopsin C used in this study were obtained according to procedures described by Munhoz et al (2017) and Navarro et al (2019), respectively. The F4A contains basically four compounds (mg/L): phenazine-1-carboxylic acid (14 mg), phenazine-carboxamide (9 mg), indol-3-one (1 mg), and Fluopsin C (6 mg) (Bedoya et al, 2019).…”

“…The majority of dead bacteria reflect the antibiotic effect of the tested compounds, but the remaining organisms present different stages of cell destruction, allowing the detailed observation of their mechanisms of action. For the scanning electron microscopy assay (SEM), bacterial suspensions (10 10 CFU) of each strain were incubated for 60 min with and without IC 90 concentrations of F4A (0.25 µg mL −1 for X. citri) (Murate et al, 2015) and Fluopsin C (2 µg mL −1 for K. pneumoniae, 1 µg mL −1 for E. faecium, and 0.5 µg mL −1 for S. aureus) (Navarro et al, 2019) were spotted onto poly-L-lysine-coated slides and stored at 28 • C for 1 h for drying. The slides were fixed with a solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.0) for 12 h. After that, the slides were washed with 0.1 M sodium cacodylate buffer (pH 7.0) and post-fixed in a solution of 1% OsO 4 for 2 h. The samples were dehydrated in ethanol at concentrations of 70, 80, 90, and 100% and then dried by critical point in CO 2 (BALTEC CPD 030 Critical Point Dryer).…”

“…In previous studies, it was observed that pathogenic bacteria treated with the semi-purified fraction F4A fraction or Fluopsin C presented complete disruption of cell membrane and frequent presence of cells that stopped septation (Vasconcellos et al, 2014;Murate et al, 2015;de Oliveira et al, 2016;Kerbauy et al, 2016;Munhoz et al, 2017;Navarro et al, 2019). However, to the best of our knowledge, the major mechanism of action of these compounds has not yet been defined.…”