Bacterial Outer Membrane Permeability Increase Underlies the Bactericidal Effect of Fatty Acids From Hermetia illucens (Black Soldier Fly) Larvae Fat Against Hypermucoviscous Isolates of Klebsiella pneumoniae

Abstract

Behind expensive treatments, Klebsiella pneumoniae infections account for extended hospitalization’s high mortality rates. This study aimed to evaluate the activity and mechanism of the antimicrobial action of a fatty acid-containing extract (AWME3) isolated from Hermetia illucens (HI) larvae fat against K. pneumoniae subsp. pneumoniae standard NDM-1 carbapenemase-producing ATCC BAA-2473 strain, along with a wild-type hypermucoviscous clinical isolate, strain K. pneumoniae subsp. pneumoniae KPi1627, and an environmental isolate, strain K. pneumoniae subsp. pneumoniae KPM9. We classified these strains as extensive multidrug-resistant (XDR) or multiple antibiotic-resistant (MDR) demonstrated by a susceptibility assay against 14 antibiotics belonging to ten classes of antibiotics. Antibacterial properties of fatty acids extracted from the HI larvae fat were evaluated using disk diffusion method, microdilution, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), half of the inhibitory concentration (MIC50), and bactericidal assays. In addition, the cytotoxocity of AWME3 was tested on human HEK293 cells, and AWME3 lipid profile was determined by gas chromatography-mass spectrometry (GC-MS) analysis. For the first time, we demonstrated that the inhibition zone diameter (IZD) of fatty acid-containing extract (AWME3) of the HI larvae fat tested at 20 mg/ml was 16.52 ± 0.74 and 14.23 ± 0.35 mm against colistin-resistant KPi1627 and KPM9, respectively. It was 19.72 ± 0.51 mm against the colistin-susceptible K. pneumoniae ATCC BAA-2473 strain. The MIC and MBC were 250 μg/ml for all the tested bacteria strains, indicating the bactericidal effect of AWME3. The MIC50 values were 155.6 ± 0.009 and 160.1 ± 0.008 μg/ml against the KPi1627 and KPM9 isolates, respectively, and 149.5 ± 0.013 μg/ml against the ATCC BAA-2473 strain in the micro-dilution assay. For the first time, we demonstrated that AWME3 dose-dependently increased bacterial cell membrane permeability as determined by the relative electric conductivity (REC) of the K. pneumoniae ATCC BAA-2473 suspension, and that none of the strains did not build up resistance to extended AWME3 treatment using the antibiotic resistance assay. Cytotoxicity assay showed that AWME3 is safe for human HEK293 cells at IC50 266.1 μg/ml, while bactericidal for all the strains of bacteria at the same concentration. Free fatty acids (FFAs) and their derivatives were the significant substances among 33 compounds identified by the GC-MS analysis of AWME3. Cis-oleic and palmitoleic acids represent the most abundant unsaturated FAs (UFAs), while palmitic, lauric, stearic, and myristic acids were the most abundant saturated FAs (SFAs) of the AWME3 content. Bactericidal resistant-free AWM3 mechanism of action provides a rationale interpretations and the utility of HI larvae fat to develop natural biocidal resistance-free formulations that might be promising therapeutic against Gram-negative MDR bacteria causing nosocomial infections.