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.

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.

Fatty Acids-Enriched Fractions of Hermetia illucens (Black Soldier Fly) Larvae Fat Can Combat MDR Pathogenic Fish Bacteria Aeromonas spp.

Aeromonas spp. cause many diseases in aquaculture habitats. Hermetia illucens (Hi) larvae were used as feed-in aquacultures and in eradicating pathogenic fish bacteria. In the present study, we applied consecutive extractions of the same biomass of BSFL fat using the acidic water-methanol solution. The major constituents of the sequential extracts (SEs) were free fatty acids (FFAs), and fatty acids derivatives as identified by gas chromatography spectrometry (GC-MS). Our improved procedure enabled gradual enrichment in the unsaturated fatty acids (USFAs) content in our SEs. The present study aimed to compare the composition and antimicrobial properties of SEs. Among actual fish pathogens, A. hydrophila and A. salmonicida demonstrated multiple drug resistance (MDR) against different recommended standard antibiotics: A. salmonicida was resistant to six, while A. hydrophila was resistant to four antibiotics from ten used in the present study. For the first time, we demonstrated the high dose-dependent antibacterial activity of each SE against Aeromonas spp., especially MDR A. salmonicida. The bacteriostatic and bactericidal (MIC/MBC) activity of SEs was significantly enhanced through the sequential extractions. The third sequential extract (AWME3) possessed the highest activity against Aeromonas spp.: inhibition zone diameters were in the range (21.47 ± 0.14-20.83 ± 0.22 mm) at a concentration of 40 mg/mL, MIC values ranged between 0.09 and 0.38 mg/mL for A. hydrophila and A. salmonicida, respectively. AWME3 MBC values recorded 0.19 and 0.38 mg/mL, while MIC50 values were 0.065 ± 0.004 and 0.22 ± 0.005 mg/mL against A. hydrophila and A. salmonicida, respectively. Thus, the larvae fat from Hermitia illucens may serve as an excellent reservoir of bioactive molecules with good capacity to eradicate the multidrug-resistant bacteria, having promising potential for practical application in the aquaculture field.

Fatty Acids from Hermetia illucens Larvae Fat Inhibit the Proliferation and Growth of Actual Phytopathogens.

The rapid increase of plant diseases caused by bacterial phytopathogens calls for an urgent search for new antibacterials. Antimicrobial compounds of natural origin stand up as frontiers in the attempts of the antibiotic overuse replacement. With this in mind, the Hermetia illucens (H. illucens) larvae have recently gained attention as a promising approach to fulfill this need. This study aimed to isolate the active constituents of H. illucens larvae fat and to estimate its antimicrobial capacity. We discovered the best composition of extracting solution retaining the pronounced antimicrobial activity of the extract. Using gas chromatography-mass spectrometry (GC-MS), we identified the unique natural array of fatty acids as the major constituents of the acidified water-methanol extract (AWME) as having new antimicrobial potency. In standard turbidimetric assay, the minimum inhibitory concentration (MIC) of the AWME was 0.78 mg/mL after 24 h of incubation for all five tested phytopathogenic bacteria strains: Pantoea agglomerans, Xanthomonas campestris, Pectobacterium carotovorum subsp. carotovorum, Pectobacterium atrosepticum, and Dickeya solani. The minimum bactericidal concentration (MBC) ranged from 0.78 to 1.56 mg/mL against all tested strains after 24 h of incubation. The inhibition zone size of AWME (INZ) at 50 mg/mL concentration was in the range 12.2 ± 0.56 to 19.0 ± 0.28 mm, while zone size for the positive control (penicillin-streptomycin) (5000 IU/mL-5000 µg/mL) was in the scale of 20.63 ± 0.53 to 24.0 ± 0.35 mm as revealed by standard disk diffusion assay. For the first time, our findings indicated the substantial antibacterial potential of AWME of H. illucens larvae fat against these actual phytopathogens, thus paving the way for further research to determine the mechanism of action in crop protection.