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.

Keywords: H. illucens; Klebsiella pneumoniae; MDR bacteria; NDM-1 carbapenemase-producing Enterobacteriaceae (CPE); colistin; free fatty acids; hypermucoviscous; nosocomial isolates.

FIGURE 1

Schematic diagram for isolation of bioactive compounds from H. illucens larvae fat using sequential extraction procedure.

FIGURE 2

Antimicrobial sensitivity of AWME3 against (A) K. pneumoniae ATCC BAA-2473, (B) K. pneumoniae KPM9, and (C) K. pneumoniae KPi1627 strains. The bacteria strains were subjected to concentrations of 1.25, 2.5, 5, 10, and 20 mg/mL of AWME3 from BSFL fat. The IZD values were measured after 12 and 24 h of incubation at 37°C. Doxycycline (DOX) used as a positive antibacterial control. All values are represented as mean ± SD, in triplicate (n = 3). Data were analyzed by two-way ANOVA, followed by Dunnett’s Test. Data represented as significant difference as compared to positive control and p-value was ranged between. *p = 0.0138, **p = 0.0062, ****p < 0.0001.

FIGURE 3

The MIC₅₀ (IC₅₀ value in the figure legends) of K. pneumoniae strains treated with AWME3 from the larvae fat compared to the doxycycline (DOX) as a positive control. The MIC₅₀ values were calculated based on the turbidimetric assay data and compared to the positive control (DOX). The planktonic bacteria turbidity was assessed for (A,B) K. pneumoniae ATCC BAA-2473; (C,D) K. pneumoniae KPM9; (E,F) K. pneumoniae KPi1627 strains at 12 and 24 h incubation with (A,C,E) DOX; (B,D,F) AWME3. The MIC₅₀ values were calculated using the non-linear regression mode of Graph pad Prism 7 (Graph Pad Software Inc., San Diego, CA, United States). The (IC₅₀)MIC₅₀ values are the average of three independent experiments ± standard deviation error mean (SEM).

FIGURE 4

Klebsiella pneumoniae strains resistance assessment. Resistance acquisition monitored during 16 serial passages (16 days) in the presence of sub-MIC (0.5 × MIC) of AWME3, and positive control (P/S) for (A) K. pneumoniae KPi1627, (B) K. pneumoniae KPM9, and (C) K. pneumoniae ATCC BAA-2473. The Y-axis represents the highest bacterial concentration during cell passaging. The figures are representative of three independent experiments.

FIGURE 5

Effect of AWME3 concentrations on the cell membrane perme ability of K. pneumoniae ATCC BAA-2473 strain. Planktonic bacteria sus pension at 10⁸ CFU/mL was subjected to various concentrations of AW ME3 ranged from 0.5 (125 μg/mL) to 4x MIC (1,000 μg/mL), and incubated for 8 h at 37°C. REC was calculated at 0, 1, 2, 4, and 8 h based on the values of electrical conductivity. Bacteria without AWME3 treatment was considered as negative control. All values presented as the mean of three independent experiments ± SD.

FIGURE 6

Cytotoxicity of AWME3 fat on HEK-293 cell lines. Cells were treated with serial of AWME3 dilutions for 24 h. Then, viability of AWME3 treated-cells was measured using MTT assay (A,B). Where, (A) cell lines were treated with AWME3 for 24 h (X-axis: log concentrations of AWME3 extract from 0 to 1,000 (μg/mL) and Y-axis: the percentage of normalized absorbance). (B) The of average OD₅₇₀ of the HEK-293 cell line treated with AWME3 for 24 h (Y-axis: the normalized absorbance of HEK-293 at OD₅₇₀), X-axis: AWME3 concentrations from 0 to 1,000 μg/mL. The IC₅₀ values were calculated using the non-linear regression mode of Graph pad Prism7 (Graph Pad Software Inc., San Diego, CA, United States). All Results are the mean (±SEM) from three independent experiments performed in triplicates (n = 8). Statistical values are indicated.

FIGURE 7

The composition of AWME3 extracted from H. illucens larvae fat under optimal conditions. (A) The percentages and the identity of the AWME3 chemical compounds detected by GC-MS analysis using the NIST-08 library; (B) The FAs profile that includes SFAs, USFAs, and FAs derivatives (FADs) statistically analyzed (***p = 0.0002, ****p < 0.0001) by one-way ANOVA.