Fat isolated from alive H. illucens larvae provided by «NordTechSad, LLC» company (Arkhangelsk, Russia) was used for this study. The HEK-293 cell line (human embryonic kidney) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States). Cells were cultured in T75 flasks with Dulbecco’s Minimal Essential Medium (DMEM) containing 10% fetal calf serum and penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C in 5% CO₂ until confluence (Gibco TM; Thermo Fisher, Waltham, MA, United States). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, United States).

Reagents for extraction solution, such as hydrochloric acid (HCl) and methanol (CH₃OH), were purchased from Thermo Fisher Scientific (Waltham, MA, United States), and Milli-Q water was obtained from Water System, Ultrapure, Millipore, and Direct-Q^® 3 with UV. Luria-Bertani (LB) broth, LB agar, Mullar Hinton agar (MHA), and nutrient agar (NA) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Tissue culture 96-well plates (TPP; Trasadingen, Switzerland), Petri dishes (90 mm) (Pertin, Saint Petersburg, Russia), paper disks with a size of 6 mm diameter (Himedia, Mumbai, India), sterile swab (Nigbo Greetmed Medical Instruments Co., Ltd., Nigbo, China) were used for this study.

Antimicrobial susceptibility disks (6 mm) with penicillin-streptomycin (10 μg/disk), chloramphenicol (Ch) (30/disk), gentamycin (G) (10 μg/disk), and doxycycline (Dox) (30 μg/disk) were purchased from, Thermo Fisher Scientific (Waltham, MA, United States). Discs with penicillin (P) (2 U/disk), vancomycin (VA) (5 μg/disk), erythromycin (E) (60 μg/disk), rifampicin (RD) (15 μg/disk), kanamycin (K) (1,000 μg/disk), colistin (CT) (10 μg/disk), ciprofloxacin (Cip) (5 μg/disk), levofloxacin (Lev) (5 μg/disk), cefepime (Cef) (30 μg/disk), and ampicillin (Amp) (200 μg/disk) purchased from Oxoid (Basingstoke, Hampshire, United Kingdom). The different antibiotics used in this study belonged to 10 classes of antibiotics, namely, aminoglycosides (G, streptomycin, and K), ansamycins (RD), cephems (Cef), glycopeptides (VA), macrolides (E), tetracyclines (Dox), fluoroquinolones (Cip and Lev), phenicols (Ch), penicillins (P and Amp), and lipopeptides (CT).

The standard strain Klebsiella pneumoniae ATCC BAA-2473 used in this study was purchased from ATCC (American Type Culture Collection, United States). Environmental isolate K. pneumoniae KPM9 and clinical isolate K. pneumoniae KPi1627 stains were obtained from the State Collection of Pathogenic Microorganisms and Cell Cultures (SCPM, Obolensk, Russia). The bacterial strains were stored in sterile glycerol stock (30%, v/v) at –80°C. A single colony from each strain was inoculated in 10 ml of the LB broth, incubated overnight at 37°C by shaking at 210 rpm/min. The overnight culture was adjusted to half of the McFarland standard (1 × 10⁸ CFU/ml) and used in susceptibility assays. All the experiments were implemented in a sterile cabinet (Safe Fast Elite, Ferrara, Italy).

Fat isolated from alive H. illucens larvae with age of 15 days old was provided by the NordTechSad, LLC company (Arkhangelsk, Russia) and used for this study. The H. illucens larvae fat was extracted according to our previous study (Mohamed et al., 2021). Briefly, 3 g of larvae fat was subjected to three rounds of extraction using an extraction solution composed of water (Milli Q quality), methanol (99.9%, HPLC grade), and hydrochloric acid (37%) with a ratio of 90:9:1, v/v/v at very low acidic pH (<1, 0). Since our previous study demonstrated that AWME3 was the most potent among other extracts against pathogenic Aeromonas sp. (Mohamed et al., 2021), the third extract AWME3 was selected for our experiments in this study.

Antimicrobial susceptibility was determined using the disk diffusion technique according to the National Committee for Clinical Laboratory Standards 2018 guidelines (Clinical and Laboratory standards Institute [CLSI], 2018; Algammal et al., 2020d). All strains were obtained from a single colony, and the overnight cultures were adjusted to 0.5 McFarland standard (1 × 10⁸ CFU/ml) and tested against a panel of 14 antibiotics belonging to 10 different classes using antibiotic disks in Mueller-Hinton (MH) agar. The antimicrobial agents included penicillin-streptomycin (200 U/ml-200 μg/ml), gentamycin (200 μg/ml), doxycycline (600 μg/ml), chloramphenicol (600 μg/ml), ciprofloxacin (100 μg/ml), levofloxacin (100 μg/ml), cefepime (600 μg/ml), and ampicillin (4,000 μg/ml), where 50 μl of each antibiotic was loaded on 6 mm disks, and then the disks were dried under sterilized cabinet conditions for 45 min and transferred to agar plates. Disks loaded with penicillin (2 U/disk, P), vancomycin (5 μg/disk, VA), erythromycin (60 μg/disk, E), rifampicin (15 μg/disk, RD), kanamycin (1,000 μg/disk, K), and colistin (10 μg/disk, CT) were picked up and transferred to bacterial agar plates and pressed gently. Additionally, the minimum inhibitory concentration for these antibiotics was determined according to a microdillution assay; therefore, the minimum bactericidal concentration was determined based on the MIC assay. The strains of K. pneumoniae were classified as sensitive (S), intermediate (I), and resistant (R) according to the Clinical and Laboratory standards Institute [CLSI] (2018) breakpoints for Enterobacteria. Inhibition zone diameters (IZD) were measured and compared to measures of the CLSI guidelines (Clinical and Laboratory standards Institute [CLSI], 2018). Strains resistant to at least one agent in three or more antimicrobial categories were defined as multidrug-resistant (MDR), strains which are resistant to all categories except one or two were defined as extensively drug resistant (XDR), while strains show resistance to all categories of antibiotics was defined as pandrug resistant (PDR) (Magiorakos et al., 2012). All data values were recorded at 12 and 24 h of incubation.

Minimum inhibitory concentration (MIC) was assessed using a 2-fold broth micro-dilution assay according to the guidelines of the Clinical Laboratory Standards Institute (Clinical and Laboratory standards Institute [CLSI], 2015; Popović et al., 2020). Experiments were carried out on 96-well microtiter plates using a series of 2-fold dilutions of the AWME3 extract as follows: 8 mg/ml AWME3 was tested in a range from 2,000 to 15.63 μg/ml. Likewise, the positive control (doxycycline) that was susceptible to the all the strains was serially 2-fold diluted in the range of 50–0.195 μg/ml. Microplates were sealed and incubated at 37°C for 24 h with shaking at 200 rpm/min. The value of MIC was recorded as the lowest concentration of the AWME3 extract, showing no detectable bacterial growth in the wells. All the experiments were performed in triplicate.

Minimal bactericidal concentration (MBC) value was defined as the minimum concentration of the AWME3 extract, which kills 99.9% of the starting inoculum. For MBC determination, an aliquot of 40 μl of bacteria from the wells corresponding to 1x MIC, 2x MIC, and 4x MIC were plated and then incubated for 48 h at 37°C (Popović et al., 2020). MBC was defined as the lowest concentration of the tested AWME3 of BSFL fat that showed no growth on the surface of the Petri dishes with agar. The MBC of AWME3 was compared with the MBC of the positive control (DOX) used against the K. pneumoniae ATCC BAA-2473, K. pneumoniae KM9, and K. pneumoniae KPi1627 bacteria. All the tests were performed in triplicate. Every experiment was repeated at three different times.

Half of the minimum inhibitory concentration (MIC50) was defined as the minimum concentration of AWME3, which inhibits 50% of total bacterial growth. A total of 100 μL of the AWME3 extract was serially diluted in the MH broth and ranged from 2,000 to 15.63 μg/ml in a 96–well microtiter plate and had a final volume of 200 μl (Balkrishna et al., 2019). The reference control doxycycline was tested against clinical strains and was serially diluted in the range of 50–0.195 μg/ml. MIC₅₀ was determined at 12 and 24 h by measuring the OD₆₀₀ for tested strains using a CLARIO star microplate reader (BMG LABTECH, Ortenberg, Germany). All the experiments were performed in triplicates. MIC₅₀ was determined using the GraphPad Prism 7 software using the non-linear regression mode.

Minimum Inhibitory Concentration (bacteriostatic) and MBC (bactericidal) methods were used to evaluate the antimicrobial effect of AWME3 on laboratory K. pneumoniae ATCC BAA-2473 and clinical K. pneumoniae KPM9, and K. pneumoniae KPi1627 strains. If the ratio of MBC:MIC values was equal to 1 or 2, the antimicrobial effect was considered as bactericidal. However, if the ratio of MBC:MIC value was equal to 4 or above, AWME3 activity was considered as bacteriostatic. The activity of the reference control (DOX) was evaluated with the same token (Konaté et al., 2012).

Resistance to AWME3 isolated from H. illucens larvae fat was determined according to Duan et al. (2021), with minor changes (Duan et al., 2021). The tested microorganisms K. pneumoniae ATCC BAA-2473, K. pneumoniae KPM9, and K. pneumoniae KPi1627 were subcultured with a sub-MIC (125 μg/ml) concentration of AWME3 and the positive control (P/S) for 16 consecutive days to investigate their ability to develop resistance to the tested drugs. Briefly, bacterial cultures were harvested in the logarithmic phase, washed three times with PBS (pH 7.4), and diluted to 5 × 10⁵ (CFU/ml) in a fresh LB medium containing 0.5x, 1x, 2x, and 4x MIC concentrations of AWME3 or an antibiotic, and incubated at 37°C for 24 h. Aliquots of cultures from the second highest concentration (0.25x MIC) of AWME3 or an antibiotic that allowed growth (OD₆₀₀ > 0.1) were diluted 1:100 in fresh an LB medium containing the same set of MIC concentrations based on the most recently observed MIC. With increasing concentrations of AWME3 or the antibiotic, serial passaging was repeated for 16 days for every strain.

Relative electric conductivity (REC) value was calculated according to Kong et al. (2008), with minor changes, and used as a measure of bacterial membrane permeability. K. pneumoniae ATCC BAA-2473 was cultured in 10 ml of the LB broth at 37°C for 10 h under shaking conditions, and then centrifuged at 4,200 rpm/min for 10 min. Then, the pellet of the bacteria was washed with 5% of glucose in water until its REC value was equal or near the REC value of the washing buffer (5% glucose), generating a so-called isotonic bacterial solution. The AWME3 at four different concentrations (0.5x MIC, 1x MIC, 2x MIC, and 4x MIC) were diluted in 5% glucose, and electric conductivities of the mixtures were measured using a conductometer (Ionomer, Moscow, Russia) and marked as L₁. Then, the same concentrations of AWME3 were added into the isotonic suspension of K. pneumoniae ATCC BAA-2473 at 10⁸ CFU/ml. After intensive mixing, the samples were incubated at 37°C for 8 h, and the conductivities were measured and marked as L₂. The bacteria 5% glucose were boiled in the water bath for 5 min and served as the positive control, using their conductivity marked as L₀. Relative electric conductivity was calculated according to the formula (%) = (L₂ -L₁)/L₀ × 100, reflecting bacterial membrane permeability value.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were carried out on a 96-well plate based on the method described by Somaida et al. (2020). Briefly, 100 μL of AWME3 was serially diluted in 2 folds of dilution in the DMEM medium to get the final concentrations (1,000, 500, 250, 125, 62.5, 31.25, 15.625, and 7.813 μg/ml), and then added to a 96-well plate, which contained 100 μl of HEK293 cells, and the plate was incubated for 24 h at 37°C and 5% CO₂. After that, 20 μl of 5 mg/ml MTT was added to each well (1 × 10⁴ cells/well). MTT specific activity was determined after 2 h of incubation at 37°C and 5% CO₂; then, 100 μl of DMSO was added into each well of the 96-well plate. The plate was shaken for 10 min to dissolve formazan crystals. Data were presented as a percentage of viability, when compared with untreated cells. Furthermore, the blank included during MTT incubation included wells without cells (DMSO + MTT). Viability was calculated based on comparison with the absorbance of untreated and treated cells. IC₅₀ values were obtained from the concentration of the AWME3 of H. illucens larvae fat that induced 50% inhibition of cell growth using the Graph Pad prism 7 software. The optical density (OD) in each well was determined with a microplate reader, Clariostar, using an absorption spectrum of 580 nm.

Gas Chromatography-Mass Spectrometry (GC-MS) analyses of AWME3 were carried out using a GC-MS-QP2010 ultra mass spectrometer (Shimadzu, Canby, OR, United States) equipped with a fused silica column, capillary column DB-5 ms measuring 30 mm × 0.25 mm with a thickness of 0.25 mm coated with a non-polar silphenylene polymer with polarity of 5% diphenyl and 95% dimethylpolysiloxane in stationary phase (Restek, Bellefonte, PA, United States). Pure helium gas (99.99%) was used as the carrier gas with a linear flow rate ranging from 1 to 15 ml/min, and column head pressure was 50.4 kPa. For GC–MS spectral detection, an electron ionization energy method was adopted with high ionization energy of 70 eV (electron Volts), with 0.2 s of scan time and fragments ranging from 40 to 600 m/z. Analysis procedure was as follows: 1 μl of AWME3 extract was injected with an autosampler injector automatically. Injector and detector temperatures were maintained at 280 and 250°C, respectively. Temperature program was initially set at 40°C, held for 1 min, and then increased to 210°C at a rate of 15°C/min, held for 0 min, and then it was increased to 216°C at a rate 5°C/min and held for 0 min. Then, the temperature was increased to 300°C at a flow rate of 40°C/min and held for 14.87 min. The contents of AWME3 present in the sample were identified based on comparison of their retention time (min), peak area, peak height, and mass spectral patterns with those in spectral databases of authentic compounds stored in the National Institute of Standards and Technology (NIST-08) library. Compounds with chromatogram peaks matched with similarity index (SI) ≥ 70% in the NIST-8 library were ascertained.

Statistical analysis performed by two-way analysis of variance of repeated measurements (Two-way RM-ANOVA) to determine the statistically significant differences between treatments of the AWME3 susceptibility against tested bacteria strains compared to positive control. Differences between means were compared by Dunnett’s post-hoc test. Results of fatty acid profiles were analyzed by ordinary one-way ANOVA, and Tukey’s post-hoc test was conducted to determine significant differences between treatment means. All the data were assessed by the standard deviation (SD) using software GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, United States). Statistical significance level was P < 0.05.

The antibiotic resistance phenomenon represents one of the most critical public health issues, and antibiotic-resistant genes are considered the emerging pollutant of the environment (Sabri et al., 2020). The disk diffusion method was used to determine the antimicrobial AWME3 susceptibility effect on three bacterial strains: K. pneumoniae ATCC BAA-2473, K. pneumoniae KPM9, and K. pneumoniae KPi1627. IZD values were interpreted by comparison to E. coli values, because there are no guideline breakpoints for K. pneumoniae in the CLSI guidelines. The breakpoints of E. coli are recommended and used for other Enterobacteriaceae such as the Klebsiella species (Le et al., 2021). To evaluate the susceptibility of the three tested strains of K. pneumoniae, we determined IZDs by disk assay, MIC. MBC values were determined by microdilluition method (Supplementary Table 1), which is recommended by Clinical and Laboratory standards Institute [CLSI] (2018), K. pneumoniae ATCC BAA-2473 showed resistance to 12, while K. pneumoniae KPi1627 and K. pneumoniae KPM9 were resistant to only six of 14 tested antimicrobials (Supplementary Figure 1, Table 1, and Supplementary Table 1).

All the K. pneumoniae strains demonstrated sustainable 100% resistance to penicillin, vancomycin, erythromycin, and rifampicin strains during 12 h and 24 h of incubations. All the tested K. pneumoniae strains isolated from different sources exhibited high susceptibility to doxycycline (Supplementary Figure 1 and Supplementary Table 1), being the rationale of using it as a positive control in this study. K. pneumoniae ATCC BAA-2473 was 100% resistant to gentamicin and penicillin, where no IZD occurred around the discs at 12 and 24-h of the incubations and possessed high resistance to kanamycin and chloramphenicol confirmed by the IZD in the disk diffusion assay (Supplementary Figure 1). Moreover, the MIC determined via the microdillution assay shows that K. pneumoniae KPi1627 and K. pneumoniae KPM9 were resistant to six antimicrobial drugs belonging to five classes, and that K. pneumoniae ATCC BAA-2473 exhibited highest resistance compared to the other two strains, where it was resistant to all the tested antibiotics except for two categories (colistin and doxycycline) (see Supplementary Table 1). Of note, both the wild-type nosocomial KPi1627 strain and the environmentally isolated KPM9 strain were resistant to colistin, the antibiotic of last resort that is broadly active against Gram-negative bacteria despite having side effects such as nephrotoxicity and ototoxicity. In contrast, the K. pneumoniae ATCC BAA-2473 strain was susceptible to this antibiotic but show high resistance to the fluoroquinolone (ciprofloxacin and levofloxacin), and cephem (cefepime) groups. On the other hand, K. pneumoniae KPi1627 and K. pneumoniae KPM9 showed significant susceptibility to the same categories of antibiotics (Cip, Levo, and Cef) (Supplementary Table 1). Based on the ratio of MBC/MIC, all the antibiotic categories were classified as bactericidal or bacteriostatic. Thus, if the ratio MBC/MIC ≥ 4, bacteria were bacteriostatic, while if MBC/MIC ≤ 2, bacteria were bactericidal. In our study, chloramphenicol, doxycycline, rifampicin, ampicillin, and cefepime showed bacteriostatic activity against K. pneumoniae KPi1627. The antibacterial agents chloramphenicol and doxycycline displayed bacteriostatic properties against the K. pneumoniae KPM9 strain, while five important antibiotics, namely, chloramphenicol, doxycycline, colistin, levofloxacin, and cefepime were bacteriostatic against K. pneumoniae ATCC BAA-2473 (Supplementary Table 1). Based on our findings, colistin and doxycycline were recommended for treatment of K. pneumoniae ATCC BAA-2473, while gentamycin, kanamycin, penicillin-streptomycin, ciprofloxacin, levofloxacin, and cefepime were recommended for treatment of both the K. pneumoniae KPi1627 and K. pneumoniae KPM9 strains. The percentage of antibiotic resistance of K. pneumoniae ATCC BAA-2473, K. pneumoniae KPi1627, and K. pneumoniae KPM9 to the 14 different antibiotics were 83.33, 42.86, and 42.86%, respectively. Hence, the K. pneumoniae KPi1627 and K. pneumoniae KPM9 strains were classified as multidrug-resistant (MDR) because they were resistant to two or more antibiotics. K. pneumoniae ATCC BAA-2473 was classified to be extensive drug resistant (XDR), because it was resistant to all the antibiotics except for two categories (doxycycline and colistin) of the total 14 used in our study (Supplementary Table 2).

Recently, we demonstrated the advantage of using consecutive extracts of the HI larvae fat where bioactive compounds from the AWME3 extract showed higher level of antimicrobial activity against pathogenic fish bacteria than the first two extracts (Mohamed et al., 2021). The schematic diagram of the workflow shows the developed methodology for obtaining AWME3 using sequential extraction approach from the same biomass of the HI larvae fat (Figure 1).

Different concentrations of AWME3 were tested against the K. pneumoniae strains by disk diffusion assay to assess antibacterial activity. All the tested Gram-negative bacterial strains were treated with 1.25, 2.5, 5, 10, and 20 mg/ml concentrations of AWME3, which were loaded as 50-μl aliquots in 6-mm-diameter discs. IZD was determined by measuring the inhibition halos induced by AWME3 around the discs at 12 and 24 h of incubation time (Table 2 and Supplementary Figure 2). For the first time, AWME3 extract activity was evaluated against clinical and environmental isolates of K. pneumoniae, such as K. pneumoniae KPi1627 and K. pneumoniae KPM9 strains, and compared with the standard K. pneumoniae ATCC BAA-2473 strain.

The ANOVA results indicated a significant difference among the mean inhibition halos obtained by treating the different human pathogenic bacterial strains with AWME3 when compared to the IZD caused by the reference antibiotic (Dox) (p ≤ 0.05). The most significant inhibition zone size belonged to K. pneumoniae ATCC BAA-2473 and K. pneumoniae KPM9, indicating their highest susceptibility to the AWME3 obtained from the natural fat of H. illucens larvae. Their IZD value was 19.72 ± 0.51 and 16.52 ± 0.74 mm, respectively, when treated with 20 mg/ml at 24 h of incubation; compared to the positive control (Dox) it was 18.1 ± 1.2 and 18.62 ± 0.35 mm, respectively. The K. pneumoniae KPi1627 strain was the least susceptible and recorded a significant difference (^****p < 0.0001) compared to the reference control at 12 and 24 h of incubation time (Figure 2). Our results show that all these MDR strains isolated from different sources can be inhibited in a dose-dependent manner by AWME3. Moreover, all the tested K. pneumoniae strains were eradicated and killed at a 20 mg/ml concentration of AWME3.

Table 3 shows the MICs of AWME3 for all the tested bacterial strains obtained after 24 h of incubation. AWME3 inhibited K. pneumoniae ATCC BAA-2473, K. pneumoniae KPM9, and K. pneumoniae KPi1627 growth at a MIC concentration 250 μg/ml. Doxycycline (positive control) inhibited the growth of K. pneumoniae ATCC BAA-2473 bacteria at MIC 6.25 μg/ml, while inhibition of K. pneumoniae KPM9 and K. pneumoniae KPi1627 growth was obtained at MICs 3.12 and 1.56 μg/mL, respectively. All the tested strains were treated with different concentrations of the positive control in the range 0.195–50 μg/ml. MBC is defined as the lowest concentration, killing 99.99% of the tested bacteria strains after 48 h of incubation time. AWME3 demonstrated equal MBC 250 μg/ml for all the tested human K. pneumoniae pathogens. In contrast, MBC 50 μg/ml for doxycyclin was the highest for the K. pneumoniae ATCC BAA-2473 strain compared to the MBC 12.5 μg/ml for K. pneumoniae KPM9 and MBC 6.25 μg/mL for the K. pneumoniae KPi1627 isolates. The ratio of MBC/MIC did not exceed 2 for AWME3 while ranging between 4 and 8 for the positive control (DOX). These results demonstrated that AWME3 has sustainable bactericidal activity comparable to the reference antibiotic control (Dox) that exhibited bacteriostatic activity against all the tested K. pneumoniae strains. Furthermore, the MBC (250 μg/ml) of AWME3 was lower than the MBC of nine antibiotics (gentamycin, penicillin-streptomycin, kanamycin, rifampicin, erythromycin, penicillin, ampicillin, levofloxacin, and ciprofloxacin) used for testing against K. pneumoniae ATCC BAA-2473, where their MBC ranged between 312.5 and ≥1,250 μg/ml (Supplementary Table 1).

Half of the minimal inhibitory concentration (MIC50) values of the AWME3 and the positive control against human K. pneumoniae strains are shown in Table 4 and Figure 3.

Acidic Water Methanol Extract 3 showed MIC50 values ranging from 149.5 ± 0.013 to 160.1 ± 0.008 μg/ml after 24 h of incubation against all the tested bacterial strains. Of note, the MIC50 values of AWME3 decreased from 12 (169.2 ± 0.006–157.1 ± 0.009 μg/ml) to 24 h (160.1 ± 0.008–155.6 ± 0.009 μg/ml) the against K. pneumoniae KPM9 and K. pneumoniae KPi1627 strains, respectively. The MIC50 values of doxycycline increased by 60% from 12 to 24 h against clinical KPi1627 and environmental KPM9 isolates, while the reference strain KP ATCC BAA-2473 demonstrated only a 10% MIC₅₀ increase (Table 4 and Figure 3). K. pneumoniae ATCC BAA-2473 was the most resistant to different concentrations of the positive control (Dox) compared to both wild-type K. pneumoniae isolates (Table 4). AWME3 possessed significant concentration-dependent inhibition, indicating high efficacy against MDR K. pneumoniae strains.

Antibacterial resistance was determined by sub-culturing the tested MDR bacteria strains with the sub-MIC (125 μg/ml) concentration of the AWME3 extract or the antibiotics for 16 consecutive days. The development of antimicrobial resistance is defined as more significant than a 4-fold increase in its initial MIC (Duan et al., 2021). Our results demonstrated that the MIC values of AWME3 remained unchanged for the tested bacterial strains, namely, K. pneumoniae ATCC BAA-2473, K. pneumoniae KPM9, and K. pneumoniae KPi1627, indicating that the human pathogens did not acquire resistance to active compounds in the AWME3 extract (Figure 4). In contrast, serial passaging of all the tested bacterial strains treated with the reference antibiotic (P/S) showed more than 4-fold higher than the initial MICs (Figure 4), demonstrating a significant increase in antibiotic resistance. Indeed, the P/S initial MIC (9.77 μg/ml) raised to 256x MIC (2,500 μg/ml) the for K. pneumoniae KPi1627, and K. pneumoniae KPM9 strains. For the K. pneumoniae ATCC BAA-2473 strain, resistance increased to 4× MIC (>5,000 μg/ml) from the initial MIC (1,250 μg/mL). Thus, K.pneumoniae ATCC BAA-2473, K. pneumoniae KPM9, and K. pneumoniae KPi1627 did not show resistance in response to AWME3 extended treatment.

Cell membrane permeability was determined based on relative electric conductivity (REC). Figure 5 shows the effect of AWME3 from the H. illucens larvae fat on the REC of the K. pneumoniae ATCC BAA-2473 strain. The REC of the control (non-treated) planktonic bacteria recorded a marginal increase, which might be due to the standard lysis and death of the bacteria. AWME3 treatment in the concentration range of 0.5–2x MIC demonstrated a biphasic effect on REC with a smooth lift by the second hour followed by a more pronounced increase by the fourth hour with almost flattened change within the next 4 h. Indeed, AWME3-induced REC gradually increased within the first 4 h, reaching a 1.5-fold rise to 13.62 ± 1.29 and 19.38 ± 0.87% against 0.5 and 1x MIC, respectively, in 8 h. A more pronounced REC increase of up to 45.57 ± 0.87 and 69 ± 1.21% was caused by the 2x MIC and the 4x MIC, respectively, with the same incubation time. Notably, the 4x MIC induced a rapid sharp increase by the first hour of incubation, recording an REC of 31.82 ± 1.21%. By the eighth hour of AWME3 exposure, the REC of K. pneumoniae ATCC BAA-2473 at the control 0.5, 1, 2, and 4× MIC was 1.86 ± 0.12, 13.62 ± 1.29, 19.38 ± 0.87, and 69 ± 1.21%, respectively. Hence, the AWME3 dose-dependently increased the REC of the treated bacteria suggesting leakage of bacterial electrolytes due to disruption of cell permeability caused by AWME3 treatment with the incubation time.

Assessment of the AWME3 sequentially extracted from H. illucens larvae fat for potential cytotoxicity (Figures 6A, B) is considered as an important step to evaluate its properties for further applications. The cytotoxic effect of AWME3 at 7.813–1,000 μg/ml concentration was investigated using HEK293 cells by MTT cell viability assay, which relies on mitochondrial metabolic capacity of viable cells. Cell viability results were obtained after 24 h of incubation with AWME3. It is obvious that cell viability decreased with increased concentration of the extract. AWME3 was toxic at a concentration higher than 500 μg/ml. However, cells exposed to lower extract concentrations (250 μg/ml) retained higher than 70% cell viability. The cytotoxic activity of AWME3 is capable of killing human pathogenic bacterial strains at low concentrations but did not kill eukaryotic HEK293 cells.

This study evaluated the chemical composition of a new AWME3 batch obtained from the H. illucens larvae using our previously developed extraction and GC-MS analysis methods (Mohamed et al., 2021). The free fatty acids (FFAs) and their derivatives represented significant substances among 33 compounds identified by the GC-MS analysis of AWME3 (Supplementary Table 3 and Figure 7).

The major antibacterial components found in AWME3 were free fatty acids divided into saturated fatty acids (SFAs) and unsaturated fatty acids (USFAs). SFAs include palmitic (C16:0, 21.76%), lauric (C12:0, 17.66%), stearic (C18:0, 5.82%), myristic (C14:0, 5.27%), arachidic (C20:0, 0.31%), capric (C10:0, 0.3%), and pentadecyclic (C15:0, 0.2%) acids. USFAs contain three major free USFAs; moreover, cis-oleic acid (C18:1, 26.28%) is the most abundant among all the FA profiles followed by palmitoleic (C16:1, 3.15%) and linoleic acid (C18:2, 0.21%), as shown in Supplementary Table 3 and Figure 7.

The GC-MS analysis shows that the AWME3 extract is rich in molecules containing a hydrocarbon (hydrophobic) in the side chain in addition to cis or trans double bonds and polar functional groups (OH, NH2) that are hydrophilic. The AWME3 constituents contain OH^– and Cl^– ions believed to increase antibacterial activity. These ions are distributed in FA derivatives such as glycerol that was found in high concentration (7.87%); oxiraneundecanoic acid, 3- pentyl-, methyl ester, cis- (1.14%), lauric acid beta-monoglycerol (1.08%), 9,12-hexadecadienoic acid, methyl ester (0.25%), cis-9-hexadecenal (0.13%); and 2,4-dodecadienal, (E, E)- (0.13%).