Atomic force microscopy reveals antibacterial mechanisms of Hermetia illucens fatty acids against MDR bacteria

Abstract

Abstract

Acinetobacter baumannii and Staphylococcus aureus are major multidrug-resistant (MDR) pathogens frequently associated with healthcare-acquired infections. The emergence of antimicrobial resistance underscores the urgent need for alternative therapeutics. This study explores the antimicrobial potential of fatty acids (FAs) extracted from Hermetia illucens (HI) larvae fat (AWME3) against MDR strains A. baumannii ATCC 19606 and S. aureus ATCC 55804. AWME3 exhibited potent inhibitory effects, with minimum inhibitory concentrations (MICs) of 0.38 mg/mL for A. baumannii and 0.19 mg/mL for S. aureus. Bactericidal activity occurred within 5–10 min at 0.75 mg/mL. Broth microdilution and propidium iodide uptake assays manifested FA-induced membrane permeabilization (55–70%) within 5 min, supporting a rapid membrane-targeting mechanism. Disruption of membrane integrity was accompanied by significant intracellular ATP depletion, cytoplasmic protein leakage, and altered cellular ultrastructure. AFM imaging showed significant morphological damage, with increased cell surface roughness in both bacterial strains. A. baumannii showed a significant height reduction (51–80%), while S. aureus had a reduction of 26–38% after exposure to 1 × MIC and 2 × MIC. AFM visualizations indicated severe cell envelope damage, including pore formation, blebbing, and surface collapse, consistent with membrane lysis. These findings reveal the swift and membrane-disrupting effects of AWME3 fatty acids on MDR nosocomial pathogens, underscoring their potential as a natural antimicrobial agent.

Key points

• Fatty acids from H. illucens fat show strong activity against MDR pathogens.

• Rapid bactericidal effect via membrane disruption and cytoplasmic leakage.

• AFM reveals nanoscale cell damage confirming membranolytic action.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00253-025-13631-x.

Introduction

The black soldier fly (Hermetia illucens) is considered a promising source of antibacterial lipids, drawing significant attention due to its potential applications in combating MDR bacteria. As the global health crisis surrounding antimicrobial resistance rises, there is an urgent need for alternative antimicrobial agents derived from natural sources. Lipids extracted from H. illucens larvae have demonstrated notable antimicrobial properties, particularly against a variety of pathogenic bacteria. Recent studies have demonstrated that the fatty acid composition of lipids from H. illucens varies depending on the substrates on which the larvae are reared (Spranghers et al. 2017; Mohamed et al. 2021; Tognocchi et al. 2024). Studies highlighted that specific fatty acids, such as lauric acid and capric acid, are particularly effective in inhibiting the growth of pathogens like Micrococcus flavus and E. coli (Marusich et al. 2020; Franco et al. 2024).

The antibacterial mechanisms of these lipids are believed to involve disruption of bacterial cell membranes, interference with biofilm formation, and modulation of bacterial virulence factors. This multifaceted approach not only enhances the efficacy of the lipids but also offers a potential pathway for developing novel antimicrobial agents that can be employed in agriculture and medicine (Ma et al. 2021; Almeida et al. 2022).

Atomic force microscopy (AFM) is a powerful tool in studying the mechanism of action of antibacterial fatty acids against MDR bacterial pathogens like Staphylococcus aureus and Acinetobacter baumannii. AFM enables the visualization of fatty acid interactions at the nanoscale by providing high-resolution imaging and manipulation capabilities (Yuan et al. 2020; Fukuma 2022). AFM is particularly effective in studying lipid membranes, offering direct imaging and mechanical probing of lipid phase structures in liquid environments down to the nanometer level (Oh And Hinterdorfer 2019). By utilizing AFM techniques such as frequency modulation and three-dimensional tip scanning, researchers can directly visualize the 3D distributions of mobile water, hydration structures, and flexible molecular chains, allowing for detailed studies on biological interfaces like lipid membranes and proteins (Yuan et al. 2020; Fukuma 2022). Additionally, AFM’s capability to measure stiffness, forces, and dissipation at the nanoscale through electrostatic interactions provides a comprehensive characterization of interactions, further enhancing the understanding of fatty acid interactions at the molecular level (Leonenko et al. 2008).

Fatty acids have long been recognized as potent antimicrobial agents, exhibiting broad-spectrum activity against a variety of bacterial pathogens (Casillas-Vargas et al. 2021; Borreby et al. 2023). This study aims to elucidate the mechanistic underpinnings of the antibacterial action of fatty acids against two standard significant multidrug-resistant bacteria, S. aureus ATCC 55804 and A. baumannii ATCC 19606. The cytotoxic effects of fatty acids were investigated through a combination of microbiological assays, proteomic analyses, and atomic force microscopy imaging. Exposure to fatty acids resulted in a significant reduction in bacterial viability for both S. aureus ATCC 55804 and A. baumannii ATCC 19606, with potent bactericidal activity observed even at low concentrations (Eder et al. 2017; Kim et al. 2018). Proteomic profiling revealed that fatty acid treatment led to the release of key ATP-generating and membrane-associated proteins, suggesting disruption of cellular bioenergetics and membrane integrity as potential mechanisms of action (Cartron et al. 2014).

Interestingly, the antibacterial activity of fatty acids was more pronounced against A. baumannii compared to S. aureus (Casillas-Vargas et al. 2021). This differential susceptibility may be attributable to the unique cell envelope structures and membrane properties of these pathogens, with A. baumannii potentially more vulnerable to the membrane-perturbing effects of fatty acids. S. aureus and A. baumannii are two of the most prevalent and concerning pathogens in the clinical setting. Understanding the mechanisms by which these organisms evade host immune defenses and withstand antimicrobial agents is crucial for developing effective treatment strategies (An et al. 2024; Bereanu et al. 2024). These bacteria are responsible for a range of infections, often leading to severe complications due to their ability to form biofilms and develop resistance to multiple antibiotics. Recent investigations into the antimicrobial properties of fatty acids have shown promising results, suggesting that they may serve as effective agents for the eradication of these resistant strains. Fatty acids, particularly monounsaturated and saturated types, have demonstrated selective bactericidal activity against S. aureus. Researchers have identified specific fatty acids, such as lauric acid and palmitoleic acid, which exhibit potent antibacterial properties. For instance, lauric acid has been shown to effectively inhibit the growth of S. aureus at low concentrations, with studies reporting minimum inhibitory concentrations (MIC) as low as 18.8 µg/mL (Watanabe et al. 2019). Additionally, sapienic acid, a fatty acid naturally present in human skin, has been recognized for its ability to selectively target S. aureus, thereby controlling its colonization without adversely affecting beneficial skin flora (Kikukawa et al. 2023). Similarly, the potential of fatty acids to disrupt the membrane integrity of A. baumannii has been explored. These fatty acids can alter membrane permeability and inhibit biofilm formation, which are critical factors in the pathogen’s survival and resistance mechanisms. The incorporation of fatty acids into the bacterial membrane has been shown to destabilize the structure, leading to cell lysis and enhanced susceptibility to other antimicrobial agents (Parsons et al. 2012).

The exploration of fatty acid extracts as a therapeutic modality against A. baumannii and S. aureus not only highlights their antimicrobial potential but also supports the development of novel treatment strategies that could mitigate the challenges posed by antibiotic resistance.

Fatty acids also influence the development of antibiotic resistance in A. baumannii. Studies have demonstrated that supplementation with certain fatty acids can reduce the mutation rates associated with antibiotic resistance. For example, polyunsaturated fatty acids like arachidonic and docosahexaenoic acids have been shown to decrease the rate at which A. baumannii acquires resistance to antibiotics such as erythromycin and tetracycline. This effect is closely linked to the primary antimicrobial efflux systems, such as the AdeABC and AdeIJK systems, which are responsible for pumping out antibiotics and are major contributors to resistance (Zang et al. 2021).

Uncovering the specific molecular pathways and virulence factors underlying the antibacterial activity of fatty acids against these pathogens could lead to the development of novel therapeutic approaches.

Materials and methods

H. illucens larvae fat

H. illucens larvae 15 days old were subjected to mechanical pressing under heating to produce oil, which was isolated, and then cooled to be used as solid fat and provided by NordTechSad, LLC (Arkhangelsk, Russia).

Microorganisms and growth conditions

Acinetobacter baumannii ATCC 19606 and Staphylococcus aureus ATCC 55804 strains were purchased from the American Type Culture Collection (ATCC), Manassas, USA. The studied bacterial strains were kept in glycerol stock (30%, v/v) at − 80 °C. To obtain a pure culture, a single colony from each strain was introduced and inoculated in 10 mL of the LB broth and then incubated overnight at 37 °C under shaking at 200 rpm/min. The overnight cultures were adjusted to be used in the next experiments.

Chemical reagents and media

Hydrochloric acid (HCl), methanol (CH₃OH), ethanol (C₂H₅OH), glutaraldehyde, and phosphate buffer saline (PBS) were purchased from Thermo Fisher Scientific, Waltham, MA, USA. The Muller Hinton (MH) and Lauri Brentani (LB) agar (Sigma-Aldrich, St. Louis, USA) were used for bacterial culturing.

Fatty acid extraction

The third acidic water–methanol extract (AWME3) was prepared sequentially from Hermetia illucens larvae fat (3 g), and the fatty acid composition was subsequently characterized by GC–MS as described previously (Mohamed et al. 2021). Briefly, 3 g of melted larvae fat (52 °C, 5 min, hot tap water) was extracted with an aqueous methanol–HCl solution (MQ water/methanol/HCl, 90:9:1, v/v). The mixture was vortexed to form an emulsion and subjected to continuous extraction for 24 h at room temperature using a Mixmate (Eppendorf AG, Hamburg, Germany). The solidified fat was remelted (52 °C, 5 min) and further homogenized by sonication (10 min, 35 °C, Elmasonic S 30H, Singen, Germany), followed by vigorous homogenization (ULTRA TURRAX-25, IKA, Staufen, Germany, 10 min). The resulting emulsion was centrifuged at 4000 × g for 20 min (Centrifuge 5804, Eppendorf AG, Hamburg, Germany) at room temperature, yielding a supernatant fraction designated AWME1. The residual oil phase was subjected to two additional extraction cycles using the same procedure to obtain AWME2 and AWME3, respectively. The AWME3 fraction was concentrated under vacuum (Concentrator Plus, Eppendorf AG, Hamburg, Germany) at 45 °C for 13 h and stored at 4 °C until further use. To ensure reproducibility, the extraction process was repeated three times, and the concentrated extract was adjusted to the required working concentration.

Determination of MIC and MBC of AWME3 extract

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of AWME3 and penicillin–streptomycin (P/S) were determined using the broth microdilution method as described previously (Mohamed et al. 2025). Briefly, 100 µL of AWME3 was serially diluted with twofold in LB broth in a 96-well microtiter plate to obtain final concentrations ranging from 48 to 3000 µg/mL. Each well was then inoculated with 100 µL of bacterial suspension adjusted to ~ 10⁶ CFU/mL. In parallel, P/S was serially diluted to yield final concentrations of 0.6–78.125 µg/mL. Plates were sealed and incubated at 37 °C for 24 h with agitation (200 rpm). Wells without visible bacterial growth were recorded as the MIC.

For MBC determination, 10 µL from wells corresponding to the MIC and higher concentrations was spotted onto Mueller–Hinton (MH) agar plates and incubated at 37 °C for 48 h. The lowest concentration showing complete absence of bacterial growth was recorded as the MBC. All MIC and MBC determinations were performed in triplicate across three independent experiments.

Time-killing assay

The antimicrobial activity of AWME3 over time was determined against S. aureus ATCC 55804 and A. baumannii ATCC 19606 according to Ramchuran et al. (2018) with minor changes. Overnight bacterial cell cultures were suspended in LB broth and adjusted to an absorbance of 10⁶ CFU/mL. Different concentrations of the AWME3 extract of H. illucens larvae fat or positive control (P/S) were added to the inoculum suspensions with final concentrations 0.5 MIC, MIC, 2 MIC, and 4 MIC (4.88–78.125 µg/mL) and incubated at 37 °C for 24 h under shaking at 180 rpm/min. Aliquots were removed from the inoculum cultures after 0, 5, 10, 20, and 60 min and then extended to 12 h and 24 h of incubation. After incubation, the bacterial cultures were serially diluted, plated on MH agar, and incubated for 24 h at 37 °C. The viable bacterial cells (CFU) were counted, and the degree and level of killing were determined by plotting the log CFU/mL against time with at least three independent experiments and analyzed with one-way ANOVA followed by Dunnett’s test to determine the significance relative to the untreated bacteria (p < 0.05).

Propidium iodide uptake (PI-uptake) assay

Membrane permeability is considered significant evidence for the disruption or cell membrane leakage due to the activity of FA in AWME3. PI is generally utilized as a DNA dye that can enter cells with damaged cell membranes, but it cannot enter cells with an intact cell membrane. PI-uptake was determined based on (Wu et al. 2019) with slight changes. Briefly, S. aureus ATCC 55804 and A. baumannii ATCC 19606 were grown on MH broth overnight at 37 °C under shaking at 180 rpm/min. Cells were washed three times with a buffer containing 5 mM HEPES and 20 mM glucose at pH 7.2, then adjusted to obtain a bacterial suspension with densities equivalent to a 0.5 McFarland turbidity standard, and then incubated with 7.5 µg/mL propidium iodide (Sigma-Aldrich) at 37 °C for 10 min. After incubation, the S. aureus ATCC 55804 and A. baumannii ATCC 19606 cells were treated with different concentrations of AWME3 (0.095–0.76 mg/mL) for 10 h, and the fluorescence intensity of the PI dye was monitored every 5 min using the CLARIOstar microplate reader at an excitation wavelength of 543 nm and an emission wavelength of 615 nm. The PI-uptake (%) was calculated based on the normalized data which are the mean ± SD of three independent experiments. One-way ordinary ANOVA Dunnett’s multiple comparisons test (*p < 0.05) was significant.

Cytoplasmic content leakage assay

The measurement of the release of 260 and 280 nm absorbing materials from S. aureus ATCC 55804 and A. baumannii ATCC 19606 was determined by measurement of the cytoplasmic materials released according to Nguyen et al. (2017) and Zhang et al. (2020) with minor modifications. Cells from the 5 mL of bacterial suspension were collected by centrifugation at 5000 × g for 10 min, washed three times with 0.1 M PBS (pH 7.4), and resuspended in PBS (0.1 M, pH 7.4) to a bacterial density of 10⁶ CFU/mL. The 3.0 mL of cell suspension was incubated at 37 °C under agitation in an environmental incubator shaker for 0 h, 1 h, and 2 h in the presence and absence (control) of AWME3 of two concentrations (MIC, 2 MIC). Then, the suspensions were centrifuged at 4000 × g for 5 min. After that, the absorption at 260 nm and 280 nm of supernatants was measured after filtration with a 0.22-µM syringe filter (Sartorius, Kings Norton, Birmingham, UK) using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Results were presented in terms of the optical density of 260 nm and 280 nm absorbing materials. Mean OD values of respective treatments and vehicle control were compared independently at each time point.

Intracellular ATP assay

The effect of AWME3 on cellular ATP levels of S. aureus ATCC 55804 and A. baumannii ATCC 19606 was determined using the ViaLight MDA Plus assay kit (Lonza Rockland, Inc., USA) based on (Lin et al. 2017) assay with minor modification, following the manufacturer’s instructions. The assay was performed in a 96-well U-bottom opaque white microtiter plate in triplicate. S. aureus ATCC 55804 and A. baumannii ATCC 19606 were grown in MH broth at 37 °C overnight, and cells were harvested by centrifugation, washed, and resuspended in fresh medium. Next, 50 µL of MH broth was added to a 96-well opaque plate, and then, 50 µL of AWME3 was diluted in twofold dilutions at different concentrations (0.75, 0.38, 0.19, 0.095 mg/mL). Likewise, positive control (P/S) was diluted in twofold dilutions under the same conditions at concentrations (4.88–39 µg/mL); after that, 50 µL of 106 CFU/mL was added to each well and incubated for 24 h at 37 °C. Next, 50 µL of bactolyse plus was added to each well and then incubated at room temperature for 10 min without shaking. Next, 100 µL of AMR plus was added to each well and incubated at room temperature for 2 min. The opaque microtiter 96-plate (Thermo Scientific, USA) was inserted into the BMG CLARIOstar Reader, and the ATP luminescence was recorded. Signals represent the mean of three replicates for each measurement.

Atomic force microscopy analysis

To study the possible effects of AWME3 on Gram-positive S. aureus ATCC 55804 and Gram-negative A. baumannii ATCC 19606 cells at the level of micro- or nano-scale, we used atomic force microscopy (AFM). AFM is an extremely useful tool for analyzing the two and three-dimensional topography of biological samples, including bacteria. The technique allows the characterization of the bacterial cell surface, producing high-resolution topographical imaging with minimal sample disruption (Müller and Dufrêne 2011). In this study, we analyzed and detected the morphological characteristics of S. aureus ATCC 55804 and A. baumannii ATCC 19606 based on the measurement of the calculated seven selected parameters including, length (µm), width (µm), height (nm), surface roughness (nm), root mean square (RMS) of surface, kurtosis (Ska), and the skewness (Ssk).

The samples of S. aureus ATCC 55804 and A. baumannii ATCC 19606 were prepared similarly to the method described in Sahu et al. (2009), Santana et al. (2012), and Campos et al. (2020) with some modifications. All bacterial cultures were harvested at checkpoints and centrifuged at 5000 × g at 4 °C for 10 min; then, all bacterial pellets were washed 3 times with PBS (10 mM, pH 7.2). After that, 100 µL of every culture was spread on a glass slide (Thermo Scientific, USA) and dried under a sterile condition in a cabinet for 20 min. Subsequently, the bacterial cells were fixed overnight with 500 µL of 2.5% (v/v) glutaraldehyde at 4 °C. The fixed samples were washed twice with 10 mM PBS, followed by dehydration in gradient ethanol solutions (50, 70, 90, and 95%) for 10 min. The surface topography of treated and untreated (control) bacteria was imaged using AFM (NT-MDT, Moscow, Russia). Air-dried samples were used for AFM imaging, where this mode of imaging is generally performed to evaluate the alterations in the bacterial morphology caused by AWME3.

V-shaped cantilevers with oxide-sharpened Si3N4 tips were used with spring constants. The silicon nitride tip was irradiated with ultraviolet in the air for 15 min to remove any organic contaminants prior to use. The curvature radius of the tips is less than 10 nm, and the length, width, and thickness of the cantilevers are 183, 34, and 3.0 µm, respectively, with an oscillation frequency of 144 kHz and a force constant of 6.0 N/m. AFM, images, and force measurements were recorded in contact mode at room temperature. Different areas were scanned, and the images were analyzed with Image Analysis P9 (IAP9) application software (NT-MDT Co., Moscow, Russia) to obtain the morphological characteristics (length, width, height, roughness, root mean square of surface (RMS), kurtosis (Ska), skewness (Ssk)). In each sample (control as well as treated), an average of 35–50 cells were selected randomly from two independent experiments and were imaged to ascertain the effect of AWME3 treatment on cell surface morphology.

Statistical analysis

Statistical analysis was performed by ordinary one-way ANOVA, and Dunnett’s post-hoc test was conducted to determine significant differences between treatment means. All experimental determinations represent observations from at least triplicate samples obtained from three experiments performed independently. All the data were assessed by the standard deviation (SD) and standard error means (SEM) using the software GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, USA). Differences between means were considered statistically significant at p < 0.05.

Results

Composition and activity of AWME3 against S. aureus ATCC 55804 and A. baumannii ATCC 19606

The chemical composition of AWME3 was investigated and identified using GC–MS; in addition, the fatty acid profile in AWME3 was described previously (Mohamed et al. 2021) (Table S1 and Fig. S1). The antibiotic sensitivity was evaluated against MDR Gram-negative A. baumannii ATCC 19606 and Gram-positive S. aureus ATCC 55804, where S. aureus was resistant to kanamycin, colistin, and vancomycin, while A. baumannii ATCC 19606 showed high resistance against 50% of the total ten tested antibiotics. The antibacterial activity of AWME3 was also estimated against the same tested strains, where IZD recorded 18.43 ± 0.35 mm and 18.1 ± 0.35 mm at a concentration of 20 mg/mL against S. aureus ATCC 55804 and A. baumannii ATCC 19606, respectively. In addition, MIC of AWME3 recorded 190 µg/mL and 380 µg/mL against S. aureus ATCC 55804 and A. baumannii ATCC 19606, respectively, while MBC recorded 380 µg/mL against both tested bacterial strains (Mohamed et al. 2025). After confirming the outstanding antimicrobial activity of AWME3 against S. aureus ATCC 55804 and A. baumannii ATCC 19606, the mechanism of action of AWME3 was assessed based on several assays. The time-kill assays were performed to elucidate the rate and extent of antibacterial activity and distinguish bacteriostatic from bactericidal activity (Fig. 1).

Fig. 1.

Time-kill curves of AWME3 isolated from HI larvae fat performed in LB broth at 0.5 MIC, MIC, 2 MIC, and 4 MIC levels of AWME3 against a A. baumannii ATCC 19606, c S. aureus ATCC 55804, b penicillin–streptomycin (P/S), against the A. baumannii ATCC 19606, and d against S. aureus ATCC 55804. Viability was assessed by determining CFU/mL at incubation times (0, 5, 10, 20, 60 min) during the first hour after AWME3, positive control (P/S) exposure, and following 12 h and 24 h of incubation. Statistical differences (*p < 0.05) between CFU recovered from treated and untreated cultures are shown (non-parametric one-way ANOVA with Dunnett’s multiple comparisons test). The results are expressed as mean ± SEM of three independent experiments

In controls, the bacterial counts increased by 2.41 log₁₀ CFU/mL after 12 h of incubation. The killing assay of AWME3 and the reference antibiotic (P/S) was conducted in parallel to determine their bactericidal and bacteriostatic behavior. AWME3 showed the killing of MDR S. aureus ATCC 55804 and A. baumannii ATCC 19606 in a concentration-dependent manner. Thus, at 2 MIC (0.75 mg/mL), 4 MIC (1.5 mg/mL) of AWME3 showed fast bactericidal activity capable of eliminating a high starting inoculum of A. baumannii ATCC 19606 (5.17 × 10⁵ CFU/mL) within 5 min (Fig. 1a). At the MIC (0.38 mg/mL), a slower bactericidal activity with complete eradication of A. baumannii ATCC 19606 was observed after 10 min of incubation. On the other hand, the S. aureus ATCC 55804 strain was eliminated after exposure to 4 MIC (0.75 mg/mL) of AWME3 within 10 min. S. aureus ATCC 55804 planktonic cells were eliminated after treatment with 2 MIC (0.38 mg/mL) within 60 min. In contrast to AWME3, standard antibiotic (P/S) demonstrated slower killing log reduction after 12 h of exposure and 24 h spent to eliminate S. aureus ATCC 55804 cells when treated with 1, 2, and 4 MIC of AWME3. The rapid bactericidal activity of AWME3, when compared to the positive control (P/S), is mainly attributed to its ability to more rapidly permeabilize the S. aureus ATCC 55804 and A. baumanni ATCC 19606 membranes. AWME3 with fast bactericidal activity against MDR strains of A. baumannii ATCC 19606 and S. aureus ATCC 55804 reduces the potential emergence of bacterial resistance and the duration of treatment. The low MIC concentrations and the high bactericidal effect of AWME3 against S. aureus ATCC 55804 and A. baumannii ATCC 19606 encouraged us to more deep investigation of the mechanism of AWME3 action, which could explain the high efficacy of AWME3 against a broad spectrum of bacterial pathogens.

Assessment of the bacterial cell membrane integrity

Based on the enhanced toxicity of AWME3, we hypothesized that the ingredient compounds of AWME3 extract are particularly effective at compromising the integrity of the bacterial membrane, causing toxicity to bacterial cells. To support this hypothesis, we employed a propidium iodide (PI) staining assay. PI can only penetrate bacterial cells with compromised membranes and binds nucleic acids of the planktonic S. aureus ATCC 55804 and A. baumannii ATCC 19606 cells. The PI uptake assay was performed on A. baumannii ATCC 19606 and S. aureus ATCC 55804, used as representatives of Gram-negative and Gram-positive strains, respectively, and exposed to the same concentrations of AWME3 (0.19–0.75 mg/mL). AWME3 increased the number of PI-positive cells within a few minutes and in a concentration-dependent manner (Fig. 2), indicating that AWME3 has the capability of permeabilizing bacterial cells at the same or even lower concentrations.

Fig. 2.

Membrane disruption and permeabilization. Membrane disruption induced by AWME3 as detected by PI uptake at 0.5, 1, and 2 MIC in a, b S. aureus ATCC 55804 and c, d A. baumannii ATCC 19606. The permeabilization assay with AWME3 has been performed in a mixture (1:1) of 20 mM glucose and 5 mM HEPES. Bacterial cells were treated for different incubation times, with indicated concentrations of AWME3. The PI-uptake (%) was calculated based on the normalized data which are the mean ± SD of three independent experiments. One-way ordinary ANOVA Dunnett’s multiple comparisons test (*p < 0.014) was significant

AWME3 permeabilized more than 60% of S. aureus ATCC 55804 cells after 5 min of treatment at the MIC concentration (0.19 mg/mL); the permeability increased to reach more than 70% at 2 MIC (Fig. 2a and b). An even higher degree of permeabilization was observed in S. aureus ATCC 55804 cells at 2 MIC to reach more than 90% after 600-min incubation. Likewise, A. baumannii ATCC 19606 cell membrane permeability increased by more concentrations of AWME3 and the time of incubation. The MIC (0.38 mg/mL) and 2 MIC (0.75 mg/mL) of AWME3 permeabilized 55% and 70%, respectively, within 5 min of treatment. The treatment with sub-MIC (0.095 mg/mL) of AWME3 achieved 30% of permeabilized S. aureus ATCC 55804 cells within 5 min, while the sub-MIC of AWME3 (0.19 mg/mL) caused permeability with 20% of the A. baumannii ATCC 19606 after 5 min exposure (Fig. 2c and d). All AWME3 treatments have a great significant permeabilization (p = 0.0001) of the cell membrane of both S. aureus ATCC 55804 and A. baumannii ATCC 19606 at different time exposures. These results clearly show that the addition of the AWME3 dramatically kills and inhibits the growth of S. aureus ATCC 55804 and A. baumannii ATCC 19606 strains.

Leakage of cellular cytoplasmic materials

Nucleic acid and protein of macromolecules in the cell have maximum absorption peaks at 260 nm and 280 nm, respectively. Another strategy for determining the antimicrobial mode of action of AWME3 against Gram-positive and Gram-negative human pathogenic bacteria was performed based on the release of 260 nm absorbing materials from the S. aureus ATCC 55804 and A. baumannii ATCC 19606 cells treated with AWME3. Figure 3 shows the leakage of nucleic acid and proteins of the bacteria treated with different concentrations of AWME3 (MIC, 2 MIC). The OD₂₆₀ value of the control group showed few changes during the first hour, while intracellular released materials increased to (OD₂₆₀ = 1) after 2 h of incubation due to disrupted cells during washing, centrifugation, and osmotic effect of PBS buffer (pH = 7.2 ± 0.2) on the cell membranes. On the other hand, minor changes were observed in the released genetic material at 280 nm in the negative control during incubation time. After treatment, an approximately more than threefold increase in the optical density of the bacterial cell culture filtrates treated with 2 MIC of AWME3 was observed compared to the negative control (Fig. 3A and C). A similar effect was observed when A. baumannii ATCC 19606 cells were treated with MIC (0.38 mg/mL) and 2 MIC (0.75 mg/mL) of AWME3 during the same interval of incubation time (Fig. 3A and B), while significant increase (****p < 0.0001) in released genetic materials occurred when S. aureus ATCC 55804 cells exposed to MIC (0.19 mg/mL) and 2 MIC (0.38 mg/mL) of AWME3 during the first hour of incubation (Fig. 3C and D). These findings directly indicate the confirmation of leakage of 260 and 280 nm absorbing materials from the bacterial cells treated with MIC and 2 MIC of AWME3. Altogether, cytoplasmic material leakage and disruption of cytoplasmic membrane integrity allow for the leakage of cytoplasmic contents out of the cell can be significant evidence for killing bacteria after AWME3 treatment.

Fig. 3.

Loss of 260 and 280 nm absorbing material. A. baumannii ATCC 19606 cells were treated with AWME3 (A, B) at MIC (0.38 mg/mL) and 2 MIC (0.75 mg/mL), respectively. S. aureus ATCC 55804 cells were treated with AWME3 (C, D) at MIC (0.19 mg/mL) and 2 MIC (0.38 mg/mL), respectively. Data represent mean ± standard error of the mean from three independent experiments. Two-way RM ANOVA Dunnett’s multiple comparisons test was used for data analysis. The asterisks denote statistical significance (p < 0.05) between the control and the treatments (MIC and 2 MIC) of AWME3 obtained from Dunnett’s test. OD, optical density

Intracellular ATP leakage

Previous studies demonstrated that intracellular ATP provides energy for normal physiological activities in microorganisms (Shi et al. 2016; Fei et al. 2019). High-sensitivity Microbial Detection ViaLight® MDA Plus Kit is used to measure the bioluminescent ATP molecules that are present in all metabolically active cells. Figure 4A and C show that the intracellular ATP concentrations of A. baumanii and S. aureus ATCC 55804 cells treated with various concentrations of AWME3 at MIC and 2 MIC incubated for 24 h at 37 °C were significantly reduced (p < 0.05) compared to those of the control group (untreated bacteria). In addition, with the increase of AWME3 concentrations, the intracellular ATP concentration of tested cells was significantly reduced, and p-values were in the range (0.0017–0.0001). Likewise, intracellular ATP levels in both A. baumannii ATCC 19606 and S. aureus ATCC 55804 cells were significantly reduced (****p < 0.0001) when treated with different concentrations of P/S (4.88–39.06 µg/mL), as obvious in Fig. 4B and D.

Fig. 4.

Change in intracellular ATP concentrations presented in metabolically active cells of A, BA. baumannii ATCC 19606 and C, D S. aureus ATCC 55804. Bacterial cells treated with 0.0, 0.5, 1, and 2 MIC of AWME3 extracted sequentially from A, C Hi larvae fat and B, D bacterial cells treated with 0.0, 0.5, 1, and 2 MIC of the positive control (P/S) using a bioluminescent method based on the conversion of ATP by luciferase. Values represent the means of independent triplicate measurements. Bars represent the mean values ± standard error of the mean (SEM) from three independent experiments; one-way ordinary ANOVA Dunnett’s multiple comparisons test (p-values were **p = 0.0017, ***p = 0.0001, ****p < 0.0001) were significant

AFM images of S. aureus ATCC 55804 control cells

The AFM images of freshly prepared untreated S. aureus ATCC 55804 show a typical spherical or cocci morphology, with a relatively smooth surface and no alterations (Fig. 5a). The cell wall and cell membrane appeared intact with no ruptures or large pores (black arrows) (Fig. 5a and d).

Fig. 5.

Morphological changes induced by AWME3 against S. aureus ATCC 55804 bacteria. Control (a), treated MIC (e) for 12 h, 2 MIC for 20 min (i), and with 4 MIC for 10 min (m) of AWME3: topography (a, e, i, m), cross-section (b, f, j, n), three dimensional (c, g, k, o), and edge enhancement, Laplacian 5 × 5 (d, h, I, p) images. Blue arrowheads show the degradation of the cell walls; green arrowheads indicate the lysed cells and debris of cells in 2D images at the left panel. Green cycles indicate the lysed cells in 3D images, and the transparent black arrowheads refer to the cell wall damage of S. aureus ATCC 55804 exposed to different concentrations (0.19–0.75 mg/mL) of AWME3 at the right panel

AFM images of S. aureus ATCC 55804 cells exposed to low and high doses of AWME3

At a low concentration of AWME3 (0.19 mg/mL), the initial morphological changes observed are that of indentations appearing on the surface of some cells as well as some micelle-like structures or outer membrane residues found around or anchored on the cells (blue arrows, Fig. 5e). This indicates the disruption of the outer membrane of the bacteria, probably due to direct interaction and binding of AWME3 to the peptidoglycan, which is specific to Gram-positive bacteria. This perturbation may have damaged the outer membrane because of exposing the peptidoglycan wall to 0.19 mg/mL of AWME3. The bacterial cultures treated with MIC (0.19 mg/mL) of AWME3 and examined by AFM showed evidence of morphological changes induced at the membrane level by fatty acids action, which were the most abundant in the AWME3 (Fig. 5e).

The AFM images allowed us to determine and compare the bacterial cell dimension but also provided details on cell surface topology. A significant decrease (****p < 0.0001) was observed in the cell dimension of S. aureus ATCC 55804 when it was treated with 0.19 mg/mL of AWME3 for 12 h. The cell length and width of treated S. aureus cells were 0.94 ± 0.19 µm and 0.89 ± 0.17 µm, respectively, compared to the untreated S. aureus ATCC 55804 cells, which were 1.1 ± 0.16 µm and 1.15 ± 0.14 µm, respectively (Table 1 and Fig. 6A and B). The AFM topography images (Fig. 5b, f, j, and n), the cellular height of two independent experiments (n = 30 bacteria per treatment) was determined. The height was estimated through the cross-section profile of each bacterium recorded. The analysis of height distribution shows that the average height of untreated S. aureus ATCC 55804 cells was 762.9 ± 99.1 nm (Fig. 5b). However, when treatments with AWME3 were applied, a high significant decrease (****p < 0.0001) to 566.5 ± 90.9 nm was recorded when bacterial cells were treated with MIC (0.19 mg/mL) for 12 h (Figs. 5b and 6C).

Table 1.

Morphological characteristics of S. aureus ATCC 55804 and A. baumannii ATCC 19606 strains at different concentrations of AWME3

Strain	AWME3 (mg/mL)	Length (µm)	Width (µm)	Height (nm)	Ra (nm)	Ska	Ssk	RMS (nm)
S. aureus ATCC 55804	Control	1.1 ± 0.16	1.15 ± 0.14	762.9 ± 99.1	43.17 ± 9.1	15.15 ± 4.3	3.3 ± 0.67	126.4 ± 22.6
	0.19	0.94 ± 0.19	0.89 ± 0.17	566.5 ± 90.9	56.91 ± 13.8	4.39 ± 0.59	1.37 ± 0.19	178.03 ± 25.6
	0.38	0.76 ± 0.22	0.68 ± 0.17	476.9 ± 32.4	73.9 ± 15.2	6.6 ± 2.64	1.81 ± 0.58	126.03 ± 44.1
	0.75	0.59 ± 0.18	0.55 ± 0.15	238.8 ± 66.5	16.59 ± 5.7	17.97 ± 2.95	3.64 ± 0.78	43.67 ± 15
A.baumannii ATCC 19606	Control	2.18 ± 0.32	1.15 ± 0.19	776.6 ± 128.3	19.9 ± 5.6	4.12 ± 2.9	1.17 ± 0.56	102.8 ± 18.5
	0.19	2.7 ± 1.4	0.93 ± 0.18	471.2 ± 108.6	49.2 ± 18.9	8.37 ± 4.2	2.29 ± 0.69	143.08 ± 35.6
	0.38	2.08 ± 0.6	0.82 ± 0.21	383 ± 81.1	56.4 ± 13.8	11.9 ± 6.4	2.9 ± 0.87	109.8 ± 16
	0.75	1.59 ± 0.67	0.79 ± 0.34	162.4 ± 75.4	14.4 ± 4.4	6.7 ± 3.8	1.59 ± 0.66	104.1 ± 40.2

Ra roughness average, Ska kurtosis average, Ssk surface skewness, RMS root mean square of surfaces

Fig. 6.

Cell dimensions: length (A), width (B), height (C), roughness (D), root mean square of surface (RMS) (E), kurtosis (Ska) (F), and skewness (Ssk) (G) measurements from multiple images of control bacteria (n = 50) and 0.19 mg/mL (MIC), 0.38 mg/mL (2 MIC), and 0.75 mg/mL (4 MIC) of AWME3 isolated from HI larvae fat treated bacteria (n = 50). All images of AFM were obtained from two independent experiments; Graph Pad Prism was used to build up all graphs and one-way ANOVA Dunnett’s multiple comparisons test for statistical comparison between treated and untreated (control) S. aureus ATCC 55804 cells; all data are the average ± standard deviation, p-value (*p = 0.041, ****p < 0.0001)

Some fluids and debris were detected around the apical end of the cells, as shown in Fig. 5e; furthermore, the cell wall and cytoplasmic membrane were damaged and collapsed in multiple locations (Fig. 5h).

At higher AWME3 concentrations (0.38 and 0.75 mg/mL), the perturbations observed on the cell surface are more evident. Bacterial strains were severely affected (Fig. 5i, k, and l), where cell walls and cell membranes were degraded and broken; lysed cells and cell debris were formed. A great significant reduction (****p < 0.0001) in the cell dimension was observed (Fig. 6), where the cell length and width decreased to 0.76 ± 0.22 µm and 0.68 ± 0.17 µm, respectively, when S. aureus ATCC 55804 cells were subjected to 2 MIC (0.38 mg/mL) of AWME3 (Table 1 and Fig. 6A and B). At high concentrations of AWME3, severe reduction in height values (476.9 ± 32.4 nm, 238.8 ± 66.5 nm) were recorded, and a great significant (****p < 0.0001) was observed when S. aureus ATCC 55804 cells treated with 2 MIC (0.38 mg/mL) and 4 MIC (0.75 mg/mL), respectively, compared to the control group (Table 1 and Figs. 5j and n and 6C). The shrinkage ratio for treated S. aureus ATCC 55804 cells was calculated based on the height values, which were recorded at 27.1%, 37.5%, and 68.7%, when bacterial cells were exposed to 0.19 mg/mL, 38 mg/mL, and 0.75 mg/mL of AWME3, respectively. Therefore, the height reduction observed in S. aureus ATCC 55804 cells after the treatment with AWME3 suggests shrinkage in the volume of S. aureus ATCC 55804 cells, probably due to loss of intracellular materials by cell collapse.

In other cases, the collapse of the whole cell was evident and observed in the 2D and 3D images (Fig. 5i, k, and l). High-resolution images of the cell surface show severe damage triggered by AWME3 exposure (Fig. 5i, k, l, m, o, and p), in comparison with those of the control cells (Fig. 5a, c, and d). There is a high shrinkage (0.47%, 53%) in the length and the width (0.59 ± 0.18, 0.55 ± 0.15 µm), respectively, of S. aureus ATCC 55804 cells, when they were subjected to 4 MIC (0.75 mg/mL) of AWME3.

Changes in bacterial surface roughness (Ra) and root mean square of roughness (RMS) values

The AFM images demonstrate that the surface roughness values of treated S. aureus ATCC 55804 cells increased, where the average roughness for the untreated cells is 43.17 ± 9.1 nm for S. aureus ATCC 55804. When S. aureus ATCC 55804 cells were exposed to 0.19 mg/mL and 0.38 mg/mL, a good significant (**p = 0.001) increase in the average surface roughness of MDR S. aureus ATCC 55804 with 1.3-fold greater than untreated bacteria recorded 56.91 ± 13.8 nm (Table 1 and Fig. 6D). The highest significant (****p < 0.0001) increase in the roughness surface of S. aureus ATCC 55804 cells was 73.9 ± 15.2 nm, which was 1.7-fold greater than the untreated cells when S. aureus ATCC 55804 exposed to 0.38 mg/mL of AWME3. Sharply significant decrease (****p < 0.0001) in the roughness recorded 16.59 ± 5.7 nm with shrinkage (62%), when S. aureus cells ATCC 55804 treated with 0.75 mg/mL of AWME3 for 10 min (Table 1 and Fig. 6D). High-resolution images of S. aureus ATCC 55804 show high disturbances and great alterations in the morphological characteristics of treated S. aureus ATCC 55804 cells (Fig. 5e, g, h, i, k, l, m, o, and p) compared to the control group (Fig. 5a, c, and d). Bacterial surface roughness, termed as RMS value, was another quantitative parameter to evaluate the cell surface morphology. The results in Table 1 showed that the tested bacteria had rough surfaces. For the control group of S. aureus ATCC 55804 strain cells, its average RMS roughness value recorded 126.4 ± 22.6 nm, while a significant increase (*p = 0.041) of RMS index was observed when S. aureus ATCC 55804 cells were treated with MIC (0.19 mg/mL) of AWME3 to be 178.03 ± 25.6 nm. At the highest concentration of 4 MIC (0.75 mg/mL) of AWME3, a great significant decrease (*p = 0.0019) of RMS value was recorded to be 43.67 ± 15 nm, while no significant difference recorded when the cells were exposed to 2 MIC (0. 38 mg/mL) compared to untreated bacterial cells (Table 1 and Fig. 6E).

Changes of S. aureus ATCC 55804 cell morphology parameters

Bacterial shape parameters include Ska and Ssk, where these parameters are able to determine the shape irregularities. Kurtosis (Ska) parameter was significantly decreased (***p = 0.0005, **p = 0.003), when S. aureus ATCC 55804 cells were treated with MIC (0.19 mg/mL) and 2 MIC (0.38 mg/mL) of AWME3, to be 4.39 ± 0.59 and 6.6 ± 2.64, respectively, compared to the untreated cells (15.15 ± 4.3). In contrast, when S. aureus ATCC 55804 cells were treated with a high concentration 4 MIC (0.75 mg/mL) of AWME3, no statistical difference for Ska (17.97 ± 2.95) was significant compared to the control group (Table 1 and Fig. 6F). In addition, skewness (Ssk) decreased sharply and recorded (1.37 ± 0.19, 1.81 ± 0.58), when bacterial cells of S. aureus ATCC 55804 were subjected to 0.19 mg/mL and 0.38 mg/mL, respectively, with a good significant difference (**p = 0.001, **p = 0.009), respectively, compared to the untreated cells (3.3 ± 0.67). On the other hand, no significant difference was observed in the Ssk parameter (3.64 ± 0.78), when S. aureus ATCC 55804 cells were treated with 0.75 mg/mL for 10 min, compared to the control group (Table 1 and Fig. 6F). Total cell damage is observed at the highest concentration of AWME3 assayed (75 mg/mL) (Fig. 5m, o, and p). High-resolution images of the cell surface show severe damage triggered by AWME3 exposure (Fig. 5m, o, and p) in comparison with those of the control cells (Fig. 5a, c, and d). Thus, the topographical changes observed in the bacterial cells become more pronounced as the concentration of AWME3 used in the treatment increases. Moreover, a clean substrate surface is observed as background around the untreated cells (Fig. 5a, c, and d). In contrast, small granules or aggregates are observed on the polymeric support close to the AWME3-treated cells (Fig. 5i, k, l, m, o, and p). This behavior is concomitant with the damage of cell integrity, suggesting that the aggregates could correspond to intracellular content and/or cellular debris.

AFM images of A. baumannii ATCC 19606 control cells

The morphological effects of AWME3 on the Gram-negative A. baumannii ATCC 19606 strain are presented in Table 1 and Fig. 7.

Fig. 7.

Representative images of morphological changes induced by AWME3 against A. baumannii ATCC 19606 bacteria. Control (a), treated with 0.5 MIC (0.19 mg/mL) for 12 h (e), MIC (0.38 mg/mL) for 20 min (i), and with 2 MIC (0.75 mg/mL) for 10 min (m) of AWME3: topography (a, e, i, m), cross-section (b, f, j, n), three dimensional (c, g, k, o), and edge enhancement, Laplacian 5 × 5 (d, h, i, p) images. Blue arrowheads show the degradation of the cell walls; green arrowheads indicate the lysed cells and debris of cells in 2D images at the left panel. Green cycles indicate the lysed cells in 3D images, and the transparent black arrowheads refer to the cell wall damage of A. baumannii ATCC 19606 exposed to different concentrations (0.19–1.5 mg/mL) of AWME3 at the right panel. Cell elongation phenomena are obvious when the A. baumannii ATCC 19606 cells are subjected to 0.5 MIC of AWME3 for a long period (12 h) (e)

High-resolution representative images were selected from two independent cultures; results show an analysis of these seven parameters, which were observed on at least 50 bacterial cells collected from two independent cultures. The threshold method was used to determine the cell dimension (length, width) of A. baumannii ATCC 19606 cells. Planktonic bacterial cells in native conditions (without treatment) show a smooth surface, with intact cell walls, and coccobacilli. Bacterial cells appeared close to each other and distinctly similar in the same image (Fig. 7a, c, and d). Cell walls and membranes were intact without any rupture or damage, distinguished by a regular septum division located in the center of the cells, and divided the cell into two symmetrical parts (Fig. 7c and d). Untreated cells were investigated with an average cell length of 2.18 ± 0.32 µm, width of 1.15 ± 0.19 µm, and a maximum height of 776.6 ± 128.3 nm for 50 cells (Table 1 and Figs. 7b and 8A, B, and C), representative of the investigated A. baumannii ATCC 19606 strain.

Fig. 8.

Cell dimensions: length (A), width (B), height (C), roughness (D), root mean square of surface (RMS) (E), kurtosis (Ska) (F), skewness (Ssk) (G) measurements from multiple images of control bacteria (n = 50) and 0.19 mg/mL (0.5 MIC), 0.38 mg/mL (MIC), and 0.75 mg/mL (2 MIC) of AWME3 isolated from HI larvae fat treated bacteria (n = 50). All images of AFM were obtained from two independent experiments; Graph Pad Prism was used to build up all graphs and one-way ANOVA Dunnett’s multiple comparisons test for statistical comparison between treated and untreated (control) A. baumannii ATCC 19606 cells; all data are the average ± standard deviation, p-value (*p = 0.041, ****p < 0.0001)

The average surface corrugation (roughness-Ra) of an untreated A. baumannii ATCC 19606 cell was determined to be 19.9 ± 5.6 nm (Table 1 and Fig. 8D). Root mean square (RMS) roughness measured by AFM was correlated with the roughness of the RMS average for untreated A. baumannii ATCC 19606, which was 102.8 ± 18.5 nm. The bacterial cell shape parameters Ska and Ssk were calculated and measured by 3D roughness analysis of images using the program 3.5.0.19426 (NT-MDT Co, Amsterdam, Netherlands) and revealed as 4.12 ± 2.9 and 1.17 ± 0.56, respectively (Table 1 and Fig. 8E, F, and G).

Effect of low AWME3 concentrations on the A. baumannii ATCC 19606 cell morphology

The 2D, 3D, and Laplacian 5 × 5 images (Fig. 7e, j, and h) show the disturbances and alterations in A. baumannii ATCC 19606 cells after exposure to 0.5 MIC of AWME3 (0.19 mg/mL). Bacterial cells appeared elongated; long bacilli, without septum (Fig. 7e), 3D, and Laplacian 5 × 5 images of AFM show cell walls; and membranes were wrinkled and rough; and multiple cleavages and splits were obvious in many positions of the cell walls (black and blue arrows) (Fig. 7g and h). Lysed and flattened cells were obvious (green arrows and circles) as shown in Fig. 7g and h. Bacterial cell dimensions (length, width), height, roughness, RMS, and shape indexes (Ska, Ss) were determined and measured after treatment with 0.19 mg/mL of AWME3 against planktonic A. baumannii cells ATCC 19606 (Table 1 and Fig. 8). A. baumannii ATCC 19606 cell length was significantly increased (p = 0.0067) to be 2.7 ± 1.4 µm compared to the untreated bacterial cells, which recorded 2.18 ± 0.32 µm (Table 1 and Fig. 8A), while a great significant decrease (p < 0.0001) in the cell width to be 0.93 ± 0.18 µm compared to the control group, which was 1.15 ± 0.19 µm (Fig. 8B). A sharp decrease was obvious in the height (471.2 ± 108.6 nm of treated A. baumannii ATCC 19606 cells with 0.5 MIC of AWME3, and a significant statistical difference (p < 0.0001) was obvious compared to untreated cells (776.6 ± 128.3 nm) (Table 1 and Figs. 7b and f and 8C). Shrinkage with 40% in the cell height of A. baumannii ATCC 19606 strain was induced when exposed to 0.5 MIC of AWME3. The bacterial surface roughness values as a function of AWME3 treatment at 0.5 MIC were quantified for the A. baumannii ATCC 19606 strain. Investigation of AFM captured height images at 2D and 3D indicated that exposure of MDR-A. baumannii cells to AWME3 at 0.5 MIC, irrespective of exposure time (12 h), increased the surface roughness significantly (p < 0.001) of cells to be 49.2 ± 18.9 nm, which was 2.47-fold greater than their corresponding control (19.9 ± 5.6 nm) (Table 1 and Fig. 8D). RMS roughness of the cell surface increased significantly (p < 0.0001) to be 143.08 ± 35.6 nm when A. baumannii ATCC 19606 subjected to 0.5 MIC of AWME3 compared to the control group (102.8 ± 18.5 nm) (Table 1 and Fig. 8E). The average of Ska and Ssk values of treated A. baumannii ATCC 19606 increased significantly (p = 0.012, p = 0.016) to be (8.37 ± 4.2, 2.29 ± 0.69) compared to the control groups, which recorded 4.12 ± 2.9 and 1.17 ± 0.56, respectively (Table 1 and Fig. 8F and G).

Effect of high AWME3 concentrations on the A. baumannii ATCC 19606 cell morphology

Extensive alterations and disturbances were detected in A. baumannii ATCC 19606 cells after exposure to MIC (0.38 mg/mL) and 2 MIC (0.75 mg/mL) of AWME3 (Fig. 7i, k, l, m, o, and p) for 20 and 10 min, respectively. Bacterial cells became shorter and rougher with an irregular septum compared with the untreated cells. Exposing to MIC (0.38 mg/mL) of AWME3 for 20 min caused several changes in the cell walls and membranes of A. baumannii ATCC 19606, where cell walls were degraded and damaged; severe cleavages, splits, pores, and serious swelling were formed on the surface of the cells (blue arrows, green circle) (Fig. 7i and k). Figure 7l shows complete degradation and rupture of cell walls, while the cell membrane appeared injured and cleaved in multiple locations (black arrows) and collapsed in several cells.

The cell length of the treated A. baumannii ATCC 19606 strain decreased lower (2.08 ± 0.6 µm) without significant (p = 0.91) statistical difference compared to the untreated bacteria (2.18 ± 0.32 µm), while cell width was significantly (p < 0.0001) decreased to record 0.82 ± 0.21 µm, corresponding to their control cells (1.15 ± 0.19 µm) (Table 1 and Fig. 8A and B). The average height index was sharply decreased with a great statistical difference (p < 0.0001) to record 383 ± 81.1 nm, compared to the untreated cells (776.6 ± 128.3 nm), which led to high shrinkage (51%) in the cell height (Table 1 and Figs. 7k and 8C). Roughness parameters (Ra, RMS) were calculated and measured (Table 1 and Fig. 8D and E), where the roughness of the surface increased significantly (p < 0.0001) to reach 56.4 ± 13.8 nm, 2.8-fold greater than untreated bacteria (19.9 ± 5.6 nm). RMS index of A. baumannii ATCC 19606 (109.8 ± 16 nm) showed no statistical difference (p = 0.32), when it was treated with MIC (0.38 mg/mL) of AWME3, compared to the control groups, which recorded 102.8 ± 18.5 nm. Shape parameters (Ska, Ssk) of A. baumannii ATCC 19606 were increased significantly (p = 0.001, p = 0.02) to be 11.9 ± 6.4 and 2.9 ± 0.87, respectively, compared to the control group, which recorded 4.12 ± 2.9 and 1.17 ± 0.56, respectively.

Great changes and irregular shapes were formed when A. baumannii ATCC 19606 subjected to the highest concentration 2 MIC (0.75 mg/mL) for a short period (10 min), as shown in topographic 2D and 3D images (Fig. 7m, o, and p). A. baumanni ATCC 19606 cell morphology was severely influenced because of AWME3 action after 10 min, where most of cells lost their cell walls and cell membranes, and, in some cases, cells were without septum, became more swelling, blebbing, and disintegrated. Figure 7m shows a great amount of cell debris formed in irregular shapes around disrupted cells, where the initial reaction was expected to be the leakage of a large amount of fluid from the partially disintegrated cells. The cytoplasmic fluid leaked out from the inner membrane of the cell. This could prove that the AWME3 caused damage to the bacterial inner membrane.

When exposed to a high AWME3 concentration of 0.75 mg/mL, a large amount of cytoplasmic fluid is exerted outside the bacteria. These bacteria appear either severely damaged or their membrane fully collapsed, and ghost cells with unclear and irregular dimensions were formed (Fig. 7m, o, and p). This indicates drastic permeabilization of the inner membrane. Bacterial cell dimensions of A. baumannii ATCC 19606 length, width, and height were evaluated, where a high significant decrease (p = 0.0019, p < 0.0001) was obvious to be 1.59 ± 0.67 µm and 0.79 ± 0.34 µm, respectively, compared to their corresponding control groups (2.18 ± 0.32 µm, 1.15 ± 0.19 µm) (Table 1 and Fig. 8A and B). High rate reduction in the height (162.4 ± 75.4 nm) of A. baumannii ATCC 19606 cells displayed a high significant difference (p < 0.0001), compared to untreated cells (776.6 ± 128.3 nm) (Table 1 and Fig. 8C). The high rate of shrinkage in the cell height (88%) of A. baumannii ATCC 19606 was obvious after exposure to 2 MIC of AWME3 within 10 min. The Ra and RMS indexes for the treated A. baumannii ATCC 19606 strain displayed no significant differences (p = 0.3, p = 0.19) to be 14.4 ± 4.4 nm and 104.1 ± 40.2 nm, respectively, when compared to untreated bacteria (19.9 ± 5.6 nm, 126.4 ± 22.6 nm) (Table 1 and Fig. 8D and E). Ska and Ssk were characterized for the cell shape of A. baumannii ATCC 19606, where both parameters did not show statistical difference (p = 0.55, p = 0.13) to record 6.7 ± 3.8 and 1.59 ± 0.66, respectively, compared to control groups (4.12 ± 2.9, 1.17 ± 0.56), respectively (Table 1 and Fig. 8F and G).

Discussion

The combination of saturated and polyunsaturated fatty acids and their glycerides in AWME3 extract was able to kill and eradicate S. aureus ATCC 55804 and A. baumanii ATCC 19606 strains in a short period (5–10 min) (Fig. 1), compared to other studies (Fischer et al. 2012; Parsons et al. 2012; Umerska et al. 2016). The low MIC concentrations and the high bactericidal effect of AWME3 against S. aureus ATCC 55804 and A. baumannii ATCC 19606 have encouraged us for more deep investigation of the mechanism of AWME3 action, which could explain the high efficacy of AWME3 against a broad spectrum of bacterial pathogens.

Results obtained from the PI assay (Fig. 2) display that the addition of AWME3 dramatically kills and inhibits the growth of S. aureus ATCC 55804 and A. baumannii ATCC 19606 strains, and suggest that cell permeabilization has become the main mechanism of killing, and this hypothesis was in agreement with several studies (Cartron et al. 2014; Armas et al. 2021). When bacterial strains are treated with fatty acids, they can incorporate them into their phospholipids, altering membrane composition and properties (Horne et al. 2020). This incorporation occurs through outer membrane transporters like the FadL gene, followed by activation of the acyl-CoA at the inner membrane (Eder et al. 2017; Horne et al. 2020). In addition, the integration of fatty acids into bacterial membranes is able to increase membrane permeability, disrupt membrane potential, and ultimately cause cell lysis. Also, fatty acids may alter the fluidity and physical properties of membranes, affecting their barrier function. Consequences of interacting with the outer membrane protein, fatty acids can increase bacterial susceptibility to antibiotics and antimicrobial peptides, reduce bacterial motility and biofilm formation, and attenuate virulence and pathogenicity (Eder et al. 2017; Ostroumova And Efimova 2023).

Treatment with AWME3 has led to cytoplasmic material leakage and disruption of cytoplasmic membrane integrity, induced the tested bacteria for the leakage of cytoplasmic contents out of the cell (Fig. 3), and can be significant evidence for killing bacteria; therefore, several studies stated the same findings (Wang et al. 2018; Yoon et al. 2018; Wong et al. 2021).

Previous studies demonstrated that intracellular ATP provides energy for normal physiological activities in microorganisms (Shi et al. 2016; Fei et al. 2019). It is obvious that a sharply decrease in intracellular ATP occurred when both strains were treated with AWME3 (Fig. 4); this decrease in intracellular ATP concentration has been related to the depletion of the intracellular ATP pool and dissipation of proton motive force components (Fei et al. 2019; Wu et al. 2019). Consequently, fatty acids can interrupt the electron transport chain in bacteria, which is essential for generating ATP via oxidative phosphorylation. By interfering with electron carriers, fatty acids make it difficult for bacterial cells to produce sufficient ATP to function, leading to growth inhibition and cell death. Direct quantification of ATP concentrations was assessed to demonstrate the effects of fatty acids on bacterial energy production, where ATP levels in S. aureus and A. baumannii were reduced when treated with various fatty acids (Cartron et al. 2014; Eder et al. 2017; Yoon et al. 2018; Zang et al. 2021; Ren And Palmer 2023). Proteomic analyses indicate that S. aureus and A. baumannii respond to fatty acid treatment by exhibiting altered metabolic states, supporting the conclusion that fatty acids disrupt energy metabolism in bacteria (Kengmo Tchoupa et al. 2020; Zang et al. 2021).

Recent advancements in microscopy techniques, particularly atomic force microscopy (AFM), have revolutionized our understanding of bacterial cell envelopes and their interactions with antimicrobials. AFM enables high-resolution imaging of bacterial membranes and live cells under physiological conditions, revealing ultrastructural features and dynamics invisible to traditional microscopy methods (Viljoen et al. 2020). This technique is beneficial to visualize and measure structural and mechanical changes in bacterial membranes upon exposure to antibiotics and other antimicrobial agents (Longo And Kasas 2014; Paiva et al. 2022). Additionally, molecular dynamics simulations complement microscopy studies by providing insights into the transport of small molecules and ions through various compartments of the bacterial cell envelope, as well as the interactions of surfactants and antimicrobial peptides with cell walls and membranes (Ganesan et al. 2023). These combined approaches offer powerful tools for developing novel antimicrobials and strategies to combat bacterial infections.

The AFM images of freshly prepared untreated S. aureus ATCC 55804 show a typical spherical or cocci morphology, with a relatively smooth surface and no alterations (Fig. 5a). The cell wall and cell membrane appeared intact with no ruptures or large pores (black arrows) (Fig. 5a and d) (Eaton et al. 2008). On the other hand, severe alterations were visualized by AFM after treating bacteria with AWME3 (Figs. 5e–p and 7e–p). Taken together, these findings were in line with previous studies that demonstrated the action of the glycinin basic peptide on the cell morphology of S. aureus (Yang et al. 2016). Similarly, chitosan treatment led to cell wall collapse and morphological changes in S. aureus, as observed through AFM (Eaton et al. 2008). In another study, AFM was used to monitor the division of S. aureus cells and record spectra showing the thickening of the septum during cell division (Kochan et al. 2018). Furthermore, the degree of dimensional reduction and morphological changes mostly depends on the antimicrobial agent and its concentration, which appeared closely in Propionibacterium acnes, with the length, width, and height of bacterial cells reducing by 42.56%, 92.0%, and 41.58%, respectively, at high concentrations of essential oils (Fu et al. 2007). Roughness surface of S. aureus ATCC 55804 increased significantly (**p = 0.001, ****p < 0.0001), when it was treated with 0.19 mg/mL and 0.38 mg/mL, respectively, and these findings were in line with previous and recent studies (Shin et al. 2007; Singh et al. 2014; Juma et al. 2020).

RMS roughness of S. aureus cells value increased (*p = 0.041) after treatment with MIC (0.19 mg/mL) and these data were matched with Singh et al. (2014). RMS roughness of A. baumannii ATCC 19606 and the cell surface increased significantly (p < 0.0001) when subjected to 0.5 MIC of AWME3 compared to the untreated cells. Additionally, Ska and Ssk values of treated A. baumannii ATCC 19606 increased significantly (p = 0.012, p = 0.016) after exposure to AWME3, and these findings are in agreement with Soon et al. (2009), where A. baumannii strains were exposed to various concentrations of colistin.

The drop in pH inside the cell affects cell survival via blocking enzymes mechanistically, where numerous enzymatic processes are pH-dependent (Scrimgeour 2020). This may lead to uncoupling of the respiratory chain and disrupting the membrane-associated process, leading to cell wall division defects as observed by all microscopy techniques (Shin et al. 2007; Hyldgaard et al. 2012; Cartron et al. 2014; Wang et al. 2018; Nasompag et al. 2021) and leading to the leakage of cytoplasmic and genetic material, which were detected and confirmed at 260 nm and 280 nm (Cartron et al. 2014). Furthermore, membrane damage resulted in large losses of cytoplasmic fluid. Being devoid of intracellular fluid and organelles, the cell architectural support was compromised, and hence, the bacteria collapsed. In the third stage, the cell membranes were disintegrated, leaving behind massive amounts of membrane residues or debris. Only bacteria exposed to high concentrations of AWME3 showed the characteristics of the last stage (Yoon et al. 2018). Further studies, including in vivo safety evaluations, are required to comprehensively assess the therapeutic potential of AWME3.

Conclusions

The study shows that the fatty acids in AWME3 can disrupt the membranes of Gram-positive and Gram-negative bacteria: Acinetobacter baumannii ATCC 19606 and Staphylococcus aureus ATCC 55804. Biological assays and microscopy reveal that fatty acids make membranes more permeable and rigid, create pores, lead to cytoplasmic leakage, disrupt essential functions, and result in cell death. AFM imaging confirmed these results, showing clear surface blebs in S. aureus and noticeable cleavages, pores, and flattening in A. baumannii. The research demonstrates that fatty acids interfere with membrane integrity, elicit various bacterial responses, and validate the effectiveness of AFM in analyzing membrane damage at the nanometer scale. The results collectively highlight fatty acid-based antimicrobials as a compelling option for fighting against multidrug-resistant pathogens in healthcare settings.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

HM: conceptualized, performed all methodology, validated, conducted all formal analysis, investigated, wrote the original draft, visualized, designed, implemented of all the experiments, prepared all figures and tables, and analyzed all results and microscopy data. SV: revised, edited the manuscript, work administration, and financial support. EM: conceived, designed, supervised the study, critically reviewed, and edited the manuscript. All the authors have revised and agreed to the final version of the manuscript.

Funding

This work was supported by the Ministry of Science and Higher Education of the Russian Federation (Project No. FSMG-2024-0045).

Data Availability

Data is provided within the manuscript or supplementary information files.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.