Mechanism of bactericidal efficacy against nosocomial pathogenic Staphylococcus aureus strain caused by fatty acids from Hermetia illucens larvae fat

Abstract

Hermetia illucens (HI) is a promising insect that widely employed as a sustainable source of food and has been recently used as a successful antimicrobial agent. Fatty acids extracted sequentially from HI larvae fight against MDR nosocomial pathogenic bacteria such as Staphylococcus aureus. This strain is resistant to various antibiotics, causing many issues and deaths in healthcare sectors. The present study aimed to elucidate the mechanism of bactericidal efficacy of fatty acids (FAs) in HI larvae fat against S. aureus ATCC 55804 strain. The disk diffusion assay, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and half of the minimum inhibitory concentration (MIC50) applied in this study, proved the antimicrobial activity of fatty acids. The mechanism of FAs action was evaluated by several approaches, including inhibition of the bacterial growth curves and salt tolerance assays, scanning electron (SEM) and transmission electron (TEM) microscopies. S. aureus ATCC 55804 was resistant to 30% out of ten tested antibiotics belonging to different classes. In addition, microscopic observations showed the inhibitory effect of acidic water methanol extract (AWME3) by targeting of the S. aureus ATCC 55804 cell membrane and causing the considerable morphological alterations on the bacterial wall and destruction its cytoplasmic contents leading to the cellular content release and cell death. This study revealed the potential efficacy of AWME3 as a novel therapeutic antibacterial agent effective against resistant nosocomial bacterial pathogens.

Keywords: Hermetia illucens; AWME3 extract Fatty acids; MDR bacteria; SEM; Salt tolerance; TEM.

Fig. 1

Susceptibility of S. aureus ATCC 55804 strain to eight groups of antibiotics confirmed by disk diffusion assay. All antibiotics were applied to the discs, following the protocol in the material and methods section, and gently transferred on the surface of the Muller Hinton agar Petri dishes with cultured bacteria strain at density 10⁸ (CFU/mL). All discs were applied in duplicates. The mean of the resulting IZD ± SD of three independent experiments was calculated and presented in Table 2.

Fig. 2

The disk diffusion assay of AWME3 against pathogenic bacteria strain.

Fig. 3

Antimicrobial sensitivity of AWME3 against S. aureus ATCC 55804.

Fig. 4

The growth curves of S. aureus ATCC 55804 treated with different concentrations of either AWME3 or P/S. Each data point is the average of three independent assays ± standard deviation of the mean (SD) and *p < 0.05 was significant.

Fig. 5

Effect of AWME3 from HI larvae fat on human pathogenic S. aureus ATCC 55804. Cell viability (%) was determined using the Alamar blue method. Error bars indicate the standard deviation of % viability obtained from three separated experiments, one-way ordinary ANOVA Dunnett’s multiple comparisons test (****p < 0.0001) was significant. The MIC50 values were calculated based on the reduction of the Alamar blue assay and compared to the positive control (P/S). The pathogenic cells viability was assessed after 24 h of incubation. (A) treatment with P/S; (B) treatment with AWME3. The MIC50 values were calculated using the non-linear regression mode of GraphPad Prism 7 (Graph Pad Software Inc., San Diego, CA, USA). The MIC50 values are the average of three independent experiments ± standard deviation error mean (SEM).

Fig. 6

Effect of AWME3 extracted from H. illucens larvae fat at MIC and 2 MIC concentrations on the reduction of salt tolerance of S. aureus ATCC 55804. All values are represented as mean ± SD, in triplicate (n = 3). Data were analyzed by two-way ANOVA, followed by Dunnett’s multiple comparisons test. Data represented a significant difference as compared to the control without NaCl and p < 0.05 was significant.

Fig. 7

SEM images of S. aureus ATCC 55804 cells. (a, b) Untreated control; (c) Treated with MIC (0.19 mg/mL) for 12 h.; (d, e, f, g) Treated with 2 MIC (0.38 mg/mL) of AWME3 for 20 min; (g, h, i) Treated with 4 MIC (0.75 mg/mL) of AWME3 for 10 min. Blue arrows show small pores formed inside the cell membrane, red arrows indicate cell wall swelling and collapsing, green arrow refers to the vacuole or periplasmic space formation and cell wall deformation, violet arrows demonstrate lysed cells, cell wall ruptured completely, cell debris and yellow arrow shows blebs and loosed intracellular compartments.

Fig. 8

TEM micrographs of untreated and treated S. aureus ATCC 55804. Untreated cells looked round and intact, with a well-defined cell membrane and the red arrows indicate intact septa (a). The onset of septation (red arrows) and the cross-wall formation were noticed. After treatment for 12 h with the MIC (0.19 mg/mL) of the AWME3; (b, c) Cells appeared malformed with shrunken cytoplasmic material and aberrant septa; (d, e) Cells appeared with non-membrane-enclosed bodies (denoted by violet arrows) after treatment with 2 MIC (0.38 mg/mL) for 30 min. Some lysed cells were also noted (f), as well as null cells (g) were also noted after treatment with 4 MIC (0.75 mg/mL) for 10 min.