The antimicrobial, antibiofilm, and wound healing properties of ethyl acetate crude extract of an endophytic fungus Paecilomyces sp. (AUMC 15510) in earthworm model

Abstract

The endophytic fungus Paecilomyces sp. (AUMC 15510) was isolated from healthy stem samples of the Egyptian medicinal plant Cornulaca monacantha. We used GC-MS and HPLC analysis to identify the bioactive constituents of ethyl acetate crude extract of Paecilomyces sp. (PsEAE). Six human microbial pathogens have been selected to evaluate the antimicrobial activity of PsEAE. Our data showed that the extract has significant antimicrobial activity against all tested pathogens. However, the best inhibitory effect was observed against Bacillus subtilis ATCC 6633 and Pseudomonas aeruginosa ATCC 90274 with a minimum inhibitory concentration (MIC) of 3.9 μg/ml and minimum bactericidal concentration (MBC) of 15.6 μg/ml, for both pathogens. Also, PsEAE exerts a significant inhibition on the biofilm formation of the previously mentioned pathogenic strains. In addition, we evaluated the wound healing efficiency of PsEAE on earthworms (Lumbricus castaneus) as a feasible and plausible model that mimics human skin. Interestingly, PsEAE exhibited a promising wound healing activity and enhanced wound closure. In conclusion, Paecilomyces sp. (AUMC 15510) could be a sustainable source of antimicrobial agents and a potential therapeutic target for wound management.

Figure 1

Paecilomyces sp. AUMC 15510: (a–c) 7-day-old colonies on PDA, Cz, and MEA at 25 °C. (d) Irregularly branched conidiophores with phialides. (e) Chains of ellipsoidal and/or cylindrical, truncate conidia. (White arrows).

Figure 2

The inhibition zone (mm) of ethyl acetate crude extract (EAE) of Paecilomyces sp. (AUMC-15510) at a concentration of 5 mg/ml against (a) A. niger (b) C. albicans (c) E. coli (d) P. aeruginosa (e) S. aureus, and (f) B. subtilis. PC: Fluconazole and Gentamicin at concentration of 5 mg/ml (positive control); NC: 10% DMSO (negative control).

Figure 3

(a) Biofilm biomass assessment by crystal violet staining (OD 600 nm) of four bacterial strains P. aeruginosa, S. aureus, E. coli, and B. subtilis after 48 h in Brain Heart Infusion broth (BHIB) supplemented with 2% glucose. (b) The 96-well microtiter plate assay showed biofilm formation by four bacterial strains. (c) Antibiofilm efficacy of ethyl acetate crud extract from Paecilomyces sp. (AUMC 15510) on P. aeruginosa, S. aureus, and B. subtilis as assessed by crystal violet quantification of biofilm. (d) The 96-well microtiter plate assay depicted the effect of ethyl acetate crud extract on the biofilm formation of P. aeruginosa, S. aureus, and B. subtilis. PC: Gentamicin (Positive Control). The error bars on the graph represent standard deviations as a percentage of biofilm inhibition.

Figure 4

Macroscopic observation of the different groups of earthworms (Lumbricus castaneus) after induction of surgical wounds and examination of wound healing; (a) worms received Vaseline; (b) worms received 5 mg; (c) worms received 10 mg, and (d) worms received 15 mg.

Figure 5

Photomicrographs of the longitudinal section of the different groups of earthworms (Lumbricus castaneus) after induction of surgical longitudinal wounds and examination of wound healing; (a) normal earthworm; (b) worms received Vaseline; (c) worm on the first day of injury, and (d) worms received 5 mg, (e) worms received 10 mg, (f) worms received 15 mg, note: the cells are loosely packed at the amputated region (raw). Hematoxylin and Eosin (H&E).

Figure 6

Photomicrographs of longitudinal section of the different groups of the body wall of earthworms (Lumbricus castaneus) after induction of surgical longitudinal wounds and examination of wound healing; (a) normal earthworm; (b) worms received Vaseline; (c) worm in the first day of injured showing hemorrhage (circle), and (d) worms received 5 mg, (e) worms received 10 mg, (f) worms received 15 mg. (Red (glandular epithelial cell layer), green (circular muscle layer) and yellow (longitudinal cell layer) Hematoxylin and eosin (H&E).

Figure 7

Scanning electron photomicrograph of the anterior part of earthworms (Lumbricus castaneus) after induction of surgical wounds and examination of wound healing; (a) normal earthworm; (b) worms received Vaseline, showing fissure(raw); (c) worm in first day of injured showing the coelomic fluid emerged as well as the blood surrounds the wound appearance (circle), and (d) worms received 5 mg, (e) worms received 10 mg showing fissure(raw), (f) worms received 15 mg.

Figure 8

Photomicrographs of semithin sections of the earthworms (Lumbricus castaneus) (a) normal earthworm; (b) worms received Vaseline, showing fissure(raw); (c) worm on the first day of injured showing the coelomic fluid emerged as well as the blood surrounds the wound appearance (circle), and (d) worms received 5 mg, (e) worms received 10 mg showing fissure(raw), (f) worms received 15 mg. Cm Circular muscle, Ep Epidermis, Lm Longitudinal muscle, Wo Wound.

Figure 9

Transmission electron microscopy micrographs of earthworms (Lumbricus castaneus) (a) normal earthworm; (b) worms received Vaseline, showing fissure (raw); (c) worm on the first day of injured showing the coelomic fluid emerged as well as the blood surrounds the wound appearance (circle), and (d) worms received 5 mg, (e) worms received 10 mg showing fissure(raw), (f) worms received 15 mg. Cm Circular muscle, Ep Epidermis, BV Blood vessel, G Granules, Lm Longitudinal muscle, N Nucleus, Wo Wound.