Pioneering study of Egyptian Neem and Jojoba extracts with molecular docking combat hospital multidrug resistant bacteria

Abstract

Hospital surfaces are often contaminated with multidrug-resistant pathogenic bacteria that cause healthcare-associated infections and lead to increased mortality and morbidity. There is a need for new alternative antibacterial agents to overcome antibiotic resistance. Azadirachta indica and Simmondsia chinensis have been found to possess antibacterial activity and medicinal value. The antibacterial activity of these plant extracts against clinical isolates was investigated using the agar disc diffusion method. These clinical isolates included E. coli, Pseudomonas aeruginosa, Acinetobacter spp., Klebsiella pneumoniae, Stenotrophomonas maltophilia, and methicillin-resistant Staphylococcus aureus (MRSA), which were identified by the vitek-2 system, and resistance genes of selected bacterial strains were identified by using the bioFire FilmArray test. The most potent extract of these plants was the ethanolic extract, where the inhibition percentage of ethanolic Jojoba and Neem extracts was 90.9% and 74.5%, respectively against all the tested pathogens. On the other hand, the methanolic extracts of Neem and Jojoba have different degrees of antibacterial activity against the tested pathogens. The phytochemical components of the most potent extracts (ethanolic extracts) were investigated by gas chromatography‒mass spectrometry (GC\MS), which revealed that the ethanolic extracts were enriched in phenolics, flavonoids, and sugars. FTIR analyses of the plant extracts confirmed the presence of alcoholic, carboxylic, and aldehydic moieties. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) activity of the ethanolic extracts of Neem and Jojoba increased in a dose-dependent manner, with average IC50 values of 98.17 ± 0.85, 4.95 ± 0.06, and 4.17 ± 0.04 mg/mL, respectively, for the ethanolic Neem extract, the ethanolic Jojoba extract, and ascorbic acid (standard). Furthermore, increased cytotoxicity was demonstrated in the HFB4 cell line in a dose-dependent manner. The average IC50s of the ethanolic Neem extract and the ethanolic Jojoba extract were 18.18 ± 0.15 and 76.16 ± 1.49 mg/mL, respectively. Moreover, the results for the antibiofilm activity of the ethanolic Neem extract showed that 99.5% of the biofilms formed at 25 mg/ml. In addition, 50 mg/ml of the ethanolic extract of Jojoba had a suppressive effect of 98.2%. The significant components Nonanoic acid (21.9405%) and Palmitic Acid (16.0869%) from Neem and pinitol from Jojoba (82.85%) were selected throughout the molecular docking investigation, by which the chosen constituents inhibited the crystal structure of penicillin-binding protein 4 (PBP4) from Staphylococcus aureus (PDB ID: 1TVF) and the crystal structure of the OXA-48 beta-lactamase (PDB ID: 7AUX) from K. pneumoniae. Overall, our study reveals the effectiveness of antimicrobial plant extracts as therapeutic solutions for antibiotic resistance in Egypt and worldwide with some modifications to decrease their cytotoxicity.

Keywords: Azadirachta indica; Simmondsia chinensis; Antibacterial; Healthcare-associated infections; Molecular docking and multidrug resistant bacteria; Multidrug resistant bacteria.

Fig. 1

General outline of the work performed in this study

Fig. 2

Morphology of (a) Azadirachta indica and (b) Simmondsia chinensis.Plant materials of Simmondsia chinensis and Azadirachta indica leaves were purchased from Wadi El-Natron valley, Beheira Governorate 150 km northen-west of Cairo (Lat. = 30° 30' N, Long. = 30° 09' E & Elev. = 30 m), Egypt

Fig. 3

Showing (a) Distribution of bacterial isolates obtained from different gender (b) percentage of the pathogenic bacterial isolates causing healthcare associated infection isolated in thesis study

Fig. 4

Screening of biofilm production for (a) Acinetobacter spp., (b) K. pneumonia, (c) E. coli, (d) MRSA, and (e) Pseudomonas eruginosa. all bacterial strains inoculated on Congo agar plates, then incubated at 37 °C for 24 h, biofilm producers bacteria appear as black colonies with a dry crystalline consistency while nonbiofilm producers bacteria remained pink colonies

Fig. 5

GC‒MS spectrometry chromatograms (full-scan mode) of 500 mg\ml (a) ethanolic Neem extract and (b) ethanolic Jojoba extract

Fig. 6

FTIR spectra of (a) ethanolic Neem plant extract and (b) ethanolic Jojoba exract

Fig. 7

UV-Vis spectra of ethanolic extracts of (a) Neem, and (b) Jojoba

Fig. 8

Antibacterial activity of different plant extracts against (a) S. maltophilia, (b) MRSA, and (c) E. coli. The antibacterial activity was carried out by agar diffusion method, all Mueller-Hinton agar plates were inoculated in duplicates with 100 µL of each fresh tested culture with a sterile cotton swab and The paper disk was immersed in each sample of plant extracts for 10 s then placed on inoculated plates. The plates were incubated at 37 °C for 24 h

Fig. 9

MIC of ethanolic and methanolic extracts. MIC of plant extracts measured by broth dilution method against bacterial strains, initial concentration (1000 mg/mL) was diluted using double fold serial dilution by adding 100 µL plant extract samples to100 µL sterile nutrient broth. Each concentration inoculated in duplicates with 5 µL of the standardized bacterial suspension and incubated at 37 °C for 24 h

Fig. 10

Antibiofilm activity of Jojoba and Neem extracts against bacterial strains. The 96-well polystyrene microtiter plates were inoculated in duplicates with 100 μL of tryptone soy broth and 100μL diluted plant extracts with concentration 50 mg\mL then incubated at 37 °C for 24 h. Then, 200 μL of a 0.1% crystal violet solution was added to the wells for 15 min. After washing three times and drying, 200 μL of 30% glacial acetic acid was added to the wells then the absorbance was measured at 570 nm

Fig. 11

Antioxidant activity of Ethanolic Neem and Jojoba extracts. using 2,2 DPPH assay expressed as DPPH scavenging %, ascorbic acid used as a control

Fig. 12

Morphological observation of HBF4 treated with (b, d) ethanolic Neem extract, and (c, e) ethanol Jojoba extracts at different concentrations under an inverted microscope, while (a) control cells by using MTT assay

Fig. 13

Molecular docking determine the interaction between (a) Pinitol, (b) Palmitic acid, and (c) Nanoic acid and active sites of 1TVF protein, while (d) showing The representative key for the types of interaction between Pinitol, Palmitic acid, and Nanoic acid and protein receptors

Fig. 14

Molecular docking determine show the interaction between (a) Pinitol, (b) Palmitic acid, and (c) Nanoic acid and active sites of 7AUX protein, while (d) showing The representative key for the types of interaction between Pinitol, Palmitic acid, and Nanoic acid and protein receptors