Mechanistic insight into the synergistic antimicrobial potential of Fagonia indica Burm.f. extracts with cefixime

Abstract

Fagonia indica Burm.f. is known for its anti-infective character and has been studied in the present work as a synergistic remedy against resistant bacterial strains. Initially, phytochemicals were quantified in n-Hexane (n-Hex), ethyl acetate (E.A), methanol (MeOH), and aqueous (Aq.) extracts by Total Phenolic Content (TPC), Total Flavonoid Content (TFC) and Reverse Phase High Performance Liquid Chromatography (RP-HPLC) analysis. Later, after establishing an antibacterial resistance profile for extracts and antibiotics against gram-positive and gram-negative strains, synergism was evaluated in combination with cefixime through time-kill kinetics and bacterial protein estimation studies. Topographic images depicting synergism were obtained by scanning electron microscopy for Methicilin-resistant Staphylococcus aureus (MRSA) and Resistant Escherichia coli (R.E. coli). Results showed the presence of maximum phenolic (28.4 ± 0.67 μg GAE/mg extract) and flavonoid (11 ± 0.42 μg QE/mg extract) contents in MeOH extract. RP-HPLC results also displayed maximum polyphenols in MeOH extract followed by E.A extract. Clinical strains were resistant to cefixime whereas these were moderately inhibited by all extracts (MIC 150-300 µg/ml) except Aq. extract. E.A and n-Hex extracts demonstrated maximum synergism (Fractional inhibitory concentration index (FICI) 0.31) against R.E. coli. The n-Hex extract displayed total synergism against R.P. a with a 4-fold reduction in cefixime dose. Time-kill kinetics showed maximum inhibition of gram-negative bacterial growth from 3 to 12 h when treated at FICI and 2FICI values with > 10-fold reduction of the extracts' dose. All combinations demonstrate > 70 % protein content inhibition with bacterial cell wall disruption in SEM images. Fortunately, FICI concentrations have low hemolytic potential (<5%). Conclusively, F. indica extracts can mitigate antimicrobial resistance against cefixime and can be investigated in detail by in vivo and mechanistic studies.

Keywords: Antibiotic; Antimicrobial potential; Bacteria; Biocompatibility profile; Cefixime-resistant strains; Eescherichia coli; Fagonia indica; Plant extract; Scanning electron microscopy; Synergism potential.

Fig. 1A

Total phenolic content (TPC) and total flavonoid content (TFC). Note; n-Hex = n-hexane, E.A = ethyl acetate, MeOH = methanol, Aq. = aqueous extract. Values are presented as mean ± standard deviation from the triplicate investigation. The columns with different superscript (a-g) letters show significant values (P < 0.05).

Fig. 1B

Pearson Corelation between TPC, TFC and Cumulative HPLC polyphenols quantified, Note; From graph it is shown, the graphical representation of the results clearly indicates a linear correlation among all three components with non-significant p-values (P > 0.05).

Fig. 1C

HPLC chromatograms of standard polyphenols.

Fig. 1D

HPLC fingerprints of F. indica where each graph presents individual extracts. Note: where n-Hexane (a), ethyl acetate (b), methanol (c) and distilled water (d). 1. Vanillic acid 2. Rutin 3. Catechin 4. Gallic acid 5. Syringic acid 6. Coumaric acid 7. Emodin 8. Gentisic acid 9. Caffeic acid 10. Ferulic acid 11. Apigenin 12. Myricetin 13. Quercitin 14. Kaemferol.

Fig. 2

Time-kill kinetics curves for Gram-positive bacterial strains. Note: where cefixime-resistant gram-positive bacterial strains were (A) MRSA and (B) R.S. hemolyticus were treated with F. indica extracts, cefixime, and their combination. The bacterial growth was monitored for 0, 3, 6, 9, and 12 h. Colored lines indicate the amount of the treatment used in the experiment where light blue is the untreated control, yellow is a positive control, purple is 1x MIC of cefixime, black is 1x MIC of extract, red is 2x MIC of extract, dark blue is 1x FICI, and green is 2x FICI.

Fig. 3

Time-kill kinetics curves for Gram-negative bacterial strains. Note: Where cefixime-resistant gram-negative bacterial strains (A) E. coli (B) R.P. aeruginosa were treated with F. indica extracts, cefixime, and their combination. The bacterial growth was monitored for 0, 3, 6, 9, and 12 h. Colored lines indicate the amount of the treatment used in the experiment where light blue is untreated control, yellow is positive control, purple is 1x MIC of cefixime, black is 1x MIC of extract, red is 2x MIC of extract, dark blue is 1x FICI, and green is 2x FICI.

Fig. 4

Scanning electron micrographs of bacterial strains. Note: Where, (A) R.E. coli (a) untreated (magnification × 25000x); (b) treated with the cefixime (magnification × 25000x); (c) treated with n-Hex extract alone (magnification × 15000x); (d) treated with combinations of n-Hex and cefixime (magnification × 10000x); (e) treated with E.A extract alone(magnification × 25000x); (f) treated with combinations of E.A and cefixime (magnification × 50000x. (B) MRSA. (g) untreated (magnification × 25000x); (h) treated with the cefixime (magnification × 25000x); (i) treated with MeOH extract alone (magnification × 25000x); (j) treated with combinations of MeOH and cefixime (magnification × 25000x).