Molecular Profiling, Characterization and Antimicrobial Efficacy of Silver Nanoparticles Synthesized from Calvatia gigantea and Mycena leaiana against Multidrug-Resistant Pathogens

Abstract

The use of natural products isolated from mushrooms against infection, cancer diseases and other oxidative-stress-related diseases is one of the cornerstones of modern medicine. Therefore, we tried to establish a combination of medicinal mushrooms and nanotechnology possibly with the field of medicine for the development of antibacterial agents against these MDR strains. The aim of the research was to understand the molecular identification, characterization and antibacterial action of Calvatia gigantea and Mycena leaiana. The identification of fruiting body species via morpho-anatomical and molecular methods was necessary to analyze the genetic variability and phylogenetic relationships of mushrooms. Phylogenetic analysis revealed that Calvatia from Hunza, Pakistan, exhibited 98% resemblance to the previously discovered Langermannia gigantean (DQ112623) and L. gigantean (LN714562) from northern Europe, and Mycena (Pakistan) showed a 97% similarity to M. leaiana (MF686520) and M. leaiana (MW448623) from the USA. UV-vis, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) were used for AgNPs' characterization. The UV-vis absorption peak of 500-600 nm indicates the AgNPs' presence. XRD results determined Calvatia gigantea AgNPs were nanocrystals and Mycena leaiana seems to be amorphous. In addition, SEM results showed the cubic morphology of C. gigantea with a diameter of 65 nm, and the FTIR spectra of fruiting body revealed the presence of functional groups-carboxyl, nitro, and hydroxyl-in AgNPs, which catalyzed the reduction of Ag+ to Ag0. Further antibacterial activity of mushrooms against MDR strains was determined via agar well diffusion assay, and Minimum Inhibitory Concentration (MIC) was estimated by qualitative experimentation using the broth dilution method. All experiments were conducted in triplicate. The results showed that the mushroom AgNPs, along with their synergy and nano-composites (with the exception of Ethyl-acetate), were shown to have zones of inhibition from 4 mm to 29 mm against multidrug-resistant pathogens such as Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, Proteus mirabilis, Enterobacter cloacae and Escherichia coli. The mushroom composites were active against most of the tested microorganisms whilst the lowest MIC value (10-40 mg/mL) was recorded against MDR strains. Hence, the present study suggested the possibility of employing compounds present in mushrooms for the development of new antibacterial agents, as well as efflux pump inhibitors.

Keywords: Klebsiella pneumonia; Proteus mirabilis; Pseudomonas aeruginosa; mushroom.

Figure 1

(A) Basidiocarp = 9 cm; (B) spores = 0.2 µm; (C) eucapillitial threads = 0.9 µm; (D) peridial threads = 0.7 µm.

Figure 2

Using Maximum Likelihood technique with Tamura 3-parameter model to analyze C. gigantea ITS sequences. The final data set includes 30 nucleotide sequences and 293 positions.

Figure 3

(A) = (pileus 1.5 μm); (B) = (lamellae & stipe) (1.5 μm); (C) = Ballistoconidia (10 μm); (D) = Basidium (5 μm); (E) = Cheilocystidium (12 μm); (F) = cuticle (8 μm).

Figure 4

ITS sequences of M. leaiana were analyzed using the Tamura 3-parameter model with Maximum Likelihood technique. The final data set includes 30 nucleotide sequences and 290 variables.

Figure 5

XRD analysis of AgNPs. m.l means Mycena leaina and c.g means Calvatia gigante.

Figure 6

UV–vis analysis of AgNPs. M.l means Mycena leaina and c.g means Calvatia gigante.

Figure 7

SEM analysis of C. gigantea and M. leaiana. (A)—Calvatia gigantia SEM analysis ans (B)—Mycena leaina SEM analysis.

Figure 8

FTIR analysis of synthesized AgNPs from C. gigantea. M.L means Mycena leaina and c.g means Calvatia gigante.

Figure 9

Antibacterial action of methanolic composite of C. gigantea and M. leaiana against MDR pathogens.

Figure 10

Antibacterial potential of silver NPs of C. gigantea (C) and M. leaiana (M) against MDR bacteria.

Figure 11

Antibacterial action of C. gigantea composites against K. pneumoniae (A) and P. aeruginosa (B) N-H = n-hexane (19 mm) and (22 mm); NP = nanoparticles (18 mm) and (25 mm); M = methanolic (23 mm) and (12 mm); Aw = aqueous water (16 mm) and (16 mm); P = pure water extract (20 mm) and (19 mm); E-A = ethyl acetate (0) and (0).

Figure 12

Antibacterial potential of M. leaiana composites against E. coli (A) and E. cloacae (B) N-H = n-hexane (19 mm) and (13 mm); NP = nanoparticles (15 mm) and (9 mm); M = methanolic (20 mm) and (20 mm); Aw = aqueous water (8 mm) and (14 mm); P = pure extract (19 mm) and (19 mm); E-A = ethyl acetate (10 mm) and (8 mm).

Figure 13

The synergetic effect of AgNPs against (A) = E. cloacae (E.B = 21 mm) and (B) = S. aureus (staph. = 29 mm).

Figure 14

MIC values of C. gigantea against MDR bacteria.

Figure 15

MIC values of M. leaiana against MDR pathogens.