Molecular Characterization of spa, hld, fmhA, and l ukD Genes and Computational Modeling the Multidrug Resistance of Staphylococcus Species through Callindra harrisii Silver Nanoparticles

Abstract

The problem of multidrug resistance in bacterial pathogens is significant and is related to the high morbidity and death rates of living things due to increased levels of beta-lactamases. Plant-derived nanoparticles have gained a great significance in the field of science and technology to combat bacterial diseases, especially multidrug-resistant bacteria. This study examines the multidrug resistance and virulent genes of identified pathogenic Staphylococcus species obtained from Molecular Biotechnology and Bioinformatics Laboratory (MBBL), culture collection. The polymerase chain reaction-based characterization of Staphylococcus aureus and Staphylococcus argenteus having ON875315.1 and ON876003.1 accession IDs revealed the presence of the spa, LukD, fmhA, and hld genes. The green synthesis of silver nanoparticles (AgNPs) was carried out by utilizing the leaf extract of Calliandra harrisii, of which metabolites act as capping and reducing agents for the precursor of nano-synthesis, i.e., AgNO₃ of 0.25 M. The synthesized AgNPs were characterized via UV-vis spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscopy, and energy-dispersive X-ray analysis which inferred the bead-like shape of our nanoparticles with the size of 2.21 nm with the existence of aromatic and hydroxyl functional groups at surface plasmon resonance of 477 nm. The antimicrobial activity by AgNPs showed 20 mm inhibition of Staphylococcus species as compared to the vancomycin and cefoxitin antibiotics along with crude plant extract, which showed a minimum zone of inhibition. The synthesized AgNPs were also analyzed for various biological activities like anti-inflammatory with 99.15% inhibition in protein denaturation, antioxidant with 99.8% inhibition in free radical scavenging, antidiabetic with 90.56% inhibition of alpha amylase assay, and anti-haemolytic with 89.9% inhibition in cell lysis which shows good bioavailability and biocompatibility of the nanoparticles with the biological system of the living being. The amplified genes (spa, LukD, fmhA, and hld) were also analyzed for their interaction with AgNPs computationally at the molecular level. The 3-D structure of AgNP and amplified genes was retrieved from ChemSpider (ID: 22394) and Phyre2 online server, respectively. The binding affinities of AgNP with spa, LukD, fmhA, and hld were -7.16, -6.5, -6.45, and -3.3 kJ/mol, respectively, which infers a good docking score except of hld which is -3.3 kJ/mol due to its small size. The salient features of biosynthesized AgNPs proved to be an effective approach in combating the multidrug-resistant Staphylococcus species in the future.

Figure 1

Amplification of virulence and resistance [Delta Hemolysin, FmhA, Spa (proteinA), and LukD], genes of (A) S. aureus and (B) S. argenteus.

Figure 2

Chromatogram of different genes of S. aureus and S. argenteus, (A) Delta Hemolysin (B) fmhA, (C) spa, and (D) LukD.

Figure 3

Determined reducing power of leaves extract with control, i.e., ascorbic acid showing the strong relationship, thus showing the capability of leaf extract to reduce the AgNPs.

Figure 4

Plant’s leaf extract transformation from pale orange to dark brown upon adding AgNO₃ precursor indicates the creation of AgNPs.

Figure 5

UV–visible spectrophotometry of AgNO₃ that was synthesized using C. harrisii leaf extracts from a 0.25 M precursor solution of AgNO₃.

Figure 6

SEM micrograph of AgNPs synthesized by using the 25 mM solution of AgNO₃ and leaf extract of C. harrisii.

Figure 7

EDX spectrum of AgNPs depict Ag peak in between 2 and 3 KeV.

Figure 8

FTIR analysis of AgNPs synthesized by leaves extract of C. harrisii.

Figure 9

Antimicrobial activity comparison with nanoparticle (a) S. aureus and (b) S. argenteus.

Figure 10

Graphical presentation of % inhibition of protein denaturation at specific concentrations of control (aspirin), leaf extract, and AgNPs, thus showing 99.15% anti-inflammatory activity by AgNPs at 500 μg/mL.

Figure 11

Graphical representation for the antioxidant activity showing the highest scavenging percentage of AgNPs (99.8%) at 1000 μg/mL concentration.

Figure 12

Graphical Representation of the hemolysis activity showing 89.9% inhibition of hemolysis by AgNPs.

Figure 13

Graphical representation of antidiabetic activity showing 90.56% inhibition of alpha amylase assay by AgNPs.

Figure 14

Cell viability of compound W1 on HEK 293 and U87 cell lines on 24 h. There was a dose-dependent killing observed with the cytotoxicity of drug increasing at lower concentrations for both cancer and non-cancer cell lines.

Figure 15

Density profiling of AgNPs at the ratio 1:1, 1:2, 1:4, and 1:6 of the water.

Figure 16

Mathematical model of specific heat capacity of AgNPs at a particular temperature range of (15–40 °C) along with the solvent mixing at 1:1, 1:2, 1:4, and 1:6.

Figure 17

Mathematical model of thermal conductivity of AgNPs at a particular temperature range of (15–40) along with the solvent mixing at 1:1, 1:2, 1:4, and 1:6.

Figure 18

3-D structure prediction of the amplified genes via Phyre2. (A) Spa, (B) fmhA, (C) LukD, and (D) hld.

Figure 19

3-D structure validation by Ramachandran Plot of ROCHECK. (A) Spa, (B) fmhA, (C) LukD, and (D) hld.

Figure 20

Phylogenetic analysis of the amplified genes via MEGA-X. (A) spa, (B) hld, (C) LukD, and (D) fmhA.

Figure 21

Interactome prediction through STRING. (A) fmhA, (B) spa, (C) hld, and (D) LukD.

Figure 22

Docking analysis of AgNPs with amplified virulent genes: (A) spa: (a) 3-D complex and (b) 2-D complex; (B) LukD: (a) 3-D complex and (b) 2-D complex; (C) fmhA: (a) 3-D complex and (b) 2-D complex; and (D) hld: (a) 3-D complex and (b) 2-D complex.

Figure 23

3-D QSAR analysis of docked complexes. (A) spa, (B) LukD, (C) fmhA, and (D) hld.

Figure 24

Boiled egg showing that our nanoparticle molecule is BBB-permeant and is not a substrate of PGP, hence making it efficient enough to work in the CNS.