Antimicrobial Potential of Biosynthesized of Nanoparticles

As discussed earlier, neem (A. indica) has been employed for the bio-fabrication of several metals, metaloxide, and hybrid metallic NPs. The metallic NPs have attracted the attention of researchers worldwide due to their amazing antimicrobial application. The antimicrobial potential of various metal (Ag, Cu, Au, Pt) and metal oxide NPs (CuO, ZnO, ТЮ₂, Fe₂0,) has been reported in numerous studies. Therefore, the same has also been implemented in various biomedical applications such as wound dressings, bone cement, and dental materials (Wang et al., 2017). The NPs synthesized using neem extract in various studies have shown excellent results against various pathogenic microorganisms, including the multi- drug-resistant bacteria as well. Algebaly et al. (2020) reported the biogenic synthesis of silver NPs using neem leaf extract and the antimicrobial activity of the NPs were tested against Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus. The synthesized NPs were potentially able to inhibit the growth of the bacteria completely and the SEM analysis of the treated bacteria showed significant membrane damage to the bacteria. In another study to explore the microbicidal potential of AgNP. Saranya et al. (2016) phytogenically synthesized AgNPs using A. indica leaf extract. The 40- to 50-nm-sized NPs were then used for testing antimicrobial activity against pathogenic bacteria, viz. Salmonella enterica, S. aureus and Streptococcus agalactiae. and fungi, viz. Malassezia pachydermatis and M. globosa. The synthesized NPs were able to completely inhibit the growth of tested bacteria as well as fungi. Roy et al. (2017) described in their study that silver NPs synthesized using neem leaf extract were more effective against E. coli as compared with gram-positive bacteria. Silver has been known to inhibit microbial growth since ancient times, and in the form of NPs, the antimicrobial potential of the same is enhanced manifolds. Not only silver but also copper, copper oxide, iron, iron oxide, zinc oxide, and titanium oxide NPs have also been synthesized using neem extract and evaluated for their antimicrobial potential.

FIGURE 3.2 Schematic diagram showing green synthesis and the stabilization of metal nanoparticles using neem extract.

The copper NPs (CuNPs) synthesized by Abhiman et al.(2018) using A. indica leaf extract were found to inhibit the growth of pathogenic bacteria, viz. with Bacillus cereus, S. aureus, E. coli, and Klebsiella pneumonia used as test organisms. Similarly, copper oxide NPs (CuONPs) synthesized using neem leaf extract completely inhibited the growth of E. coli (Sharma and Oudhia, 2016). Bhuyan et al. (2015), explored the synthesis of ZnONPs and their potential to inhibit the growth of pathogenic bacteria. It was concluded in their study that biosynthesized ZnO significantly inhibited the growth of E. coli, Streptococcus pyogenes, and S. aureus, and the efficacy increased with increasing the NP concentration used. In addition to this, the grampositive bacteria appeared to be more susceptible to ZnONPs than were gram-negative bacteria. Poopathi et al. (2015) revealed that the high viability of the toxic action of silver NPs synthesized from A. indica against mosquito vectors (Aedes aegypti and Culex. quinquefasciatus). Table 3.2 lists the NPs synthesized using neem extract and their antimicrobial activity against various microorganisms.

Mechanism of Antimicrobial Activity

The growing use of metallic NPs in biomedical applications has led the scientists to explore the science behind their bactericidal action. The ability of NPs to alter the metabolism of the bacteria in several ways (Slavin et al., 2017) implies that they can be used in eliminating disease-causing bacteria. The potential of NPs to inhibit 5. aureus biofilm by altering gene expression was also shown in a study published by Zhao and Ashraf in 2016. This implies how useful metal NPs can prove to be in biomedical applications. Recently, NPs have also emerged as a potential candidate to combat multidrug-resistant microorganisms caused due to improper/excessive use of antibiotics (Singh et al., 2014).

TABLE 3.2

Antimicrobial Activity of Azadirachta indica Mediated Biosynthesized Nanoparticles (NPs)

For effective bactericidal action, the contact of NPs with the bacterial cell is a prerequisite, which may be achieved by any of the forms of interaction, including van der Waals interactions, receptor-ligand binding, and hydrophobic interactions (Wang et al„ 2017). Once the NPs succeed in crossing the cell membrane, they start altering the bacterial basic metabolism by interacting with the DNA, enzymes, ribosomes, and lysosomes, leading to several outcomes, including oxidative stress and distorted cell membrane permeability, to name a few'. The exact mechanism by which the NPs act against bacteria remains to be unravelled. The following are the most hypothesized and accepted mechanisms for the antimicrobial action of the metallic NPs. Bacterial cell walls and membranes act as shields against the external environment. The bacterial cell wall particularly keeps the bacteria intact. NPs interact with bacterial cell walls according to the nature of its components. It has been seen that the NPs exert a greater inhibitory action on gram-negative bacteria as compared to the grampositive ones. This has been linked to the difference in their cell walls. Gram-positive bacteria possess thicker peptidoglycan (PG) layer studded with teichoic acid while gram-negative bacteria possess thinner peptidoglycan layer surrounded by an additional layer of lipopolysaccharides (LPS). This arrangement favours the facilitation of NPs across the PG layer. Also, due to the presence of the LPS layer, the negative charge is higher on the gram-negative cell wall, which can easily attract positively charged ions released by NPs and lead to disruption of the cell wall. In an investigation to explore the bactericidal action of AgNPs on E. coli, Li and his colleagues (2010) performed permeability studies that implied an efflux of essential sugars and proteins and the distortion of respiratory enzymes. The TEMs and SEMs depicted the destruction of bacterial cell walls, causing the death of the bacteria. Oxidative stress induced by the reactive oxygen species (ROS) generated by nanoparticles is one of the most important mechanisms rendering the bacteria susceptible to NPs. It is one of the major factors damaging the integrity of the bacterial cell membrane and altering permeability behaviour (Cheloni et al., 2016). Superoxide radical (O^-2), hydrogen peroxide (H₂0₂), the hydroxyl radical ( OH), and singlet oxygen (O,) are the ROSs that cause damage to the bacterial cell in varying amount. ZnONPs can possess the ability to produce OH and H,0₂ free radicals w hile CuO NPs can generate all of them (Malka et al., 2013). The generation and scavenging of the ROS are in equilibrium under normal conditions, but surplus production of ROS induces oxidative stress, culminating into the damaging number of cell components (Li et al., 2012; Peng et al., 2013). Metal oxides such as CuO. Ti0₂, and ZnO produce excellent antibacterial activity as they are capable of generating ROS (Singh et al., 2014). Das et al. (2017) studied the antimicrobial action of green-synthesized silver NPs on E. coli and S. aureus. It w'as concluded that ROS generation was associated with the treatment of bacteria with AgNPs and that the consequent oxidative stress caused the death of the bacteria. Another crucial mechanism involved in the lethal action of metal NPs on bacteria is based on the interference of the synthesis of DNA and bacterial proteins. It has been shown that silver nanoparticles disintegrate the bacterial DNA as they interact with phosphorus and sulfur-containing compounds. Also, nanoparticles can disrupt signal transduction pathways by dephosphorylating the peptide substrate on tyrosine residue in gram-positive bacteria, thus obstructing growth. The binding ability of AgNPs to the mercapto (-SH) group leads to the denaturation of bacterial proteins (Kim et al..

FIGURE 3.3 Mechanism of antimicrobial action of metal nanoparticles.

2009). CuNPs also act similarly by affecting DNA replication and transcription and obstructing growth by interacting with the mercapto (-SH) group (Nisar et al., 2019).

The antimicrobial activity of NPs is greatly dependent on their size as well. There is a direct correlation between the size and the antimicrobial potential of the NPs. Smaller NPs show enhanced antimicrobial activity. The larger surface area offers greater contact while the smaller size facilitates the penetration into the bacterial cell wall (Gurunathan et ah, 2014). NPs having size of less than 50 nm possess efficient antimicrobial activity while those having a size if 10 to 15 nm possess superior microbicidal action (Roy et ah, 2019). A study by Panacek et ah (2006) reported the synthesis of silver NPs using four saccharides, viz. glucose, galactose, maltose, and lactose. The antimicrobial activity of the NPs synthesized using glucose and galactose was found to be higher than those synthesized using maltose and lactose. This might have resulted due to the smaller size of NPs synthesized using glucose and galactose than those synthesized using maltose and lactose.