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Pharmaceutics 2020, 12, 1044 9 of 19 other hand, that of Gram +ve bacteria is basically thicker. Gram +ve bacteria has a thick layer of peptidoglycan within their cell walls while Gram −ve bacteria have a thin layer of peptidoglycan with an extra outer membrane embedded lipopolysaccharide. This additional membrane in Gram −ve bacteria means that there is also an extra membrane layer termed periplasm (Figure 4). Several research works have reported that Gram +ve bacteria are more resistant to MNPs mechanisms of action [83–87]. This is due to the different cell wall structure. In Gram −ve bacteria, such as E. coli, a 1–3 μm layer thick of lipopolysaccharides cover the cells, in addition to 8 nm thick layer of peptidoglycans. This facilitates the passage of ions from NPs into the cell whereas Gram +ve bacteria like S. aureus have a thicker peptidoglycan layer, which stretches over 80 nm with covalently bound teichoic and teichuronic acids. The damage to the cell membrane of bacteria that happens from the interaction between the cells and MNPs becomes more harmful to the Gram-negative bacteria. This is due to the absence of a thick protective layer of peptidoglycan as seen with Gram +ve bacteria Furthermore, Gram −ve bacteria susceptibility to MNPs is due to their negatively charged lipopolysaccharide. This causes an attraction to the positive ions released by most MNPs. The consequent effect is an accumulation of ions that leads to intracellular damage. However, it is known that both Gram +ve and Gram −ve bacteria have a negatively charged cell wall that allows for interactions between the cell wall and the MNPs or its ions [88]. A study of Gram −ve Salmonella typhimurium revealed that a mosaic of anionic surface domains present on the cell wall in an abundant measure [89]. Thus, increased toxicity is observed when a high concentration of NPs binds to these negative anionic domains. Additionally, through mathematical calculations and electrophoretic mobility study, it was found that E. coli is more negatively charged and rigid than S. aureus [90]. The outer membrane comprising of proteins and lipids is the first barrier encountered by AgNPs. Silver forms a complex with electron donors like nitrogen, oxygen, sulphur atoms or phosphorus in the interactions with the proteins in the outer membrane. This interactions leads to the inactivation of proteins and membrane bound enzymes of the bacterial cell wall [91–93]. The bactericidal mechanism of AgNPs biosynthesized with turmeric against E. coli O157:H7 and Listeria monocytogenes was elucidated by Alsammarraie et al. [94]. Microscopic images of cells treated with AgNPs showed cell membrane damage with irregular shapes, protrusions and fragmentations. The cytoplasmic membrane of both cells was separated from their cell walls and completely damaged. This led to their rupture and release of cell constituents due to the physical impacts of the AgNPs. In addition, deposits of AgNPs were seen around severely damaged bacterial cells, both in the cell membrane and cytoplasm of the bacteria, especially in E. coli O157:H7 (Figure 5B). The treated cell shown in Figures 5D and 6D reveals a big hole and fragmented cell membrane that resulted in a totally lysed cell. Figure 5D further reveals severe shrinkage cytoplasmic constituents’ leakage of E. coli O157:H7. The TEM and SEM micrographs confirm that the antibacterial activity of biosynthesized AgNPs by turmeric was obviously bactericidal and not bacteriostatic. SEM-EDS analysis showed that a strong signal of elemental Ag was present in the treated cells confirming that AgNPs were responsible for the observed damages in the cells. The Fourier transform infrared (FTIR) microspectroscopic method was used to study the bactericidal mechanism of garlic acid (Ga) conjugated AuNPs (AuNPs-Ga) against Plesiomonas shigelloides and Shigella flexneri [95]. The results were analyzed by a principal component analysis (PCA). There were two regions of interest in the PCA. Firstly was the biochemical print region for stretching vibrations of esters found in lipids (1800–1000 cm−1), amide I and II groups belonging to peptides and proteins (1655–1637 cm−1), P=O stretching of nucleic acids (1250–1220 cm−1 and 1084–1088 cm−1) and the typical bands for polysaccharides and carbohydrates (1200–900 cm−1). Secondly was the wavenumber from 3000 to 2850 cm−1 that is attributed to known functional groups of specific amino acid side chains and membrane fatty acids. The PCA plot revealed the differences in the spectra of the treated and untreated bacterial cells. Each of the representative loading plots had a change in their lipid, protein and cellular phosphorylation signal [95–98]. Significant changes in the lipid and protein signal signifies a destruction of the cell membrane and biochemical alteration of the bacteria cells.PDF Image | Bactericidal Antibacterial Mechanism of Plant Nanoparticles
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