**3. Some mechanisms of bactericidal action of silver nanoparticles (AgNPs)**

#### **3.1 Electrostatic attraction**

*Pathogenic Bacteria*

ripening [10–12].

microorganisms.

of the nanoparticle [21, 22].

these contaminations [13, 14].

**2. Silver nanoparticles for pathogen control**

The main triggers for invasion by microorganisms are physiological changes that activate ethylene synthesis or that cause changes to the cuticle or cell walls (loosening), or declines in natural antifungal compounds or high contents of carbohydrates and other nutrients and water. These changes usually occur naturally during

Postharvest contamination of fruit by human pathogens can be another key issue in the supply chain. The most commonly reported human pathogen contaminants causing disease outbreaks are bacteria such as *Escherichia coli* (*E. coli*), *Salmonella* spp., *Mycobacterium* spp., *Brucella* spp. and *Pseudomonas aeruginosa* (*P. aeruginosa*)*.* However*,* good manufacturing and handling practices can significantly reduce

Because of the behavior of microbial populations, including fungi and bacteria, an initial infection may originate new infection foci that appear near the primary one, so increasing disease incidence and/or severity [15, 16]. Quality deterioration and loss of fresh fruit and vegetables during storage have an exceptionally high economic impact because by this stage high costs have been incurred in harvesting, grading, packaging, freighting and storage. All these reasons emphasize the importance of defining new practices to reduce populations of the postharvest

Silver nanoparticles (AgNPs) offer oligodynamic action which is also of low toxicity and broad spectrum [17–19]. Moreover, compared with synthetic biocides, there is also only a low chance that microbial resistance might develop. These AgNPs have been exploited against Gram-negative bacteria, such as *Acinetobacter, Escherichia, Pseudomonas, Salmonella* and *Vibrio,* and against Gram-positive bacteria including *Bacillus, Clostridium, Enterococcus, Listeria, Staphylococcus* and *Streptococcus* [20]. A number of research reports have demonstrated that their antimicrobial nature depends on the surface-capping agent and the size and shape

The effectiveness of AgNPs also depends on bacterial dose [23]. Silver nanoparticles affect the growth of bacteria in a dose-dependent manner. In a study conducted by Agnihotri et al. [23], concentrations of 10 and 20 μg/ml Ag (10 nm) caused reductions of ~18 and ~53% in *E. coli*, respectively. Meanwhile, AgNP

Silver nanoparticles smaller than 100 nm, and containing between 10,000 and 15,000 silver atoms, are effective as antibacterial agents [20]. The AgNPs' antibacterial potential increases as size decreases. This effect is more pronounced for AgNPs of size <10 nm, because contact with the bacterial cell is direct [24].

Research into the antimicrobial activity of AgNPs against Gram-positive and Gram-negative bacteria shows Gram-negative bacteria are more sensitive to AgNPs than Gram-positive ones [23, 25], although their relative sensitivity cannot be explained based only on a difference in the composition of the cell membrane. In studies using discs impregnated with AgNP in culture media with bacteria, the formation of a clear zone of inhibition around the impregnated discs is an indicator of bactericidal potential of AgNP > 15 nm [21]. Bacteria are unable to survive in this area, possibly because of the release of silver in the form of nanoparticles or

In addition, nanoparticle silver can be released by the mobility of small size AgNPs through the semisolid agar, whereby a zone of inhibition is observed.

concentrations at 30 and 40 μg/ml eliminated all bacterial growth.

**204**

of silver ions.

A way to transport active silver cations to the bacteria can occur on the cell membrane or within the cell. When combined with protonated chitosan, the positively charged AgNPs bind well to the negatively charged bacterial membrane proteins through electrostatic attraction [23].

#### **3.2 Alterations in the bacterial membrane**

The first bacterial contact with AgNP can trigger an antibacterial mechanism by facilitating the entry of AgNPs into the bacterial cells. This is followed by an explosive release of silver ions inside the bacterial cells causing the bactericidal effect.

The nature of the AgNP, bacteria interaction and its antibacterial effect have been analyzed by a number of methods. Bacteria exposed to AgNPs show high protein leakage and morphological changes [26]. As an example, *E. coli* treated with AgNPs (∼10 nm) appeared to shrink and develop an irregular shape. Micrographs show AgNPs on the cell membrane attached to the lipopolysaccharide layer of the cell wall, and a proportion of AgNPs were found inside the bacterial cell [23].

Biao et al. [21] noticed that bacterial strains have intact membranes and smooth surfaces in the absence of silver colloid, whereas after exposure to chitosan-silver colloid, the cell membrane and surface become shriveled, invaginated and disrupted. This cell membrane damage indicates the mode of action of chitosan-silver colloid. Its bactericidal effect is attributed to the release of silver cation from AgNPs and to alteration of the bacterial cell wall structure and associated physicochemical changes.

Osmoregulation of the bacterial cell can also be affected causing extrusion of intracellular material and hence cell death. The deformed or wrinkled cell wall can also cause leakage of cytoplasmic contents.

In addition, AgNPs can penetrate bacterial membranes, facilitating internalization. The rupture of perforation of the cell wall is an evidence of internalization of AgNP and of uncontrolled transport through the cytoplasm resulting in cell death [27] (**Figure 1**).

#### **3.3 Silver nanoparticles internalization: effects on DNA**

Multiple pathways of AgNP can occur after internalization. Silver atoms in nanoparticles are characterized by a high affinity with sulfur and phosphoruscontaining compounds such as DNA. In this way, they readily combine with cell constituents and so destroy the cell.

Silver ions can also inhibit bacterial replication by binding and denaturing bacterial DNA. Silver ions react with the thiol groups of enzymes, followed by DNA condensation resulting in cell death [28–29].

Blocking of respiration is also a result of the interaction with cell membranes [30].

**Figure 1.** *Mode of action of silver nanoparticles in the bacterial cell.*

Disruption of biofilms is another effect of AgNPs. The anti-biofilm action of ~8 nm AgNPs on Gram-negative bacteria has been demonstrated [31]. The outer membrane of Gram-negative contains aquaporins (water-filled channels) which are involved in the transport of Ag ions into the cell where they exert their antibacterial effects [32].
