The Effect of Silver Nanoparticles on Listeria monocytogenes PCM2191 Peptidoglycan Metabolism and Cell Permeability

An ionic form of silver has been used for centuries to cure several diseases which causative agents were bacteria such as Staphylococcus aureus, Klebsiella sp. and Pseudomonas sp. (Rai et al. 2009). It was shown that silver nanoparticles, AgNPs, have higher antibacterial activity than silver ions (Ingle et al. 2008). Nanoparticles are defined as the clusters of atoms of size from 1 to 10 nm with a large surface area to volume ratio, what is proportionally correlated with AgNPs antibacterial activity (Morones et al. 2005). Several studies demonstrated that antibacterial effect of silver nanoparticles is based on their interaction and subsequent damage of cell membranes and on the induction of reactive oxygen species (ROS), including free radicals. The interaction of AgNPs with cell membranes is promoted by their strongly positive zeta potential, which is a difference of an electric potential between the particle and the surrounding solution (Stapsford et al. 2011). This interaction leads to membrane disruption, bacterial flocculation, efflux of cytoplasm, and as a consequence reduction in viability. Formation of ROS is responsible for oxidation, subsequent inactivation and damage of cellular proteins and DNA and peroxidation of lipids (Singh et al. 2008).

It was also proved that AgNPs are active against bacterial biofilms, which are complex bacterial communities resistant to antibiotics and the human immune system. Biofilm resistance is very important and now constitutes a medical challenge as recently the number of infections associated with antibiotic-resistant bacteria living in biofilms has been increased exponentially. These included infections caused by Pseudomonas aeruginosa, the causative agent of nosocomial respiratory tract pneumonia, infections of burn wounds, and chronic lung infections of patients with cystic fibrosis. Biofilm formed by S. aureus also constitutes a very important clinical problem being responsible of e.g. osteomyelitis, periodontitis and chronic wound infections. Well-defined diseases are also caused by biofilms formed by gastrointestinal tract pathogens belonging to Enterobacteriaceae family (for review see Markowska et al. 2013; Wolska et al. 2015). Beside their intrinsic antibacterial activity silver nanoparticles were proved to enhance the effect of conventional antibiotics, such as: vancomycin, amoxicillin, gentamycin, ampicillin, streptomycin in curing bacterial infections (Shahverdi et al. 2007; Birla et al. 2009).

Listeria monocytogenes is a Gram-positive pathogen able to survive in a variety of environments including food, soil and humans. It constitutes very severe problem for food industry because it can survive and multiply even in low temperature; moreover, it forms biofilm and therefore is hard to be eradicated from food production lines. This species is characterized by a unique life mode; it grows in the cytoplasm of the host cell and spreads between cells utilizing actin-based motility (Gray et al. 2006). This pathogen has an ability to cross three human barriers: intestinal, bloodbrain and fetoplacental. L. monocytogenes is a causative agent of listeriosis, which can be a fatal infection especially for elder people, immunocompromised individuals, and pregnant women (Alleberger and Wagner 2010). The fatality rate achieves 30%, so this disease represents a major public health concern. Listeriosis in neonates is one of three principal causes of bacterial meningitides. The infections of central nervous system are also described in adults with the mortality rate reaching even 60% (Vázquez-Bolland et al. 2001). L. monocytogenes produces several virulence factors; the major one is listeriolysin O (LLO), a pore-forming toxin belonging to the family of cholesterol-dependent cytolysins that is crucial for escape from vacuole after entry to the mammalian cell (Hamon et al. 2012). The activity of LLO is vital for inducing diarrhea and an inflammatory response after reaching intestinal tract (Barbuddhe and Chakraborty 2009).

The peptidoglycan (murein) constitutes the main compound of L. monocytogenes cell wall and plays a major role in L. monocytogenes pathogenesis (Boneca 2005). Its structure is unusual for Gram-positive bacteria, remaining this of Gram-negative species (e.g. E. coli) because of the presence of partially deacetylated N-acetyloglucosamine residues (Boneca et al. 2007). Another exceptional future of this pathogen is its ability to encode a high number of surface proteins what reflects the ability of L. monocytogenes to survive in a range of diverse environments (Bierne and Cossart 2007). L. monocytogenes is still susceptible to a variety of antibiotic but it should be mentioned that it is intrinsically resistant to a broad spectrum of cephalosporins commonly used in the therapy of many bacterial infections. The resistance to cephalosporins is based on several mechanisms including multidrug transporters and envelope proteins with a detoxification function (Krawczyk-Balska and Markiewicz 2016). In view of expanded resistance to antibiotics, the search for alternative therapies seems to be an urgent task.

The aim of the present study was to investigate the antibacterial effect of silver nanoparticles towards L. monocytogenes in order to identify the cellular target and mechanism of their activity.

MIC of AgNPs was 8 μg/ml, and susceptibility of Gram-positive L. monocytogenes to AgNPs was higher than that observed for one of Gram-negative pathogens Pseudomonas aeruginosa ATCC 10145, for which MIC value was equal to 1 μg/ml (Markowska et al. 2014). The results presented in Fig. 1 showed that neither growth nor survival of L. monocytogenes was substantially diminished in the presence of AgNPs in a concentration of 0.25 MIC or 0.5 MIC. In the subsequent experiments, AgNPs were used in these concentrations.

Fig. 1.

The effect of AgNPs on the autolysis/lysis of L. monocytogenes cells was studied in the cultures treated with nonionic surfactant Triton X-100 or lysozyme. The nonionic detergent Triton-X100 and lysosyme are the commonly used agents for induction of autolysis or lysis of L. monocytogenes cells, respectively (Smith et al. 1991; Popowska et al. 2009). When used in the moderate concentration, they can be applied to study the effect of various substances on their activity. The results of the experiments are presented in Fig. 2. In the control cultures, treated only with lysosyme or Triton X-100, 49% and 89% of cells survived, respectively. The observed killing effect of lysosyme was much weaker than that of Triton X-100 as it had already been presented by Kurek and coauthors (2010). The observed result can be due to the low level of glucosamine acetylation in L. monocytogenes peptidoglycan (Amano et al. 1977). The addition of AgNPs at a concentration of 0.25 MIC resulted in very high, over 1000-fold increase of the lysis of cultures treated with lysozyme or Triton X-100. AgNPs added in a concentration of 0.5 MIC caused further drop in the number of living cells.

Fig. 2.

The addition of nanosilver to peptidoglycan also caused the enhancement of peptidoglycan autolysis; however, this effect was not as pronounced as the effect observed for the whole cells. The drop in absorbance (A₆₀₀) of the control peptidoglycan sample was 33% after 2 h incubation in the buffer. In samples treated with AgNPs at the concentration of 0.25 MIC or 0.5 MIC the observed drop was 49% and 53%, respectively. Only the last value was statistically relevant, as it was shown in Fig. 3. It can be speculated that the AgNPs enhanced the ability of autolysins – peptidoglycan hydrolyzing enzymes which catalyse polymer destruction (Rice and Bayles 2008). Five L. monocytogenes autolysins have been identified (Popowska 2004); however, the analysis of the bacteria genome revealed the presence of more than twenty proteins with the putative peptidoglycan hydrolase domains (Bierne and Cossart 2007).

Fig. 3.

The effect of AgNPs on L. monocytogenes cells permeability was estimated by two methods. First, the efflux of cF dye was measured and after 10 min of treatment with AgNPs at a concentration of 0.5 MIC the efflux of cF was enhanced by 14% in comparison to the control sample, which was 79% relative to the total leakage determined after cell lysis with DMSO (Table I). In the second set of experiments the efflux of macromolecules, DNA and proteins, was studied by measuring respectively A₂₆₀ and A₂₈₀ of the supernatants of cultures treated with AgNPs at a concentration of 0.25 MIC or 0.5 MIC, in the control culture, and in the culture treated with lysozyme (positive control).

It was demonstrated that AgNPs in a concentration of 0.5 MIC enhanced DNA efflux by 48% after 60 min of treatment with AgNPs in comparison to control culture (Fig. 4). The efflux of proteins was enhanced by 30% after 60 min exposure to AgNPs at a concentration of 0.5 MIC. The maximal efflux of DNA and proteins caused by 60 min exposure to lysozyme amounted to 171% and 191% of the control sample without AgNPs. The observed enhancement of cF, DNA and proteins efflux as a result of AgNPs treatment points to the damage of cell wall. It was previously demonstrated that AgNPs are able to cover cells surface and to induce the formation of the hollows in cell envelopes what can results in the enhancement of cell permeability (Chwalibóg et al. 2010). In turn, AgNPs, when adsorbed on the cell surface, modify membranes potential what stimulates nanoparticles transport to the cytoplasm (Morones et al. 2005; Marambio-Jones and Hoek 2010).

Fig. 4.

Until now there have been only few papers describing the effect of silver nanoparticles on L. monocytogenes membranes. Microscopic analysis demonstrated deformation, disintegration and decrease in cell surface roughness of L. monocytogenes treated with silver nanoparticles synthesized by Jatropa curcas (Chauhan et al. 2016). It has also been demonstrated that AgNPs released from nanocomposites can penetrate the cell wall and plasma membrane of L. monocytogenes what results in separation of the cytoplasmic membrane from the cell wall (Tamayo et al. 2014). The results of our group demonstrated that AgNPs caused the decrease in L. monocytogenes cell length even by 50% what may also indicate their interaction with the cell wall (data not shown) (Milczarek 2015). It has been postulated recently that the activity of AgNPs against L. monocytogenes and the other foodborne pathogens make them useful in food industry, particularly in food packaging and food preservation (Patra and Baek 2017).

In conclusion, the original results presented here show that L. monocytogenes peptidoglycan is the target of AgNPs activity. This effect is demonstrated by the increase of cell autolysis and autolysis of the extracted peptidoglycan and also by the enhancement in cell permeability. Interference with L. monocytogenes cell wall integrity and functionality constitutes the important mechanism of nanosilver antibacterial activity towards this Gram-positive pathogen.