Elucidation of biogenic silver nanoparticles susceptibility towards Escherichia coli : an investigation on the antimicrobial mechanism

Abstract

Elucidation of the role of silver nanoparticles (AgNPs) in combating bacterial infection is important for the development of new antimicrobial compounds. In this study, several key factors underlying biological effects of biogenic AgNPs were investigated on recombinant Escherichia coli (XL1‐Blue) which contains a reporter gene encoding β ‐galactosidase enzyme. Biogenic AgNPs were prepared from the tea decoction. Cytotoxicity effects were profound on the bacteria tested by the synthesised NPs. The β ‐galactosidase activity of the released intracellular proteins in the supernatant of E. coli was used as a measure of membrane damage and cellular leakage. Occurrence of a significant amount of β ‐galactosiadase activity in the supernatant of treated cells clearly demonstrated the formation of holes in the bacterial membrane. Scanning electron microscope pictures visibly indicated destruction of the membrane of the bacteria, which further confirmed membrane damage. The synthesised NPs caused damage of E. coli genomic DNA in a dose dependent manner.

1 Introduction

A major cause of death in developing countries is due to infectious diseases caused by dreadful microorganisms [1, 2]. To overcome such infectious diseases, antimicrobial molecules that kill or inhibit the growth of microbial population have been discovered, since 1939. An antimicrobial molecule works by binding to important components of bacterial metabolism, thereby inhibiting functional biomolecules synthesis or disturbing regular cellular activities. Despite rapid development in antibacterial therapy, many infectious diseases, particularly intracellular infections, are hard to treat. One of the reasons is that many antimicrobials are difficult to be transported through cell membranes and have low activity towards the cells imposing negligible inhibitory or bactericidal effects on the intracellular bacteria [3, 4]. Many microbes have developed resistance to almost all antibiotics used routinely [5, 6, 7]. Hence, there is need for new antimicrobials like silver nanoparticles (AgNPs). It has been reported that AgNPs has strong cytotoxic and antimicrobial property against wide range microbes which are pathogenic [8, 9, 10, 11]. Apart from medical application of these NPs, there were reports of disinfection of drinking water [12, 13]. Though AgNPs have numerous antimicrobial applications, the mechanism of killing microbes is not completely comprehended. It has been hypothesised that AgNPs can cause cell lysis or inhibit cell signal transduction [14]. Nanoparticles have the ability to anchor to the bacterial cell wall and subsequently penetrate it, enabling structural changes in the cell membrane like modifying the permeability of the cell membrane and resultant death of the cell. The formation of free radicals by the NPs is believed to be another mechanism of cell death [15]. Electron spin resonance spectroscopy studies confirmed formation of free radicals by the NPs when in contact with the bacteria and these free radicals have the ability to damage the cell membrane and make it porous that may ultimately lead to cell death [16]. The nano‐sized metal particles additionally enhance many of these properties because of the increase in surface area and surface to volume ratio [17]. Some studies nevertheless reported that the positive charge on the Ag ion is crucial for its antimicrobial activity through the electrostatic attraction between negatively charged cell membrane of microorganism and positively charged NPs [18]. Although this involved binding, the mechanism of the interaction between NPs and component(s) of the outer membrane is still unclear. Environmental signals are sensed by the membrane‐bound signal transduction system. Disturbing the membrane integrity is thus a crucial strategy of an antimicrobial drug. Conventional antibiotics aim at inhibiting the synthesis of protein, RNA, DNA, folic acids or peptidoglycan and thus act on growing bacteria. Therapeutic molecules having potential to damage membrane may thus be used as antimicrobial even for bacteria causing persistent infection. In our previous communication, we had reported antimicrobial and antibiofilm activity of AgNPs synthesised with the help of tea leaf extracts against pathogenic bacteria, but cell killing strategies by NPs was not fully studied [19]. So, in this study, we have focused on biogenic AgNPs highlighting the intricate cell killing strategy. The reducing property of the tea leaf was utilised for synthesising biogenic AgNPs. The cellular leakage owing to membrane damage due to AgNP treatment was observed in different bacteria. For this, recombinant Escherichia coli (XL1‐Blue) expressing reporter enzyme (β ‐galactosidase) was used to confirm cellular leakage created by NPs on the bacterial cell membrane. In vivo DNA damage and production of reactive oxygen species (ROS) in bacteria were demonstrated and it was shown that one of the possible ways by which NPs kill bacteria is by the production of ROS which fragments DNA.

2 Experimental

2.1 Materials

Silver nitrate (AgNO3, >99.9% pure) was purchased from Sigma Aldrich. Orthonitrophenyl‐β ‐D‐galactopyranoside, 2, 3, 5‐triphenyl tetrazolium chloride (TTC), antibiotics (erythromycin and norfloxacin) and all other reagents were purchased from Himedia, India. Semi‐fermented tea (Camelia sinensis) leaf was purchased from Castle Vintage Darjeeling Tea, Goodricke group Ltd., Kolkata. All reagents used were of analytical grade.

The E. coli XL1‐B strain having reporter gene encoding β ‐galactosidase enzyme, was a kind gift from Dr. S. Datta, Haldia Institute of Technology, Haldia (India) and was used as a model organism for this study.

2.2 Synthesis and characterisation of AgNPs

Biogenic AgNPs were synthesised according to Kumar et al. [20]. Tea leaf extract was prepared by taking 10 g of thoroughly washed leaves in a 250 ml flask with 100 ml of sterile distilled water and then boiling the mixture for 2 min before finally filtering it through Whatman No.1 filter paper. The extract was stored at 4°C till further use. Silver NPs were synthesised according to standard reported methods. The reduction of pure Ag ion (Ag⁺) into Ag (Ag⁰) was monitored by measuring the ultraviolet–visible (UV–vis) spectrum of the reaction medium (sample) by Shimadzu UV spectrophotometer (PharmaSpac, UV‐1700) using a quartz cell (1 cm path). The transmission electron microscopy (TEM) study was carried out in JEOL, JEM 2100 electron microscope, operating at an acceleration voltage of 200 kV. The NPs suspension was thoroughly sonicated before TEM analysis.

2.3 Antimicrobial efficacy of AgNPs

In vitro antimicrobial activity of the colloidal NPs was screened against a recombinant E. coli (XL1‐Blue). Stock culture of bacterial strain was maintained at 4°C on nutrient agar medium. Active cultures were prepared by inoculating fresh nutrient broth medium with a loopful of cells from the stock cultures at 37°C overnight. To get desirable cell counts for bioassays, overnight grown bacterial cells were subcultured in fresh nutrient broth at 37°C. Minimum inhibitory concentrations of NPs against the bacterial strains were determined using the growth indicator tetrazolium/formazan test (TTC) according to Omeroglu et al. [21]. In the presence of viable bacteria, TTC is reduced to red formazan and thus the change from colourless to red colour indicates the viability of the bacterial cells. All bacterial strains were grown in 10 ml of Luria Broth (LB) with shaking at 225 rpm overnight (37°C). To each well, 100 μl (1 × 10⁵ cells/ml) of bacterial suspension was added and then 100 μl of different concentrations of NPs (0–100 μg/ml) was transferred into a well of microplate. The final volume of each well of microplate was adjusted to 1 ml by LB medium. The microplates were incubated at 37°C overnight. 25 μl of sterile TTC (5 mg/ml) was put in each well and then the microplates were incubated at 37°C again. After overnight incubation, the minimum inhibitory concentration (MIC) of NPs against the isolates was determined by observing the colour change in each well. The concentrations at which no changes of colour occur indicate complete cell death.

2.4 Membrane damage

For the study of the release of cytoplasmic material, bacterial cells were harvested from 10 ml of culture by centrifugation at low speed and the pellet was washed with phosphate buffer saline (PBS) buffer (1×, pH 7.2). Next, the pellet was suspended in 1 ml of PBS buffer and the cell concentration was adjusted 1 × 10⁵ cell/ml. Different aliquots of the cell suspension were treated with and without NPs (MIC₅₀) and incubated at room temperature for different time periods (0, 1, 3, 4, 7 h). The samples were centrifuged to remove bacterial cells, and absorbance values of supernatants were recorded at 260 nm for nucleic acid release and protein leakage was estimated by Lowry's method [22]. Determination of β ‐galactosidase activity was based on the hydrolysis of the substrate ortho‐nitrophenyl‐β ‐D‐galatopyranoside to the yellow product ortho‐nitrophenol and β ‐D galactose. Ortho‐nitrophenol was spectroscopically quantified at 420 nm. The amount of cytoplasmic material released was recorded by subtracting the optical density (OD) values obtained from cells without treatment and was compared with OD values of Triton X‐100 treatment (positive control) to get cytoplasmic leakage index. The interactions between microbial cell membrane and NPs were also observed using a scanning electron microscope. The experiment was repeated three times.

2.5 Effect of AgNPs on E. coli genomic DNA

E. coli (1 × 10⁵ cells/ml) was treated with and without NPs (2.5–10 μg/ml) and incubated at 37°C overnight. After the incubation is over, cells were treated with 200 μl cell lysis buffer (50 mM TrisHCl, pH 8.0, 10 Mm ethylene diamine tetra acetic acid, 0.1 M sodium chloride, and 0.5% sodium dodecyl sulphate) for 1 h at 37°C. The lysate was incubated with 0.2 mg/ml proteinase K at 40°C for 2 h. After completion of incubation, the sample was centrifuged for 10 min at 10,000 g. The aqueous portion, containing the DNA was transferred to new Eppendorf tube. DNA was subjected to electrophoresis in 0.9% agarose gel, in 1× TAE buffer (40 mM Trise‐acatate, 1 mM ethylenediaminetetraacetic acid), followed by ethidium bromide staining (50 μg/ml) and visualised by gel documentation system. The resulting fragmentation of DNA owing to NPs treatment of bacterial cells was analysed.

3 Results and discussion

3.1 Synthesis and characterisation

The formation of AgNPs by reduction of the Ag⁺ s during the exposure to the extact of semi‐fermented leaves of tea (Camelia sinensis L) was followed by UV–vis spectroscopy. Fig. 1 a shows the UV–vis spectrum recorded from the aqueous Ag NO3‐tea leaf extract reaction medium with the surface plasmon resonance band of Ag appearing at 425 nm. The spectra also indicate that the particles have an average size of 35 nm [23, 24]. The colourless aqueous Ag NO3 solution became brown after overnight incubation at 37°C with tea aqueous extract (Inset Fig. 1 a). It is well known that this change in colour of the AgNPs occurs because of the surface plasmon vibration in the NPs [25, 26]. Representative TEM images of sample are shown in Fig. 1 b. TEM micrograph reveals that the particles are spherical with average size of 35 nm diameter. The selected area electron diffraction (SAED) pattern (Inset of Fig. 1 b) with bright spots indicate that the AgNPs are crystalline in nature. Aqueous extract of tea leaf that contains phenolic compounds, proteins and carbohydrates acts as an efficient reducing agent for Ag⁺ s as well as a good capping agent for AgNP. Our previous report demonstrats that these phytochemicals and proteins are present on the NPs by Fourier transform infrared spectroscopy analysis [19]. Recently, many authors had reported the use of plant extracts for biosynthesis of AgNPs. Vaidyanathan had extensively reviewed the synthesis of metal NPs using green technology. In these reviews, the authors found that the phenolic compounds, flavonoids, terpenoids, polysaccharides, enzymes and other proteins are responsible for reduction of metal ions and stabilisation of NPs [27]. Capping is an essential requirement for stability of NPs. Stability of any NPs is prerequistive for its role in a specific environmental condition [28]. Steric stability of NPs must be due to protein and carbohydrate present in the tea extract. Silver NPs can thrive well in wide range of water chemistries through its steric stability by protein or carbohydrate [13]. Zhang et al. [28] showed high anti‐bacterial efficacy of polymer (casein, dextrin and PVP) stabilised AgNPs. Our studies corroborate these results.

Fig. 1.

Synthesis and characterisation of AgNPs

a UV–vis spectra of aqueous solution of AgNPs. The inset depicts colour change observed in the reaction mixture containing tea aqueous extract and 1 mM Ag NO3 after overnight incubation at 37°C with continuous shaking

b TEM image of AgNPs illustrating the high resolution image. The inset displays the SAED pattern of NPs

3.2 Antimicrobial activity by AgNPs

Silver NPs showing bacteriocidal activity on both Gram‐positive and Gram‐negative bacteria as earlier reported [8, 17, 20, 29] yet detailed mechanism of cell killing by these particles is not fully elucidated. Among Gram‐negative bacteria, E. coli causes various diseases affecting large population. Hence, it has been considered as an important model organism for in‐vitro analysis of antimicrobial agents. To study in more details the mode of action towards bacterial killing; we selected pUC18 containing E. coli (XL1‐Blue) as our model organism which contains a reporter gene which encodes β ‐galactosidase enzyme. The enzyme activity of the released intracellular proteins in the supernatant was used as a measure of membrane damage and leakage of cellular proteins. The antimicrobial activity of our synthesised NPs was tested against bacterial cells and found to be highly susceptible. MIC result of NPs exhibited inhibitory response against the tested bacterial strain was > 3 µg/ml.

3.2.1 Membrane damage

Silver NPs destroy permeability of E. coli membranes as evidenced by formation of pits and membrane fragmentation [9, 24]. Cell membrane leakage which contains both DNA and protein in significant amount confirmed the damage of bacterial membrane and resultant formation of pores. To examine the pore formation, E. coli XL1‐Blue strain carrying the plasmid pUC 18 was treated for different time period with NPs to confirm leakage of β‐ galactosidase enzyme with ortho‐nitrophenyl‐β ‐D‐galactopyranoside as substrate. The β ‐galactosidase enzyme activity in the supernatants of NP treated (MIC₅₀) and untreated bacterial cells was assayed using spectroscopic analysis (Fig. 2 a). The occurrence of significant amount of β ‐galactosiades activity in the supernatant of NP‐treated cells indicate the formation of holes in the bacterial membrane, since this enzyme is a complex large molecule. β ‐galactosiades is a homotetramer of 464 kD, so it is a large protein, which could be secreted through the NPs treated E. coli and not from untreated cells. Upto 5 h post treatment with NPs the β ‐galactosiades activity increased in the supernatant in a time dependent manner, but beyond this period the activity diminished. Decrease of β ‐galactosiades activity was probably due to release of proteolytic enzyme after membrane damage that degrades the protein with increasing incubation time.

Fig. 2.

Bacterial killing mechanism by synthesised AgNPs in E. coli

a Bacterial cytoplasmic leakage of β ‐galactosidase enzymes due to nanoparticles treatment for different time points (1, 3, 5, 7 h) in PBS buffer at room temperature

b SEM exhibiting the effect of NPs (panel 2) on cell morphology in comparison to the untreated cells (panel 1)

c Effect of ascorbic acid on NPs treated bacterial cell. Ascorbic acid reverses the ROS generated by NPs inside the cells

d DNA fragmentation assay. Lane M (1 kb ladder), the remaining lanes (1–5) exhibit DNA samples isolated from the untreated cells (lane 1) and NPs treated (2.5–10 μg/ml) cells (lane 2–5). Owing to the heterogeneous sizes of CT DNA sample (lanes 2–5), the DNA fragments merge into one another and appears as a smear on the gel

3.2.2 Membrane modifications

The surface morphology of NP treated (MIC₅₀) and untreated bacterial cells was studied using scanning electron microscope (SEM) (Fig. 2 b). SEM pictures indicated a morphological change after 3 h of treatment with NP (pointed by white arrows in Fig. 2 b) showing the disrupted membrane structure and the elongated shape of the bacterium E. coli. Due to leakage of the intracellular materials from the pores of NPs treated bacteria, many of the cells look slimmer and irregular in shape. Significant change in cell surface morphology is not observed in SEM image because bacterial cell has an outer skeleton (cell wall) which keeps the shape even if the cell loses cytoplasmic material. The morphological change indicates that the target of the NPs was on the microorganism's surface such as lipid bilayer or cell wall. The destabilising interaction of NPs and membrane components of bacteria is also an important reason for membrane damage.

3.2.3 ROS generation

Metal NPs and their ions can produce free radicals, resulting in induction of ROS [4, 30, 31]. A few studies reported that these nano‐sized Ag particles get converted into metal ions after entry into the bacterial cells. Moreover, these heavy metal ions generate ROS, which damages bacterial membrane and DNA [32] and thus exhibits bacteriocidal activity. To check ROS production by NP, we tested the effect of antioxidant (ascorbic acid) on NP treated cells. Here, ascorbic acid was used as antagonist which eradicates ROS generated by NP. The histogram representing E. coli cells treated with NP (MIC₅₀) and ascorbic acid (0–10 μg/ml) when compared with untreated control revealed the scavenging of NP generated ROS by the antioxidant (Fig. 2 c). Dose dependent increase in cell number was noted with ascorbic acid. Few reports on ROS scavenging by antioxidants like ascorbic acid and N‐acetyl‐L‐cysteine (NAC) in NP treated animal cell lines are reported. NAC reverses the generation of ROS in NP treated myeloid leukaemia cells [32]. However, study on bacterial system is still lacking. The increase in cell density with increasing concentration of ascorbic acid is a reflection of scavenging of ROS generated by NPs.

3.2.4 DNA damage

TTC results suggested that NPs caused cell death, and presence of protein and DNA in the culture supernatant was due to leakage of cellular materials through holes created by NPs. To further elucidate the mode of action, we profiled the DNA of E. coli with NPs treatment. A dose‐dependent analysis was performed using NPs treated and untreated E. coli cells in growing state. Four different doses (2.5–10 μg/ml) of NPs were selected. After overnight incubation of bacterial growth, the DNA profile was analysed (Fig. 2 d) and compared with untreated sample. DNA smear of the NPs treated samples confirmed necrosis and showed that the AgNPs treated cells exhibited extensive double strand breaks of various sizes, thereby yielding a smear like appearance (lane 2), while the DNA of control cells exhibited no breakage (lane 1). Study on NPs interactions with the nitrogenous bases and phosphate groups in DNA were earlier reported [33] which support necrosis like events in bacterial DNA. Reports on bacterial DNA degradation via ROS generation by AgNPs were reported by very few authors [33, 34]. The overall results revealed bacterial susceptibility, the mechanism of which might be the damage of cellular membrane and eventually genomic DNA by the ROS generated by NPs.

4 Conclusions

Surface active agents and other membrane damaging compounds are drawing significant attention in the field of antimicrobial chemotherapy. Membrane active agents generally have multiple target sites and diverse modes of action against the organism, reducing the chance of mutation at the target site. This study on eco‐friendly, biogenic AgNPs highlighted the cell killing strategy which is exhibited by the proposed model (Fig 3). Though, at this stage it is premature to suggest that NPs will ever substitute the role of antibiotics in the treatment of bacterial infection, particularly in the case of multiple‐drug resistant (MDR) strains but it is tempting to speculate that it might provoke more work in this direction to aid in medical therapy.

Fig. 3.

Schematic illustration of mechanism of bacterial killing by AgNPs. NPs is postulated to disturb cell membrane integrity

Disturbance of cell membrane causes pores through which cellular materials (DNA and protein) leakage takes place. Furthermore, ROS generation inside the cell takes place that affects gene expression and even fragmentation of genomic DNA. Finally cell lysis occurs due to pores on the cell membrane, loss of metabolic activity and cellular leakage