Culture Method-Dependent Variation in the Sensitivity of Escherichia coli to Silver Nanoparticles

Abstract

Preparation of various metal nanoparticles using plant extracts has been well studied in recent years. In this study, we found that nanoparticles synthesized using the extracts of the inflorescence of Cocos nucifera exhibited differential inhibitory activity against Escherichia coli depending on the nature of the bacterial culture source. Incorporation of silver nanoparticles (Ag-NPs) into the nutrient broth culture of E. coli resulted in poor inhibitory activity. However, when the silver nanoparticles are added to nutrient agar plates used for culture of E. coli, effective inhibition was observed. Additionally, E. coli in broth culture resisted the inhibitory effects of Ag-NPs by forming aggregates of bacterial cells. The aggregates then generated a protective zone around the colonies to prevent the entry of Ag-NPs, and the bacterial cells multiplied without inhibition by the Ag-NPs. These differential effects of Ag-NPs on E. coli culture grown in nutrient broth and on nutrient agar plates indicated that E. coli in broth culture formed aggregates of cells to develop a biofilm for protection against toxins probably via the quorum sensing mechanism.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Background

The antimicrobial potential of silver nanoparticles (Ag-NPs) in biomedical application has been reported [1–4]. Indeed, Ag-NPs have been reported to exhibit antimicrobial activities through various mechanisms including catalytic oxidation of cell surface radicals for inhibition of oxygen transfer, reaction with surface radicals on bacterial and viral cells for interference with electron transport, and binding with DNA for prevention of unwinding [5–8]. Many researchers have reported that the Ag-NPs exhibit good inhibitory activity against Escherichia coli [9–14]. More than 80% of E. coli biofilms was inhibited by silver nanoparticles through membrane damage [15–17]. The silver nanoparticles induced the ROS production like changes of cellular proteins, DNA, and lipids to create the cellular death [18]. In this study, we evaluated the antibacterial effects of Ag-NPs synthesized from extracts of the inflorescence of Cocos nucifera on E. coli cells. When E. coli cells were grown in nutrient broth, Ag-NPs could not inhibit the growth of E. coli cells. However, incorporation of Ag-NPs into agar plates on which E. coli was cultured resulted in strong inhibition of Ag-NPs. Based on these findings, we also discussed this differential mechanism of drug resistance in E. coli culture depending on the culture source.

2. Methods

2.1. Plant Source and Extraction

The inflorescences were collected from mature coconut trees in Kanyakumari district, Tamil Nadu, India. The collected inflorescences were extracted using methanol as a solvent. The extracts were filtered, and excess methanol was removed [19].

2.2. Purification of Crude Extract

Five grams of crude extract were purified using column chromatography. The ethyl acetate and methanol (EA : M of 40 : 60) fraction was selected for further analysis. Phytochemical components in the extract were recorded [19].

2.3. Preparation and Characterisation of Ag-NPs

To prepare Ag-NPs, 0.169 g of AgNO₃ (Merck, India) was dissolved in 1 L double distilled water and stored in a brown bottle under dark conditions. Ten millilitres of selected fractions of C. nucifera extracts (EA : M of 40 : 60) were added to 90 mL AgNO₃ solution and allowed to react at room temperature (30°C) without any disruption. The solution colour was changed from a milky white colour to pink colour indicating the formation of Ag-NPs. The synthesized nanoparticles were characterised using ultraviolet (UV)/visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), fluorescence spectroscopy, scanning electron microscopy (SEM), and X-ray diffraction (XRD).

2.4. Assessment of Antibacterial Activity

The antibacterial activity of green synthesized Ag-NPs was tested against E. coli using the agar well diffusion method [20, 21]. Wells (8 mm in size) were made in agar plates containing bacterial inocula. The prepared Ag-NPs (25, 50, 75, and 100 μL) were added to the wells of the culture plates. Following incubation for 24 h at 37°C, the plates were observed. The zone of inhibition was measured using a HiMedia measuring scale and expressed in millimetres.

In another experiment, fresh cultures of E. coli were inoculated into sterile nutrient broth and incubated at 37°C for 24 h. The prepared Ag-NPs (0.250, 0.500, 0.750, 1, and 2 mL) were added to the broth culture tubes. The total volume of bacterial culture with nanoparticles was 5 mL. This mixture was divided into two portions, which were incubated at 37°C for 2 h. After the incubation period, one of the portions of this mixture of bacterial culture was spread on sterile MH media (Muller–Hinton media) plates. Another portion of broth with the nanoparticles mixture was incubated for 2 h, and the bacterial growth was determined by measuring the absorbance at 605 nm using a UV/visible spectrophotometer.

Five microlitres of bacterial culture were added to a nutrient broth tube containing prepared nanoparticles (0.250, 0.500, 0.750, 1, and 2 ml). This mixture was incubated at 37°C for 24 h. After incubation, the bacterial culture was spread on a sterile plate with MH medium. The plates were then incubated at 37°C for 24 h. The culture growth rate was determined by measuring the absorbance at 605 nm using a UV-visible spectrophotometer.

Streptomycin was used as a standard for both experiments.

3. Results and Discussion

Various phytochemicals including tannins, alkaloids, carbohydrates, terpenoids, saponins, phenolic compounds, and reducing sugars are present in selected fractions of inflorescence extracts from the coconut tree [19].

In the current study, Ag-NPs were prepared by reducing AgNO₃ using the ethyl acetate-methanol extract of the coconut tree inflorescence. Synthesis of Ag-NPs from AgNO₃ was confirmed based on an observed change in colour [19]. The presence of terpenoids and reducing sugars in the extract of the inflorescence promoted the reduction of AgNO [3] to Ag-NPs within 2 h [19].

The UV/vis absorption spectrum (Figure 1) of Ag-NPs indicated that the absorption peak was in the visible range (428 nm). This result indicated that silver ions in the reaction mixture were changed to elemental silver, with size in the nanometre range.

[figure(s) omitted; refer to PDF]

Figure 2 depicts FTIR analysis of Ag-NPs synthesized using selected fractions. The figure shows a strong band at 1638.80 cm⁻¹.

[figure(s) omitted; refer to PDF]

Fluorescence spectroscopic data were evaluated to determine whether the prepared nanoparticles were fluorescent. The spectrum indicated that prepared nanoparticles were florescent particles and exhibited strong absorbance at 550 nm (Figures 3(a) and 3(b)).

[figure(s) omitted; refer to PDF]

SEM images of the Ag-NPs revealed that the particles were spherical in shape (Figure 4).

[figure(s) omitted; refer to PDF]

The XRD patterns of Ag-NPs prepared using plant extracts are presented in Figure 5. Sharp, intense peaks were observed at 39.9, 45.4, and 65.79° for the spectrum ranging from 10 to 80°, and the corresponding plane values were (1 1 1), (2 0 0), and (2 2 0). The secondary phase of Ag-NPs, e.g., Ag₃O₄, was from our spectral data, and its respective crystalline planes are measured.

[figure(s) omitted; refer to PDF]

The prepared Ag-NPs exhibited good antimicrobial activity against E. coli microbial culture using the agar well diffusion method (Figure 6).

[figure(s) omitted; refer to PDF]

In agar well diffusion assays, wells with 100 μL of Ag-NPs showed good inhibitory activity against E. coli culture (16 mm zone diameter).

When the inhibitory potential of the prepared Ag-NPs was tested on E. coli in broth culture assay, we observed poor inhibitory effects (Figures 1(a) and 1(b)). UV spectral observations of E. coli culture with Ag-NPs in broth culture showed no differences in absorbance spectra when compared with E. coli broth culture without Ag-NPs (Figure 1(c)).

This differential inhibitory potential shown by Ag-NPs in agar well diffusion and nutrient broth condition indicated that E. coli exhibited novel behaviours when cultured under broth conditions. The silver nanoparticles could not penetrate into E. coli cells in the broth state because the E. coli colonies formed a biofilm matrix (Figure 7) and prevented the effects of Ag-NPs. The biofilm matrix was confirmed using a light microscope and SEM. The poor absorbance of UV rays in the broth culture of E. coli with Ag-NPs as determined by measuring the optical density value and subsequent colony counting confirmed that the Ag-NPs had poor inhibitory effects on E. coli in broth culture. Aggregation of bacterial colonies was noted in the broth culture [19].

[figure(s) omitted; refer to PDF]

4. Conclusion

The present study confirmed the biofilm forming ability of E. coli. Incorporation of Ag-NPs into the nutrient broth culture of E. coli revealed poor inhibitory effects of Ag-NPs under these conditions. The Ag-NPs could not penetrate into the E. coli cells in broth state because the E. coli colonies formed a biofilm matrix and prevented Ag-NP activity. E. coli cell aggregates prevented the entry of Ag-NPs into the bacterial cells in the biofilm matrix. Quorum sensing among bacterial cells under broth conditions enhanced the resistance of E. coli to Ag-NPs. The prepared Ag-NPs exhibited good antimicrobial activity against E. coli microbial culture in the agar well diffusion method. Thus, the synthesized Ag-NPs may have application in biomedical equipment (such as in medical implants) to control biofilm formation.

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