Rapid biological synthesis of silver nanoparticles and their enhanced antibacterial effects against Escherichia fergusonii and Streptococcus mutans

2.1 Bacterial strains and reagents

Luria–Bertani (LB) agar was purchased from USB Corporation (Santa Clara, CA, USA). Mueller Hinton Broth (MHB), Mueller Hinton Agar (MHA), silver nitrate, and crystal violet were purchased from Sigma–Aldrich (St. Louis, MO, USA). All other chemicals were purchased from Sigma–Aldrich unless otherwise stated. The E. fergusonii, B. cereus, and S. mutans strains used in the present study were from our in-house culture collections. All three strains were routinely grown in LB broth or on LB agar at 37 °C.

2.2 Isolation and characterization of AgNP-producing bacteria

Soil samples were collected from an agricultural field in Coimbatore, Tamil Nadu, India in sterile Falcon tubes and transferred to the laboratory under aseptic conditions on ice. The sample (1 g) was suspended in 100 mL of 50 mM phosphate buffer (pH 7.0), serially diluted in the same, and plated on LB agar containing 10 g tryptone, 5 g yeast extract, 10 g/L NaCl, and 15 g/L agar). The plates were incubated at 37 °C for 2–3 days. After the incubation period, the bacterial colonies were further sub-cultured in the same medium to obtain pure colonies. The effective synthesizer of AgNPs was isolated and named GS6. The isolated bacteria were grown routinely in LB broth.

The characterization of bacteria was carried out as described previously (Kalimuthu et al., 2008). The morphological and physiological characterization was performed according to the methods described in Bergey’s Manual of Determinative Bacteriology. Further characterization was carried out using the 16s rRNA technique. The sequences have been submitted to Genbank under accession number KF944447.

2.3 Bacterial strains and growth conditions

Bacterial growth and media preparation were carried out according to a method described previously (Gurunathan et al., 2009). Briefly, B. cereus cultures were first grown aerobically at 37 °C in LB media. The cultures were maintained by streaking a colony on LB agar plates and sub-cultured every fortnight. Pure colonies were isolated and stored at −80 °C. The cells were harvested by centrifugation at 6000 rpm for 10 min, and resuspended in sterile LB medium to obtain an optical density at 600 nm (OD₆₀₀) of 1.0.

2.4 Synthesis and characterization of AgNPs

Typically, a 1-liter overnight shaker-culture of B. cereus with a cell density of 3–4 × 10⁹/mL corresponded to a pellet wet weight of approximately 3 g/L grown aerobically. A working concentration of bacterial cells (diluted to OD₆₀₀ of 1.0) was inoculated in 50 mL of LB broth and the flasks were incubated for 24 h (37 °C, 200 rpm) under aerobic conditions. After incubation, the culture was centrifuged (10,000 rpm, 10 min) and the supernatant was used for the synthesis of AgNPs. The culture supernatant was mixed with an aqueous solution of 1 mM AgNO₃ solution and kept at 40 °C for 60 min. The production yield of AgNPs was calculated according to a previously described method (Kalishwaralal et al., 2010b), and diluted to a final concentration of 1 mg/mL. The conversion efficiencies of these bacterial strains were consistent with a previous report.

The synthesized particles were characterized according to a previously described method (Gurunathan et al., 2009). UV–vis spectra were recorded using a Biochrom WPA Biowave II UV/Visible Spectrophotometer (Biochrom, Cambridge, UK). The particle sizes were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). X-ray diffraction (XRD) analyses were carried out on an X-ray diffractometer (Bruker D8 DISCOVER, Bruker AXS GmBH, Karlsruhe, Germany). The high-resolution XRD patterns were measured at 3 Kw with Cu target using a scintillation counter. (λ = 1.5406 Å) at 40 kV and 40 mA were recorded in the range of 2θ = 5–50°. Further characterization of changes in the surface and surface composition was performed by Fourier transform infrared spectroscopy (FT-IR) (PerkinElmer Spectroscopy GX, PerkinElmer, Waltham, MA, USA). Transmission electron microscopy (TEM; JEM-1200EX) was used to determine the size and morphology of AgNPs. TEM images were obtained at an accelerating voltage of 300 kV.

2.5 Determination of the minimum inhibitory concentration (MIC) and sublethal concentration of antibiotics and AgNPs

Susceptibility tests with AgNPs and various antibiotics were carried out in 96-well microtiter plates using a standard 2-fold broth microdilution of the antibacterial agents in MHB, following the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2005). To examine AgNP MICs, E. fergusonii and S. mutans were exposed to 0–10 μg/mL AgNPs prepared in phosphate-buffered saline (PBS). The appropriate concentration of AgNP solution and 1 mL of the bacterial suspension were mixed in MH media to a final bacterial concentration of 10⁵–10⁶ colony forming units (CFUs)/mL for 24 h. After the treatment, 100 μL of the reaction mixture was diluted 10-fold, 100 μL of which was used for plating on agar-containing media. The loss of viability was evaluated by the colony-counting method and compared with those on control plates, which consisted of MH media without AgNP-based materials. All treatments were prepared in triplicate, and performed in at least three independent experiments. The MIC was determined as the lowest concentration that inhibited the visible growth of the bacteria (Table 1). Control tests were carried out with solutions containing all the reaction components with the exception of AgNPs. Antibiotic or AgNP concentrations that reduced the number of susceptible cells by less than 20% after 4 h of incubation were designated as sub-lethal (Minahk et al., 2004). In addition, viability assays were carried out with different concentrations of antibiotics alone, AgNPs alone, or a combination of sub-lethal concentrations of antibiotics and AgNPs.

2.6 Antimicrobial activity of antibiotics and AgNPs against E. fergusonii and S. mutans

The assessment of AgNPs’ microbial toxicity was performed according to a previously described method (Shahverdi et al., 2007; Kora and Rastogi, 2013). To examine the effect of AgNPs on the growth of E. fergusonii and S. mutans, the overnight cultures were centrifuged at 6000 rpm for 5 min, washed with PBS (1×), and the pellet was resuspended in PBS. Finally, the OD₆₀₀ of the sample was adjusted to 0.1. The cells (5 × 10⁵ bacteria/well in 96-well round bottom plates in triplicate) were then exposed to different concentrations of AgNPs. Bacteria were harvested at the indicated time points or dose responses, and the number of CFUs was assayed. Medium or medium with AgNPs served as controls. All samples were plated in triplicate and values were averaged from three independent experiments.

2.7 Agar diffusion assay

The agar diffusion assay was performed as described previously (Shahverdi et al., 2007; Kora and Rastogi, 2013). In general, the agar diffusion method is used to assay various antibiotics for their bactericidal activity against test strains on MHA plates. In our experiment, we evaluated the antibacterial activity of antibiotics in combination with or without AgNPs. We selected conventional and broad-spectrum antibiotics to analyze the combined effect of antibiotics and AgNPs. Based on the CLSI standard, antibiotics were used at selected concentrations as follows: ampicillin (10 μg/mL), chloramphenicol (30 μg/mL), erythromycin (15 μg/mL), gentamicin (10 μg/mL), tetracycline (30 μg/mL), and vancomycin (30 μg/mL). To determine the combined effects, each standard paper disk was further impregnated with the MIC of AgNPs for each bacterial strain. A single colony of each test strain was grown overnight in MHB on a rotary shaker (200 rpm) at 37 °C. The inocula were prepared by diluting the overnight cultures with 0.9% NaCl to a 0.5 McFarland standard and were applied to the plates along with the standard and prepared disks containing different antibiotics. Similar experiments were carried out with AgNPs alone. After incubation at 37 °C for 24 h, a zone of inhibition (ZOI) was measured by subtracting the disk diameter from the total inhibition zone. The assays were performed in triplicate. The increase in antibacterial activity of different antibiotics was quantified by the equation (B − A)/A × 100, where A and B are the ZOI for antibiotic and antibiotic + AgNPs, respectively (Kora and Rastogi, 2013).

2.8 In vitro killing assays

In vitro killing assays were performed as described previously (Mohanty et al., 2012; Morones-Ramirez et al., 2013) with suitable modifications. Cells were grown overnight in MHB broth at 37 °C and regrown in fresh medium for 4 h before being collected by centrifugation and suspended in deionized water, which was used because it lacks the chloride ions that are present in PBS that would interfere with silver (Morones-Ramirez et al., 2013). A cell suspension consisting of 10⁶ cells/mL was incubated with various concentrations of antibiotics, AgNPs, or combinations of AgNPs with the respective antibiotics for 4 h at 37 °C. After incubation, bacteria were harvested at the indicated time points and 100-μL aliquots were taken from each sample to determine CFUs. The experiment was performed with various controls, including a positive control (AgNPs and MHB media without inoculum) and a negative control (MHB and inoculum without AgNPs). All samples were plated in triplicate, and values were averaged from three independent experiments.

2.9 Measurement of protein leakage

Protein leakage from bacterial cells was determined as described previously (Kim et al., 2011). The concentration of AgNPs or antibiotics was adjusted to the sub-lethal concentration for each bacterial strain and the concentration of bacterial cells was adjusted to 10⁶ CFUs/mL. Each culture was incubated in a shaking incubator at 37 °C for 4 h. One milliliter of sample was obtained from each culture, centrifuged at 4 °C for 30 min at 300×g, and the supernatant was frozen at −20 °C until assay. Protein concentrations in the supernatants were determined by the Bradford assay and the OD was measured at 595 nm.

2.10 Measurement of reactive oxygen species (ROS) generation

Quantitative assays for superoxide anions were carried out using a (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt (XTT)-based in vitro toxicology assay kit (TOX2; Sigma–Aldrich), according to the manufacturer’s instructions. E. fergusonii and S. mutans were grown in MHB medium and cells were washed with PBS buffer and resuspended in PBS at a concentration of 2 × 10⁶ viable cells (determined as CFUs) per mL. XTT was added to the cell suspension at a concentration of 125 μM from a stock solution (7.5 mM) prepared in PBS. Cell suspensions were incubated at 30 °C on a rotary shaker for 12 h, aliquots were spun in a microfuge, and the absorbance of the supernatant was measured at 450 nm. XTT reduction in the absence of cells was always determined as a control and subtracted from the values observed in the presence of cells.

2.11 Measurement of malondialdehyde (MDA)

The concentration of MDA generated in the culture media was determined using a thiobarbituric acid reactive substances assay as described previously (Dutta et al., 2012) with suitable modifications. Briefly, an aliquot of 1 mL of culture media was collected from the AgNP-treated cells and 10% SDS was added and swirled vigorously. Further, 2 mL of freshly prepared thiobarbituric acid (TBA) was added to the above mix and incubated at 95 °C for 60 min. The reaction was then cooled to room temperature and centrifuged at 5000 rpm for 10 min, and the OD of the supernatant was measured at 530 nm.

2.12 Statistical analysis

All the experiments were performed in triplicate and repeated at least three times. The results are presented as means ± SD. All experimental data were compared using the Student’s t-test. A p-value less than 0.05 was considered statistically significant.

3.1 Synthesis and characterization of AgNPs using supernatant from B. cereus culture

The AgNPs were synthesized using supernatants from B. cereus cultured with AgNO₃. The appearance of a yellowish-brown color in the AgNO₃-treated culture supernatant suggested the formation of AgNPs (Fig. 1A) (Shahverdi et al., 2007; Kalimuthu et al., 2008; Gurunathan et al., 2009). The initial pH of the culture supernatant was 6.0, which was adjusted to pH 8.0 with NaOH. The slightly alkaline pH increases the synthesis reaction rate as well as favoring synthesis of smaller-sized nanoparticles (Gurunathan et al., 2009). The synthesis was rapid and completed within 60 min. We previously synthesized AgNPs using various bacteria, for example, Bacillus licheniformis (Kalimuthu et al., 2008), Brevibacterium casei (Kaliswaralal et al., 2010a,b), Escherichia coli (Gurunathan et al., 2009), etc., and all these bacterial strains produced AgNPs within 24 h of incubation. In contrast, B. cereus produces AgNPs within 1 h of incubation, the speed of which is attributed to the type of capping agent produced by these bacteria and also it depends on type of strain.

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Figure 1 Synthesis and characterization of AgNPs using supernatant of B. cereus culture. (A) Synthesis of AgNPs using B. cereus culture supernatant. The image shows vials with samples of AgNO₃ (1), culture supernatant (2), and AgNO₃ with the culture supernatant (3). After incubation for 60 min, the color of the solution turned from colorless to brown, indicating the formation of AgNPs. (B) Ultraviolet–visible spectrum of AgNPs. The absorption spectrum of AgNPs exhibited an intense, broad peak at 420 nm. The observation of this band was attributed to surface plasmon resonance of the AgNPs particles.

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AgNPs were further characterized using ultraviolet–visible spectroscopy, and an intense, broad absorption peak was observed at 420 nm (Fig. 1B), which was assigned to surface plasmon resonance and is well documented for various metallic nanoparticles with sizes ranging from 2 nm to 100 nm (Henglein, 1993; Kowshik et al., 2003). The synthesis could be mediated by the enzyme nitrate reductase, as suggested by results obtained for B. licheniformis (Kalimuthu et al., 2008). Several methods for AgNPs synthesis and formulation have been described previously. For example, conventional physical and chemical methods have been used for the synthesis of AgNPs such as reduction of silver ions. However, the yields were low, the control of the particle size is difficult, and these methods are environmentally incompatible due to the use of toxic chemical reducing agents such as citrate, borohydride, or other organic compounds (Wani and Ahmad, 2013a; Wani et al. 2013b). Moreover, the chemical synthesis of nanoparticles requires an additional step to prevent the aggregation of the particles, whereas in the biological method, the proteins present in the media function as stabilizers and prevent aggregation. In addition, the size and shape of the particles can be controlled by pH and temperature (Gurunathan et al., 2009). Since particle size and shape is an important factor in various biomedical applications, biologically based methods are environmentally friendly alternatives for the synthesis of AgNPs with defined particle size and monodispersity. Using the proper growth medium, synthesis conditions, pH, substrate concentrations, temperature, and reaction time, it might be possible to obtain sufficient amounts of AgNPs (Gurunathan et al., 2009).

Earlier studies have proposed mechanisms for synthesis of silver nanoparticles using bacteria. Butler et al. (2004) demonstrated that MacA is an inner membrane-bound c-type cytochrome that is located in the periplasm and is proposed to transfer electrons from the inner membrane to the periplasm. It has been shown to be important in the reduction of Fe(III) citrate and U(VI) (Slawson et al., 1992, 1994), both of which could potentially enter the periplasm. MacA could also be involved in electron transfer to outer-membrane cytochromes, including those on the surface of the cell, where the Ag(0) was found to accumulate in these experiments. OmcF is an outer membrane c-type cytochrome and is thought to play a role in the reduction of extracellular electron acceptors (Kim et al., 2005). For example, Geobacter sulfurreducens reduces Ag(I) (as insoluble AgCl or Ag⁺ ions), via a mechanism involving c-type cytochromes, resulting in precipitation of extracellular nanoscale Ag(0). Further, Law et al. (2008) have suggested a role for an electron transfer chain traversing the periplasm and the outer membrane, consistent with the extracellular localization of reduced Ag(0) nanoparticles. Kalimuthu et al. (2008) reported that this reduction may occur via electron transfer from NADH, where NADH-dependent reductase can act as a carrier in B. licheniformis. In our case, an analogous phenomenon might happen in B. cereus in which nitrate reductase is present on the membrane.

3.2 X-ray diffraction analysis of AgNPs

X-ray diffraction (XRD) was employed to confirm the crystalline nature of the particles, and the XRD displayed numbers of Braggs reflections that may be indexed on the basis of the face-centered cubic structure of silver (Shahverdi et al., 2007; Matsumura et al., 2003; Kalishwaralal et al., 2010a; Gurunathan et al., 2014). A comparison between the XRD spectra of AgNPs and the standard confirmed that the silver particles formed in our experiments were in the form of nanocrystals, as evidenced by the peaks at 2θ values of 31.9, and 45.58, corresponding to (1 1 1), and (2 0 0), respectively, Bragg reflections of silver (Fig. 2). The assigned peaks at 2θ values of 29.8 (∗) may be related to crystalline and amorphous organic phases, respectively. The X-ray diffraction results clearly show that the AgNPs formed by the reduction of Ag⁺ ions in the culture supernatant of B. cereus are crystalline in nature.

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Figure 2 XRD pattern of AgNPs synthesized in the supernatant of B. cereus.

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3.3 FTIR analysis of AgNPs

FTIR spectroscopy was performed to confirm that the culture supernatant of B. cereus has the ability to perform the dual functions of reduction of Ag⁺ to Ag^o, and stabilization of AgNPs. The FTIR spectra of culture supernatant-derived AgNPs shows a broad and intense band at 3400 cm⁻¹, which is due to bound hydroxyl (–OH) or amine (–NH) groups found in the proteins of the culture supernatant. Shoulder peaks at 1600 cm⁻¹ and 1550 cm⁻¹ indicate that the amide I and amide II arise due to carbonyl and –NH stretch vibrations in the amide linkages of the proteins, respectively. The band at 1380 cm⁻¹ corresponds to C⚌C stretching of aromatic amine (Fig. 3). FTIR bands of biologically synthesized silver nanoparticles, confirms the presence of protein in the silver nanoparticles, which further confirm that biosynthesized AgNPs coated with biological molecules. This study further proves that capping protein stabilizes AgNPs and prevents agglomeration of AgNPs (Gurunathan et al., 2009, 2014; Thakkar et al., 2010; Parikh et al., 2008). Thus, these FTIR data confirm the presence of protein in the biologically synthesized AgNPs.

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Figure 3 FTIR spectra of AgNPs synthesized by the supernatant of B. cereus.

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3.4 Size and shape analysis of AgNPs

The physical characterization of nanoparticles in solution is essential before assessing their in vitro toxicity (Murdock et al., 2008). Particle size, size distribution, morphology, composition, surface area, surface chemistry, and reactivity in solution are important factors that are essential to assess nanoparticle toxicity (Murdock et al., 2008). Powers et al. (2006) suggested that DLS is a powerful technique for evaluating particle size, size distribution, and the zeta potential of nanomaterials in solution. DLS measurements were performed in aqueous solution to elucidate the size of AgNPs synthesized by B. cereus. It was found that the average hydrodynamic diameter of AgNPs was 10 nm. Fig. 4A shows that the particles synthesized by B. cereus presented sizes ranging from 2 nm to 20 nm, with an average size of 10 nm.

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Figure 4 Determination of size and shape of AgNPs. (A) The particle-size distribution revealed that the particles ranged in size from 2 to 20 nm, with an average size of 10 nm. (B) The size and morphology of AgNPs were analyzed using TEM, which confirmed an average particle size of 10 nm. (C) Percentage of particle size distribution based on TEM images.

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Next, we examined the size of the synthesized AgNPs using TEM. TEM micrographs showed that well-dispersed particles were significantly spherical in shape (Fig. 4B) and the particle size ranged from 2 nm to 16 nm, with an average size of 10 nm (Fig. 4C). Several studies reported the use of a biological system for the synthesis of AgNPs using supernatant from Klebsiella pneumoniae, B. licheniformis, and E. coli cultures, in which the particles had an average size of 52.5, 40, and 50 nm, respectively (Shahverdi et al., 2007; Gurunathan et al., 2009; Kalimuthu et al., 2008). When the spore-crystal mixture of Bacillus thuringiensis was used for synthesis of AgNPs, the average size of AgNPs was 15 nm, which was confirmed by TEM, and the structure was cubic and hexagonal. Taken together with these previous reports, our FTIR, DLS, and TEM data suggest that capping protein stabilizes AgNPs and prevents their aggregation.

3.5 Determination of MIC and sub-lethal concentration of antibiotics and AgNPs

The following experiments were performed to determine the lowest concentration that completely inhibits the visible growth of bacteria. According to previous studies, AgNPs exhibit potent antibacterial effects, particularly against Gram-negative bacteria (Kim et al., 2007; Shahverdi et al., 2007). We were interested to study the antibacterial activity of AgNPs with inhibitory concentrations and also to evaluate the enhanced effect of biologically synthesized AgNPs with antibiotics using sub-lethal concentrations on Gram-negative and Gram-positive bacteria. To examine the antimicrobial activity of the AgNPs alone or in combination with ampicillin, chloramphenicol, erythromycin, gentamycin, tetracycline, and vancomycin, antibacterial susceptibility testing was conducted against E. fergusonii and S. mutans. As shown in Table 1, AgNPs exhibited antibacterial activity against E. fergusonii and S. mutans with MIC values of 7.5 μg/mL and 9.5 μg/mL, respectively. In general, AgNPs are more potent against Gram-negative than Gram-positive bacteria. The bacterial strains showed MIC values of 2–3 μg/mL for ampicillin, 3–6 μg/mL for chloramphenicol, 2–4 μg/mL for erythromycin, 1–3 μg/mL for gentamycin, 3–5 μg/mL for tetracycline, and 7–16 μg/mL for vancomycin (Table 1). AgNPs and the antibiotics showed diverse degrees of antibacterial activity in relation to the bacterial species used in this study. E. fergusonii tended to be more sensitive to gentamycin than S. mutans. Conversely, S. mutans tended to be more sensitive to vancomycin than E. fergusonii. Hwang et al. (2012) reported that Gram-negative and Gram-positive bacterial strains showed MIC values of 0.25–2.0 μg/mL for AgNPs, 2–8 μg/mL for ampicillin, 1–4 μg/mL for chloramphenicol, and 2–4 μg/mL for kanamycin. However, the AgNPs and antibiotics showed different degrees of antibacterial activity in relation to the bacterial species. The results drawn from the present study suggest that the MICs of AgNPs synthesized from B. cereus against E. fergusonii and S. mutans are significantly lower than previously reported. The MICs of poly-(N-vinyl-2-pyrrolidone)-stabilized AgNPs against Staphylococcus aureus and E. coli are 50 and 100 μg/mL, respectively (Cho et al., 2005). Raffi et al. (2008) reported that AgNPs (mean size 16 nm) synthesized using an inert gas condensation method were effective bactericides against E. coli at concentrations ⩾60 μg/mL.

3.6 Dose-dependent antibacterial effect of AgNPs in E. fergusonii and S. mutans

The dose-dependent effect of AgNPs was assessed in representative Gram-positive and Gram-negative bacterial strains that were used to assess the relative susceptibility to AgNPs and the extent of bactericidal activity of AgNPs. Fig. 5 shows the potential toxic effect of biologically synthesized AgNPs on E. fergusonii and S. mutans. The bacterial strains were treated with various concentrations of 10-nm AgNPs at 1–10 μg/mL. The results show that the introduction of AgNPs affected the cell viability as compared to the negative control. Furthermore, cell viability decreased with increasing AgNP concentration. As determined in the MIC experiment, at their respective MIC values, no visible bacterial growth was observed above 7.5 μg/mL and 10 μg/mL in E. fergusonii and S. mutans cultures, respectively (Table 1). In the case of E. fergusonii, the introduction of 7.5 μg/mL of AgNPs reduced bacterial cell density by approximately 99% as compared to the control sample. Furthermore, increasing the concentration of AgNPs to 8 μg/mL and 10 μg/mL inhibited bacterial growth, as these concentrations represent MIC values. Interestingly, for S. mutans, the introduction of a similar concentration of AgNPs (i.e., 7.5 μg/mL) reduced cell viability by approximately 75% as compared to the control sample. However, the higher concentrations of 8 μg/mL and 10 μg/mL rapidly inhibited the growth of bacteria (Fig. 5). Nevertheless, the specific mechanism of the antibacterial effect of AgNPs against bacteria remains unknown. AgNPs may pass through the cell wall of bacteria to oxidize the surface proteins on the plasma membrane and consequently disturb cellular homeostasis (Sondi and Salopek-Sondi, 2004). Several research groups have suggested that AgNPs may attach to the surface of the cell and alter its permeability or disrupt metabolic functions such as respiration (Chauhan et al., 2013; Murray et al., 1965). Our results suggest that AgNPs produced using a biological method are smaller in size compared to chemically synthesized AgNPs, which may provide more bactericidal effects than larger particles, as the cellular uptake of particles tends to be inversely correlated with size.

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Figure 5 Effect of AgNPs on cell survival of E. fergusonii and S. mutans. All the test strains were incubated in the presence of different concentrations of AgNPs. Bacterial survival was determined at 4 h by a CFU count assay. The experiment included a positive control (AgNPs and MHB media, without inoculum) and a negative control (MHB and inoculum, without AgNPs). The results are expressed as the means ± SD of three separate experiments, each of which contained three replicates. Treated groups showed statistically significant differences from the control group by the Student’s t-test (p < 0.05).

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Similarly, Li et al. (2010) reported that 10 μg/mL AgNPs could completely inhibit the growth of 10⁷ CFUs/mL of E. coli in liquid MHB. They also suggested that the mechanism of antibacterial activity involved leakage of reduced sugars and proteins, and induced the respiratory chain dehydrogenases. AgNPs were also evaluated for use in combination with different antibiotics against S. aureus and E. coli, and were shown to increase the antimicrobial activities of these antibiotics (Shahverdi et al., 2007). Guzmán et al., 2009 demonstrated that AgNPs showed high antimicrobial and bactericidal activity against Gram-negative bacteria such as E. coli, Pseudomonas aeruginosa, than S. aureus. Similarly, our results also suggest that AgNPs show strong antibacterial activity against Gram-negative and Gram-positive bacteria.

3.7 AgNPs enhance the antibacterial effects of antibiotics

We analyzed whether the combination of antibiotics with AgNPs could increase their antibacterial activity. In this experiment, the combined effect of antibiotics with AgNPs was assessed using the agar diffusion method in E. fergusonii and S. mutans. The activity of the six antibiotics tested, ampicillin (10 μg/mL), chloramphenicol (30 μg/mL), erythromycin (15 μg/mL), gentamicin (10 μg/mL), tetracycline (30 μg/mL), and vancomycin (30 μg/mL), with AgNPs showed significant antibacterial effects against both Gram-negative and Gram-positive bacteria (p < 0.05). Fig. 6 shows that AgNPs enhanced the antibacterial activity of the antibiotics. The activity of the tested antibiotics was increased in combination with AgNPs against the tested bacterial strains. In the case of E. fergusonii, the highest increase was observed for gentamycin (p < 0.05) followed by ampicillin, chloramphenicol, erythromycin, tetracycline, and vancomycin (p < 0.05). In the case of S. mutans, the highest increase was observed for vancomycin (p < 0.05), followed by ampicillin (p < 0.05), chloramphenicol (p < 0.05), gentamycin, tetracycline, and erythromycin (p < 0.05). Among the selected antibiotics, gentamycin and vancomycin showed the highest percentage of enhancement of activity against E. fergusonii and S. mutans, respectively. These results suggested a differential susceptibility between Gram-negative and Gram-positive bacteria toward the type of antibacterial agents combined with AgNPs, which may be due to differences in cell-wall composition.

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Figure 6 Enhancement of antibacterial activity of antibiotics in the presence of AgNPs. The antibacterial activity of AgNPs was evaluated using the agar diffusion method. The MICs of AgNPs for each test strain were loaded into the wells formed on plates containing a layer of bacteria. Growth inhibition was determined by measuring the zone of inhibition after 24 h. Experiments were performed in triplicate. The percentage of enhancement in antibacterial activity was calculated using the formula (B − A/A) × 100. The results are expressed as the means ± SD of three separate experiments, each of which contained three replicates. Treated groups showed statistically significant differences from the control group by the Student’s t-test (p < 0.05).

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3.8 Enhanced antibacterial effect of antibiotics and AgNPs

Next, we explored the possibility of using AgNPs as an antibiotic adjuvant to evaluate the additive potential effect on antibiotic susceptibility. We first reasoned that Ag⁺ could potentiate the bactericidal effect of antibiotics that share a common mechanism of action involving the overproduction of ROS (Morones-Ramirez et al., 2013; Kohanski et al., 2007). The results showed that the highest enhancing effect was observed for gentamycin against E. fergusonii and for vancomycin against S. mutans. Therefore, we selected these two antibiotics to examine the antibacterial activity in Gram-negative and Gram-positive bacteria, respectively. In this experiment, bacteria in the exponential growth phase were incubated with sub-lethal concentrations of antibiotics, AgNPs, or a combination of both. The CFU number was analyzed by harvesting bacteria at different time points and the surviving colonies were enumerated after 24 h. CFU assays showed that sub-lethal concentrations of antibiotics or AgNPs alone had no significant killing effect in both Gram-negative and Gram-positive bacteria. Compared to untreated controls, the combination of gentamycin and AgNPs significantly inhibited cell viability in E. fergusonii (p < 0.05), by more than 65% (Fig. 7), whereas gentamycin combined with AgNPs showed a noticeable effect on S. mutans. In response to vancomycin, S. mutans exhibited a sharp and significant decrease in cell viability of approximately 61% (p < 0.05) (Fig. 7), whereas E. fergusonii showed a noticeable effect with vancomycin. These results suggest that E. fergusonii and S. mutans are more susceptible to gentamycin and vancomycin, respectively. However, the combination of any antibiotic with AgNPs significantly increased the effect on bacterial cell death relative to treatments with AgNPs or ampicillin alone (p < 0.05).

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Figure 7 AgNPs increase the bactericidal effect of antibiotics. Upper panel: All test strains were treated with sub-lethal concentrations of gentamycin, AgNPs, or a combination of AgNPs and gentamycin for 4 h. Lower panel: All test strains were treated with sub-lethal concentrations of vancomycin, AgNPs or a combination of AgNPs and vancomycin for 4 h. Bacterial survival was determined at 4 h by CFU assay. The experiment was performed with various controls, including a positive control (AgNPs and MHB media, without inoculum) and a negative control (MHB and inoculum, without AgNPs). The results are expressed as the means ± SD of three separate experiments, each of which contained three replicates. Treated groups showed statistically significant differences from the control group by the Student’s t-test (p < 0.05).

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3.9 Enhanced effect of antibiotics and AgNPs on membrane protein leakage

AgNPs are known to enhance protein leakage by increasing the membrane permeability in bacteria. In order to determine the impact of AgNPs alone or in combination with antibiotics on protein leakage, the cells were treated with AgNPs alone or in combination with antibiotics for 12 h. The amount of protein released in the suspension of the treated cells was estimated using the Bradford assay. When the cells were treated with a combination of AgNPs and antibiotic, the amount of protein released from the cells increased compared to treatment with AgNPs or antibiotics alone (Fig. 8). At 12 h after incubation, protein leakage from cells treated with AgNPs was considerably increased. However, there was no change in the amount of protein leakage from cells in the control group or in the groups treated with antibiotics alone. Leakage from cells treated with a combination of AgNPs and antibiotics was significantly higher than that from cells in the control group or AgNPs alone group. Leakage from cells treated with AgNPs was significantly increased compared to that from cells in the control group, indicating that AgNPs alone can increase membrane permeability. Notably, higher amounts of proteins leaked through Gram-negative bacterial membranes compared to Gram-positive bacterial membranes, suggesting that the antibacterial sensitivity of the Gram-positive S. mutans was lower than that of the Gram-negative E. fergusonii. This difference may be attributed to the thickness of the peptidoglycan layer of Gram-positive bacteria. An essential function of the peptidoglycan layer is to protect against antibacterial agents such as antibiotics, toxins, chemicals, and enzymes. This result is consistent with the results of previous studies (Kim et al., 2011; Tiwari et al., 2008). Tiwari et al. (2008) showed that protein released from the cells increased along with increasing concentration of AgNPs, and that the leakage of proteins in Bacillus subtilis was lower than that of E. coli. A similar trend was observed in our experiments, as Gram-negative bacteria showed higher amounts of protein leakage than Gram-positive bacteria. Interestingly, the combined effect of antibiotics and AgNPs exhibited significantly higher effects than AgNPs or antibiotics alone.

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Figure 8 Enhanced effect of antibiotics and AgNPs on protein leakage. For protein leakage analysis, the Bradford assay was performed on the media supernatants from cells treated with sub-lethal concentrations of antibiotics, AgNPs, or combinations of both for 12 h.

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3.10 Enhanced effect of antibiotics and AgNPs on MDA and ROS generation

MDA content was measured in cultures treated with AgNPs, antibiotics, or the combination of both. It has been shown that hydroxyl radicals generated in suspensions of AgNPs in culture media can extract hydrogen from the allylic positions of unsaturated fatty acids (Dutta et al., 2012). These allylic free radicals react with O₂ to form lipid peroxide radicals (LOO^•), which can then undergo rearrangement to form MDA (Dutta et al., 2012). Previous studies reported that MDA is produced during the lipid peroxidation of unsaturated fatty acids by oxygen-based free radicals, and the amount of MDA is related to the concentration of generated ROS (Yagi, 1998; Lefevre et al., 1998; Dutta et al., 2012). As shown in Fig. 9, compared to the control, the levels of MDA were significantly higher and increased further with combinations of antibiotics and AgNPs (Fig. 9). These results indicate that the increase in ROS generation is due to AgNPs in the culture media. It is also noteworthy that the amount of MDA was 3–4-fold higher with the combination of antibiotics and AgNPs than AgNPs or antibiotics alone. These results suggest that the inhibition of bacterial growth due to AgNPs or antibiotics is attributable to ROS formation. Our results are consistent with recent reports on the toxicity of ZnO nanoparticles toward E. coli (Dutta et al., 2012) and increased apoptosis in cancer cells induced by lipid peroxidation (Premanathan et al., 2011). To substantiate that MDA plays an important role in oxidative stress, we further investigated the generation of ROS in cells treated with AgNPs, antibiotics, or a combination of both.

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Figure 9 Enhanced effect of antibiotics and AgNPs on MDA level. All test strains were treated with sub-lethal concentrations of antibiotics, AgNPs, or a combination of AgNPs and antibiotics for 12 h. The quantity of MDA was measured as described in Section 2. The results are expressed as the means ± SD of three separate experiments, each of which contained three replicates. Treated groups showed statistically significant differences from the control group by the Student’s t-test (p < 0.05).

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Recently, several studies showed that the application of lethal doses of bactericidal antibiotics promotes the formation of highly detrimental ROS (Kim et al., 2007; Kohanski et al., 2007; Dwyer et al., 2012) which is one of the key mechanisms of toxicity. The mechanisms of cell death, oxidative stress, and ROS formation were shown to be some of the key mechanisms in cellular defense after particle uptake. Intracellular oxidative stress could be hastened by NPs through disturbing the equilibrium between oxidative and reductive processes (Nel et al., 2006). Thus, we investigated the effect of sub-lethal concentrations of antibiotics, AgNPs, or combinations of antibiotics and AgNPs on the formation of ROS in E. fergusonii and S. mutans. The ROS levels in antibiotic- or AgNP-treated cells were lower than that in cells treated with a combination of antibiotics and AgNPs (Fig. 10). Elevated ROS and free radical levels are candidate mediators for cell death. The production of ROS could be caused by an impeded electronic transport along the respiratory chain in the damaged plasma membrane (Su et al., 2009).

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Figure 10 Enhanced effect of antibiotics and AgNPs on ROS generation. ROS generation was measured by an XTT assay. The results are expressed as the means ± SD of three separate experiments, each of which contained three replicates. Treated groups showed statistically significant differences from the control group by the Student’s t-test (p < 0.05).

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Taken together, these results indicate that cell death is mediated by ROS production, which might alter the cellular redox status. In general, the cellular uptake of smaller nanoparticles is more efficient than that of larger particles. Some studies suggest that, in addition to the uptake of greater numbers, the higher cytotoxicity of smaller particles compared to larger ones is related to the amount of ROS generated on the relatively larger surface area of small nanoparticles (Carlson et al., 2008). The precise mechanism by which AgNPs exert their bactericidal effect remains unclear. Guzmán et al., 2009 proposed that AgNPs may attach to the surface of the cell membrane and alter its permeability or disturb metabolic functions such as respiration, and suggested that the binding of the particles to the bacteria depends on the surface area available for interaction. Smaller particles, having a larger surface area available for interaction, would have a greater bactericidal effect than larger particles (Guzmán et al., 2009; Carlson et al., 2008). Taken together with previous studies, the results presented here suggest that smaller-sized nanoparticles are more cytotoxic than larger particles.

Silver ions are very reactive and are known to bind to various vital components of cells and induce processes that initiate cell death. Silver ions exhibit their antimicrobial functions via binding to negatively charged DNA and to thiol-containing proteins, thereby inhibiting protein function. Finally, the induction of ROS synthesis leads to the formation of highly reactive cytotoxic free radicals (Matsumura et al., 2003; Pandian et al., 2010). However, the mechanism of action of AgNPs is not yet clearly understood. It has been reported that the mode of antibacterial action of AgNPs is probably similar to that of silver ions (Kong and Jang, 2008). Some authors have proposed that AgNPs form deposits on the microbial cell wall and exert their toxic effects by inactivating essential enzymes, for example by forming complexes with the catalytic sulfur of thiol groups in cysteine residues (Feng et al., 2000), and through the production of ROS such as superoxide anions, hydrogen peroxide, and hydroxyl radicals, all of which have potent bactericidal activity. In addition to their direct bactericidal activity, nanoparticles are also known to disrupt biofilm formation (Kalishwaralal et al., 2010a). Soo-Hwan et al. (2011) reported that S. aureus and E. coli treated with AgNPs displayed many fragments indicative of membrane damage on the cell surface. They also found that ROS were responsible for increased permeability of the cell membrane or leakage of cell contents. Liu et al. (2013) demonstrated that silver nanoparticles embedded in titanium oxide (Ag–TiO₂) are an effective biocidal agent against microbes. Transmission electron microscopy and electron energy-loss spectroscopy indicated cellular damage after co-incubation with the nanocomposite, showing that Ag–TiO₂ interaction with the bacterial membrane led to leakage of ions that are critical for cell survival, and strongly suggested that OH^• was at least partly responsible for the ROS-mediated damage (Liu et al., 2013).

Morones-Ramirez et al. (2013) suggested that the increase in ROS production is likely an indirect effect of the interaction of silver with its targets. Another possible mechanism to explain the enhanced antibacterial activity of antibiotics with AgNPs is the bonding between nanoparticles and antibiotic molecules. The active functional groups of antibiotics, such as hydroxyl and amino groups, can be chelated by silver and thereby cover a considerable portion of the surface of AgNPs (Fayaz et al., 2010). Morones-Ramirez et al. (2013) proposed a mechanism of silver-induced cell death in which silver disrupts multiple bacterial cellular processes, including disulfide-bond formation, metabolism, and iron homeostasis. These changes lead to increased production of ROS and increased membrane permeability of Gram-negative bacteria, both of which can potentiate the activity of a broad range of antibiotics against Gram-negative bacteria in different metabolic states, as well as restore antibiotic susceptibility to a resistant bacterial strain.