Bactericidal Effects of Silver Nanoparticles on Lactobacilli and the

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Bactericidal Effects of Silver Nanoparticles on Lactobacilli and the Underlying Mechanism Xin Tian, Xiumei Jiang, Cara Welch, Timothy R Croley, Tit-Yee Wong, Chao Chen, Sanhong Fan, Yu Chong, Ruibin Li, Cuicui Ge, Chunying Chen, and Jun-Jie Yin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17274 • Publication Date (Web): 26 Feb 2018 Downloaded from http://pubs.acs.org on February 26, 2018

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ACS Applied Materials & Interfaces

Bactericidal Effects of Silver Nanoparticles on Lactobacilli and the Underlying Mechanism Xin Tian,† Xiumei Jiang,‡ Cara Welch,§ Timothy R. Croley,‡ Tit-Yee Wong,⊥ Chao Chen,¶ Sanhong Fan,¶ Yu Chong,† Ruibin Li,† Cuicui Ge,† Chunying Chen†† and Jun-Jie Yin*,‡

†

State Key Laboratory of Radiation Medicine and Protection, School for Radiological and

Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123, China. ‡

Division of Analytical Chemistry, Office of Regulatory Science, and § Office of Dietary

Supplement Programs, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland 20740, United States. ⊥

Department of Biological Sciences, University of Memphis, Memphis, Tennessee 38120, United States. ¶

††

School for Life Science, Shanxi University, Taiyuan 030006, China.

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for

Nanoscience and Technology of China and Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100190, China.

*Corresponding author, E-mail: [email protected]

KEYWORDS: silver nanoparticle, antibacterial, lactobacilli, hydroxyl radical, acidic environment

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ABSTRACT While the antibacterial properties of silver nanoparticles (AgNPs) have been demonstrated across a spectrum of bacterial pathogens, the effects of AgNPs on the beneficial bacteria are less clear. To address this issue, we compared the antibacterial activity of AgNPs against two beneficial lactobacilli (Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus casei) and two common opportunistic pathogens (Escherichia coli and Staphylococcus aureus). Our results demonstrate that those lactobacilli are highly susceptible to AgNPs, while the opportunistic pathogens are not. Acidic environment caused by the lactobacilli is associated with the bactericidal effects of AgNPs. Our mechanistic study suggests that acidic growth environment of lactobacilli promotes AgNPs dissolution and hydroxyl radical (•OH) overproduction. Furthermore, increases in silver ions (Ag+) and •OH deplete the glutathione pool inside the cell, which are associated with the increase in cellular reactive oxygen species (ROS). High levels of ROS may further induce DNA damage and lead to cell death. When E. coli and S. aureus are placed in a similar acidic environment, they also become more susceptible to AgNPs. This study provides a mechanistic description of a pH-Ag+-•OH bactericidal pathway and will contribute to the responsible development of products containing AgNPs.


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1. INTRODUCTION Nanotechnology has been identified as a major source of innovation in the food industry. Increasingly, nanomaterials are being used in the food industry, which generates questions regarding their safety to human beings. Silver nanoparticles (AgNPs) have received much attention as a broad-spectrum antibacterial agent.1–4 The antimicrobial properties of AgNPs are being applied in a growing number of medical and consumer products, including many medical devices, textiles, and cosmetics.5–8 Additionally, AgNPs have often been integrated into food packing materials. Several studies have reported significant silver migration from nanosilver food containers.9–11 It’s obvious that the application of AgNPs in the food industry will increase opportunities for the general public to directly interact with AgNPs.12–14 Ingestion is one of the main routes of human exposure to AgNPs, whether occurring directly from dietary supplements or indirectly via AgNPs dissolution from consumer products, as well as from antibacterial packaging. Recently, several studies have evaluated the potential effect of AgNPs on intestinal flora after oral exposures.15–18 For example, Williams et al. investigated the effect of dietary AgNPs on the intestinal flora of rats.18 The nanoparticles can induce microbial alterations in the gut and the alterations are similar to those reported in inflammatory diseases. Similarly, oral exposure to a low dose of AgNPs can change the population of intestinal flora in rats.19 Bacteria and other microorganisms have often been considered as harmful, but many microorganisms are conducive to the human body. Many of these beneficial bacteria have been used as probiotic dietary supplements to improve activities of digestion, metabolism, and immune system.20,21 Some probiotics, particularly Lactobacillus species, can prevent the growth of pathogens by changing the pH of mucous membranes by secreting lactic acid. Some of them even accumulate H2O2.22 Lactobacillus species in the gut microbiome are an essential part of intestinal flora of healthy human bodies. Alterations in the composition and amount of these normal flora can disrupt the intestinal ecology, leading to metabolic and inflammatory disorders.23–25 To date, investigations regarding the antibacterial effects of AgNPs and



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the mechanisms by which these effects occur have been primarily conducted using pathogenic bacteria; only a few studies have been carried out using beneficial bacteria.16,17 However, the diversity of the gut microbiome and the increasing usage of AgNPs suggested that studies of AgNPs activities on a wider range of bacteria are necessary. Here we have tested the bactericidal effects of AgNPs on two common beneficial bacteria,

Lactobacillus

delbrueckii

subsp.

bulgaricus

(L.

bulgaricus)

and

Lactobacillus casei (L. casei), both of which exist in human intestine and are widely used as probiotic dietary supplements. For comparison, we also studied the effect of AgNPs on strains of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), both of which are considered pathogens under certain conditions. Data from the present work indicated that the lactobacilli were more susceptible to AgNPs than the reference strains. Through detailed toxicity testing, we found that the acidic environment created by the lactobacilli was associated with the bactericidal effects of AgNPs. This speculation was verified by showing an increase in the bactericidal effect of AgNPs on E. coli and S. aureus under acidic environment. 2. EXPERIMENTAL SECTION 2.1. Materials. L. bulgaricus strain (CGMCC 1.6970) and L. casei strain (CGMCC 1.2435) were obtained from the China General Microbiological Culture Collection Center (CGMCC, Beijing, China). E. coli strain (ATCC 25922) and S. aureus strain (ATCC 25923) were obtained from the American Type Culture Collection (ATCC, MD, USA). Spin traps 5-tert-Butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO) and 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) were obtained from Enzo Life Science, Inc. (New York, USA). LIVE/DEAD BacLight Bacterial Viability kit and CM-H2DCFDA fluorescent probe were obtained from Invitrogen. (Waltham, MA, USA). GSH-Glo Assay kit was purchased from Promega (Madison, WI, USA). 2.2. Preparation and Characterization of AgNPs. The AgNPs were prepared via the reduction of silver ions (Ag+) in water, according to procedures reported


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previously.26 Briefly, citrate-capped 4 nm AgNPs were synthesis firstly as starter seeds. Then, a 2 mL of 1% (w/v) citrate solution and an 80 mL of water were added into a three-necked round bottom flasks, equipped with a reflux condenser. The mixed reaction was heated to boiling for 15 min. Next, the 10 mL starter seeds solution was added to the mixture, followed by the addition of 1% AgNO3 solution. The reaction solution was kept stirring for 1 h and cooled to room temperature. Transmission electron microscopy (TEM, JEOL JEM-2100, Tokyo, Japan) was used to characterize the morphology of these AgNPs. The mean hydrodynamic size of our AgNPs was determined using a Zetasizer Z90 instrument. The UV-vis absorption spectra were recorded on an Agilent Cary 300 UV-vis spectrophotometer. 2.3. Bacterial Culture and Antibacterial Activity Test. E. coli and S. aureus were cultured in Luria-Bertani broth medium (LB) and Tryptone Soy broth medium (TSA), respectively. L. bulgaricus and L. casei were cultured in De Man, Rogosa and Sharpe broth medium (MRS). Bacteria were grown at 37 °C to reach the logarithmic phase (OD600 between 0.5 and 0.7), then diluted with a isotonic saline solution to 106 colony-forming units (CFU/mL) and then exposed to different concentrations of AgNPs for 2 h at 37 °C. After treatment, each set of bacteria was diluted to approximately 103 CFU/mL with the same saline solution and cultured on the appropriate agar plates overnight at 37 °C. Finally, colonies were counted and compared with the number of colonies that had not been exposed to AgNPs on control plates, to calculate changes in cell numbers. The minimum bactericidal concentration (MBC) refers to the lowest concentration of AgNPs leading to no growth of bacteria. 2.4. Cell Morphology Observation. Scanning electron microscopy (SEM) was applied to assess the surface morphological changes in bacteria after incubation with AgNPs. After treatment with Ag NPs, bacterial cells were collected by centrifugation and fixed with 2.5% glutaraldehyde. These cells were gradually dehydrated in ethanol. Then, dehydrated bacterial cell suspension was placed on a silica chip. The mounted samples were viewed under a SEM (S-4700, Hitachi, Japan). 2.5. Cell Membrane Permeability Assay. After treatment by AgNPs, bacteria were



stained by adding PI to the suspension. Fluorescent images were acquired using confocal laser microscopy (FV1200, Olympus, Japan) with excitation/emission wavelengths of 490/630 nm. 2.6. Assessment of Ag+ Release. The concentration of dissolved Ag+ from AgNPs was determined using inductively coupled plasma mass spectrometry (ICP-MS) (ELEMENT2, Thermo, USA). For the measurement, AgNPs were suspended in different media having pH 4.5 and pH 7.0 at 37 oC. After different times, the suspensions were centrifuged with 10 kDa ultrafiltration tube. A series of silver nitrate dilutions containing 0, 1, 10, 25, 50, 75, and 100 ppm silver was used as standard solutions. 2.7. Electron Spin Resonance (ESR) Measurements. Hydroxyl radical (•OH) and glutathionyl radical (•SG) were determined using a Bruker EMX ESR spectroscope according to our previous study.27 The spin traps BMPO and DMPO were used to verify the formation of •OH and •SG, respectively. All the ESR measurements were carried out at ambient temperature. 2.8. Assessment of Intracellular Reactive Oxygen Species (ROS). To determine the burst of ROS, a fluorescent ROS sensor, CM-H2DCFDA, was used. Bacterial cells were pretreated with 7 ppm AgNPs for 2 h. Then, cells were stained with 10 µM CM-H2DCFDA. Images were acquired using a confocal laser microscopy. 2.9. TUNEL Assay. The DNA damage of bacterial cells were determined by TUNEL assay. Briefly, after treatment with Ag NPs, bacterial cells were fixed in 2% paraformaldehyde for 30 min and permeabilized on ice. Then, cells were mixed with reaction solution from TUNEL assay kit (Roche, USA). Analysis was done by a flow cytometry (FACSCalibur, BD, USA) 2.10. Statistical Analysis. All experiments were performed in triplicate and the results were presented as mean ± standard deviation (SD). Statistical analysis was performed using Statistical Product and Service Solutions software (SPSS). The differences between the groups were assessed by Student’s t tests. The results were


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considered statistically significant when p value was less than 0.05. 3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of AgNPs. AgNPs were synthesized according to procedures reported previously.26 The as-synthesized AgNPs exhibited an UV-Vis absorption at approximately 400 nm (Figure 1A). TEM revealed that the AgNPs exhibited a spherical shape with an average size of 20.1 ± 4.4 nm (Figure 1A, insert). The AgNPs had a negative ζ-potential (-19.2 ± 0.7 mV) due to the citrate that was used as stabilizing and coating material in the synthesis process. The dynamic light scatting (DLS) analysis showed that AgNPs in DI water had a narrow size distribution with an average hydrodynamic size of 49.3 ± 5.7 nm (Figure 1B). In addition, DLS analysis found that there was no significant aggregation after AgNPs added into different bacterial culture media.

Figure 1. Characterization of AgNPs. (A) UV-Vis absorption spectrum of AgNPs dispersed in DI water. Inset shows their representative TEM image. Scale bar = 100 nm. (B) Hydrodynamic size distribution of AgNPs in DI water.

3.2. L. bulgaricus and L. casei are Sensitive to AgNPs. Strains of L. bulgaricus, L. casei, E. coli, and S. aureus were used to test the bactericidal effects of AgNPs. The detailed biological properties of these bacteria are listed in Table S1. Bacterial cells were incubated with AgNPs in saline solution for 2 h. After incubation, the percentage of the surviving cells was determined by bacterial plate count in agar nutrient plates.



Figure 2A shows that AgNPs exhibit a dose-dependent bactericidal effect against all four bacterial species. Notably, AgNPs exhibit a much stronger bactericidal effect against L. bulgaricus and L. casei than E. coli and S. aureus. At a concentration of 3 ppm, AgNPs show a negligible effect against E. coli and S. aureus, whereas that same concentration of AgNPs killed nearly half the populations of L. bulgaricus and L. casei (data are shown in Table S2). When the concentration of AgNPs was increased to 7 ppm, about 80% of the L. bulgaricus and L. casei were killed, in contrast to 30% of the E. coli and S. aureus. The minimal bactericidal concentration (MBC) values of AgNPs against bacterial cells are shown in Figure 2B.

Figure 2. Antibacterial activity of AgNPs. (A) Percentage survival of bacterial cells after treated with different concentration of AgNPs, determined by colony counting results. Isotonic saline solution without AgNPs was used as control. * indicates p < 0.05 compared with the E. coli group or S. aureus group. (B) MBC values of AgNPs against bacterial cells. * indicates p < 0.05 compared with the E. coli group or S. aureus group.

To further confirm the antibacterial activity of the AgNPs, a fluorescence-based Live/Dead assay was used to measure the ratio of live vs dead cells after treatment with AgNPs (Figure S1). The fluorescence-based Live/Dead assay results are consistent with the results in Figure 2. After an exposure period of 2 h, AgNPs caused more cell deaths among the L. bulgaricus and L. casei samples than in the E. coli and S. aureus samples. In addition, we investigated the uptake level of AgNPs by bacterial


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cells. There are no significant differences in the uptake level of AgNPs in four bacteria (Table S3). The shape of a bacterial cell is dictated by its peptidoglycan cell wall organization. To find out how AgNPs affect the cell morphology, we used SEM to examine any change in cell shapes that might occur due to AgNPs exposure (Figure 3 and Figure S2). Normal L. bulgaricus and L. casei cells not exposed to AgNPs exhibit as rod-shape with a smooth surface (Figure 3A). After treatment with AgNPs, the bacterial cells display serious deformation. In contrast, the cell membranes of E. coli and S. aureus exhibit only minor surface disruptions. However, the L. bulgaricus and the L. casei cells stay rod-shaped even after AgNPs treatment suggests the cell walls are still intact. Those changed cell shapes suggest that AgNPs may affect the cell membrane permeability. To explore this possibility of membrane disruption, we used propidium iodide (PI), a membrane-impermeable fluorescent dye for cell membrane permeability detection.28 As shown in Figure 3B & 3C, AgNPs-treated L. bulgaricus and L. casei have higher fluorescence intensities than the untreated cells. This directly demonstrates that AgNPs can destabilize the cell membrane permeability. In contrast, there is no significant increase of fluorescence in E. coli and S. aureus cells treated with AgNPs. Therefore, the alterations in cell membrane permeability may be restricted to certain types of bacteria – L. bulgaricus and L. casei are more susceptible to membrane damage by AgNPs than E. coli and S. aureus.



Figure 3. Interaction of AgNPs with bacterial cells. (A) SEM images showing the interaction between AgNPs with bacterial cells. Cells were treated with 7 ppm AgNPs for 2 h. Arrows show the cell wall damage after AgNPs treatment. Scale bar = 2 µm. (B) Fluorescence images of PI-stained untreated cells and cells treated with AgNPs. Scale bar = 5 µm. (C) Fluorescence fold increase of PI-stained bacterial cells after AgNPs treatment. Data is represented as the mean fluorescence intensity and standard deviation from three regions. * indicates p < 0.05 compared with the E.coli group or S.aureus group.

3.3. Acidic Conditions Increase AgNPs Dissolution and •OH Production. Many studies show that the antibacterial activity of AgNPs is depended on the concomitant release of Ag+.29,30 Ag+ can cause the irreversible aggregation of thiol- or aminebearing molecules, the so-called oligodynamic effect.31 Important categories of biomolecules, including peptides and DNA, have been identified as Ag+ targets.32 The


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oxidation of AgNPs proceeds through complex processes and is affected by several factors.31 As reported previously, AgNPs can be oxidized in aqueous solutions resulting in the release of Ag+ under acidic conditions.33 Such acidic conditions are to be expected in the presence of L. bulgaricus and L. casei, as these bacteria produce large volumes of lactic acid as the metabolite.34,35 Therefore, the low pH environment where these lactobacilli created would favor the release of Ag+ from AgNPs. We further explored the pH changes by comparing the spent media from the four bacteria over a 16-h period. Figure 4A shows that the pH of L. bulgaricus and L. casei media gradually decrease from pH 6.5 to 4.5 after 10 h of growth. In contrast, the spent media on which E. coli and S. aureus exhibit no significant changes in pH over a 16-h period, remaining steady at around pH 7.0. Additionally, an independent experiment shows that, over a 16-h period, more Ag+ were formed in a buffer at pH 4.5 than in a buffer solution kept at pH 7.0 (Figure 4B). Overproduction of ROS is recognized as another mechanism by which AgNPs kill bacteria.36,37 Many studies demonstrate that the •OH causes oxidative damage to biomolecules.38,39 It’s known that the bactericidal activity of many antibiotics against E. coli involves the generation of •OH via the Fenton reaction (Equation 1).40,41 In this reaction, intracellular ferrous ions (Fe2+) react with hydrogen peroxide (H2O2) to form •OH. Our previous study demonstrated that •OH could be generated when AgNPs were exposed to H2O2.42 Therefore, a similar mechanism may run in AgNPs induced bacterial killing (Equation 2). Fe2+ + H2O2

Fe3+ + •OH + OH-

Ag0 (AgNPs) + H2O2 + H+

Ag+ + •OH + H2O


(1)

(2)


Figure 4. AgNPs dissolution and •OH production. (A) pH changes of bacteria culture media. (B) AgNPs dissolution under pH 4.5 and pH 7.0 buffer solutions. (C) ESR spectra of BMPO/•OH adducts. (a.u. – arbitrary units). (D) Signal intensity vs solution pH from (C).

To further understand the mechanism by which AgNPs exert antibacterial activity, we used ESR technology to identify and quantify the •OH induced by AgNPs. Here, we selected BMPO as a spin trap for detection of •OH. The ESR spectrum of BMPO/•OH has four lines and hyperfine splitting parameters of αNα = 14.0 G and αHβ = 13.2 G (Figure S3).43 As shown in Figure 4C, the strong ESR signals indicate the formation of BMPO/•OH adduct (second line, and third line). The •OH is generated efficiently at pH 4.6 and its production decreases with increasing pH from 4.6 to 7.0 (Figure 4D). At pH 6.5 and pH 7.0, the spectra are similar to that of the control without AgNPs (Figure 4C, first line), and the signal is negligible (fourth line, and fifth line). This result shows that the AgNPs-induced •OH radicals are strongly


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dependent on the pH. The lactobacilli produce lactic acids and lower the pH of their growth environment that favors AgNPs dissolution and •OH formation. Our observed results are completely in line with the Fenton-like mechanism of •OH formation (Equation 2). A previous study found that Ag+ disrupted intracellular Fe-S clusters in the bacterial cells, causing them to release Fe2+, which could further stimulate the generation of •OH through the Fenton reaction (Equation 1).27 In current study, we show that the dissolution of AgNPs in an acidic environment is accompanied by the formation of •OH. 3.4. AgNPs Disturb Intracellular Redox Balance and Induce DNA Damage. The above results show that the dissolution of AgNPs in an acidic environment is accompanied by the formation of •OH. We predict that the consequences of increasing Ag+ and •OH overproduction would deplete intracellular antioxidants, eventually exhausting that means of cellular defense against oxidative stress. Glutathione (GSH) is a known antioxidant in preventing cellular damage from ROS.44 Therefore, we tested the effects of AgNPs and Ag+ on GSH oxidation. GSH was incubated with AgNPs and AgNO3 (for Ag+ form), respectively. The intermediate product of GSH oxidation, glutathionyl radicals (•SG), were determined by ESR.45,46 We used DMPO as a spin trap for detection of •SG. The DMPO is often used to capture •SG.47,48 It can form a stable adduct with •SG (DMPO/•SG) with a distinct ESR signal: a typical spectrum of DMPO/•SG adduct has four lines and hyperfine splitting parameters of αNα = 15.3 G and αHβ = 16.2 G (Figure S4). As shown in Figure 5A, the extremely weak ESR signal of DMPO/•SG indicates that the oxidation of GSH by AgNPs is very limited (second line). However, when GSH is co-incubated with AgNO3 (third line), a strong ESR signal of DMPO/•SG is found, indicating the oxidation of GSH occurs in the presence of AgNO3.



Figure 5. GSH oxidation and intracellular ROS detection. (A) ESR spectra of •SG generated in the presence of AgNPs and AgNO3. (B) Cellular GSH levels determined by the GSH-Glo assay. * indicates p < 0.05 compared with the E.coli group or S.aureus group. (C) Fluorescence images of intracellular ROS by fluorescence microscopy. Intracellular ROS were detected using the fluorescence dye CM-H2DCFDA. Scale bar = 5 µm. (D) The percentage of DNA damage in bacterial cells after AgNPs treatment. * indicates p < 0.05 compared with the E.coli group or S.aureus group.

E. coli, S. aureus, and L. casei are capable to produce GSH against oxidative damage.49 L. bulgaricus, although it cannot produce GSH, can import exogenous GSH into the cell.50 We used a GSH assay kit to analyze the intracellular GSH levels in our model bacteria with or without AgNPs treatments (Table S4). As shown in Figure 5B, after incubation with AgNPs, the levels of GSH in E. coli and S. aureus decrease by 22.2% and 31.4%, respectively. However, the GSH in L. bulgaricus and L. casei decrease much larger, about 80%. Therefore, the significant decrease of GSH in L. bulgaricus and L. casei is mainly due to the dissociation of Ag+ from AgNPs in an


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acidic environment. As an antioxidant, the central roles of GSH are to maintain the cell’s redox state and participating in the neutralization of ROS. The oxidation of GSH leads to the generation of intracellular ROS, which can result in DNA damage. In addition, the sudden increase of •SG can also affect intracellular oxidative stress of bacteria.47 To test AgNPs could increase intracellular oxidative stress in bacteria, cellular ROS and DNA damage are determined. Using the fluorescence ROS sensor CM-H2DCFDA, we detect a high level of ROS in L. bulgaricus and L. casei and little signal in E. coli and S. aureus treated with AgNPs (Figure 5C). TUNEL assay was used to detect DNA fragmentation by labeling the 3′-hydroxyl termini in the double-strand DNA breaks. As shown in Figure 5D, the percentage of bacterial cells with DNA damage in L. bulgaricus and L. casei treated with AgNPs increase nearly ten-fold higher than that of controls. In contrast, no significant differences are observed in E. coli and S. aureus compared with their controls. 3.5. Highly Efficient Killing of E. coli and S. aureus by AgNPs in Acidic Condition. In our observations, the antibacterial activity of AgNPs against the two lactobacilli is associated with their acidic growth environment. Therefore, we want to see if AgNPs could show a similar antibacterial activity against E. coli and S. aureus under acidic environment. We adjusted the growth agar plates’ pH of E. coli and S. aureus from 7.0 to 5.5 to test the antibacterial activity of AgNPs. The bacterial viability was also determined by the colony counting method. As shown in Figure 6, the killing effects of AgNPs against E. coli and S. aureus are more effective at pH 5.5 than at pH 7.0, suggesting that acidic environment can enhance the antibacterial activity of AgNPs.



Figure 6. Antibacterial activity of AgNPs against E. coli and S. aureus in acidic environment. * indicates p < 0.05 compared with the pH 7.0 group.

Physicochemical environment (temperature, osmotic pressure) plays a critical role in the toxicity of nanomaterials.51,52 In this study, we show that AgNPs exhibit markedly different effects on cell viability in the two beneficial lactobacilli (L. bulgaricus and L. casei) and the two pathogens (E. coli and S. aureus) (Figure 2 and Figure 3). Many reports show that the activation of Ag+ is a very important feature of the antibacterial activity of AgNPs.29,30 Here, we show that the acidic growth environment of lactobacilli promotes AgNPs dissolution, which led to •OH production. Furthermore, Ag+ and •OH disturb the redox balance through promoting GSH oxidation and •SG production inside the cell. These changes led to a continuous stream of ROS that cause various cellular damages. The whole process is shown schematically in Scheme 1.


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Scheme 1. Schematic of a proposed mechanism of AgNPs against L. bulgaricus and L. casei. Acidic growth environment (as low as pH 4.5) of L. bulgaricus and L. casei will enhance AgNPs dissolution and •OH production, which further lead to a lethal effect.

4. CONCLUSIONS In this study, we evaluate the bactericidal effects of AgNPs on two beneficial lactobacilli and explore the underlying mechanisms. Our results show that these lactobacilli are very susceptible to AgNPs. Our mechanistic study shows that the pH of the growth environment can affect the actions of AgNPs on different types of bacteria, as pH plays an important role affecting the AgNPs dissolution and •OH production. The subsequent induction of GSH oxidation and DNA damage are associated with the lethal effect of AgNPs on bacteria. Interestingly, we find that placing E. coli and S. aureus to an acidic environment also caused these bacteria to become more susceptible to AgNPs. Our study provides the first mechanistic description of a pH-Ag+-•OH bactericidal pathway against different types of bacteria and will contribute to a key consideration when formulating AgNPs-containing consumer products. Additionally, this study enlightens the increased antibacterial activity of AgNPs in acidic conditions and has potentials for bacterial disinfection



applications.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Biological characteristics of bacteria, antibacterial effect of AgNPs, Ag content in bacterial cells, GSH content in bacterial cells, Live/Dead assay, SEM images of bacterial cells, ESR spectral of BMPO/•OH spin adduct and DMPO/•SG spin adduct. AUTHOR INFORMATION *Corresponding Author: [email protected] Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work is partially supported by the China Postdoctoral Science Foundation (2015M571797), National Natural Science Foundation of China (31400862), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Jiangsu Provincial Key Laboratory of Radiation Medicine and Protection, and a regulatory science grant under the FDA Nanotechnology CORES Program. The authors appreciate Dr. Teresa Croce (Office of Food Additive Safety, CFSAN) and Dr. Lili Fox Vélez (Office of Regulatory Science, CFSAN) for their comment on the manuscript and scientific writing support. The views presented in this paper do not necessarily represent those of the U.S. Food and Drug Administration. No official support or endorsement by the U.S. Food and Drug Administration is intended or should be inferred.


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Bactericidal Effects of Silver Nanoparticles on Lactobacilli and the

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