Impact of graphene exposure on microbial activity and community

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Impact of graphene exposure on microbial activity and community ecosystem in saliva Yuting Shi, Wenjun Xia, Shima Liu, Jingyang Guo, Zhengnan Qi, Yan Zou, Liping Wang, ShengZhong Duan, Yi Zhou, Chenglie Lin, Jiye Shi, Lihua Wang, Chunhai Fan, Min Lv, and Zisheng Tang ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00566 • Publication Date (Web): 07 Dec 2018 Downloaded from http://pubs.acs.org on December 12, 2018

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Impact of graphene exposure on microbial activity and community ecosystem in saliva Yuting Shi1,2,3 ‡, Wenjun Xia1,2,3 ‡, Shima Liu4,5 ‡, Jingyang Guo4,5, Zhengnan Qi1,2,3, Yan Zou1,2,3, Liping Wang6, Sheng-Zhong Duan3,7, Yi Zhou8, Chenglie Lin8, Jiye Shi9, Lihua Wang4, Chunhai Fan4, Min Lv4*, Zisheng Tang1,2,3* 1. Department of Endodontics, Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China 2. National Clinical Research Center of Oral Diseases, Shanghai 200011, China 3. Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology, Shanghai 200011, China 4. Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China 5. College of Sciences, Shanghai University, Shanghai 200444, China 6. School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200011, China 7. Laboratory of Oral Microbiota and Systemic Diseases, Ninth People’s Hospital, School of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China


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8. School of Basic Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China 9. UCB Pharma, Slough, Berkshire SL1 3WE, UK

KEYWORDS graphene oxide (GO), graphene oxide-silver nanoparticles (GO-AgNPs), saliva, microbial community

ABSTRACT

Graphene-based nanomaterials (GMs) are served as great promising agents for the prevention and therapy of infectious diseases. However, their dental applications remain to be evaluated, especially under the context of the oral microbial community. Here, we examined the exposureresponse of salivary bacterial community to two types of GMs, i.e. graphene oxide (GO) and GO-silver nanoparticles (AgNPs). Both GO and GO-AgNPs showed lethal effect against salivary bacteria in a concentration-dependent manner, and the antibacterial capacity of GO-AgNPs is superior to GO. Interestingly, the salivary bacterial community enhanced the tolerance to GMs as compared to homogeneous bacteria. High-throughput sequencing revealed that both 80 μg/mL GO and 20 μg/mL GO-AgNPs significantly altered the biodiversity of salivary bacterial community. Especially, they increased the relative abundance of Gram-positive bacteria compared to the untreated sample, notably Streptococcus, suggesting that the bacterial wall


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structure plays a critical role in resisting the damage of GMs. Although GMs could effectively limit the salivary bacterial activity and cause changes in bacterial community structure, they are not toxic to mammalian cell lines. We envision this study could provide novel insights into the application of GMs as “green antibiotics” in nanomedicine.


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Introduction Saliva contains diverse biomarkers such as mRNA, miRNAs, protein, and microbiome, and has attracted increasing attention in clinical and diagnostic utilities owing to its non-invasive collection feature.1 Especially, saliva as a microbial reservoir contains numerous bacteria that outflow from microbial communities inhabiting on the surface of various oral organs and tissues such as tongue, teeth, as well as gingival crevices.2 Molecular analysis of microbial communities by 16S rDNA high-throughput sequencing has suggested that there are more than 3,000 discernible OTU level phylotypes of bacteria floating in the saliva.3 Salivary microbiota exhibit individual variation and long-term stability for up to one year,2, 3 while diet, oral disease, and medication intake can result in ecological changes. These alternations are related to oral and systemic diseases, such as caries, periodontitis, diabetes mellitus-Type 2, cancers, and even HIV.2-7 Given these health threats caused by dysbacteriosis, it is greatly important to determine whether the medication disturb the salivary ecological stability before clinical use. Graphene-based nanomaterials (GMs) exhibit the potential applications in biomedicine over past decades,8 such as drug delivery,9,

10

tissue regeneration,11,

12

biosensor,13-16 and cancer

therapy.17, 18 They also possess excellent characterization in optics, electronics, thermionics, and mechanics.8 Graphene oxide (GO), a graphene derivative, is one of the most widely used GMs in biological research due to its prominent water dispersion and easy modification, stemming from functional groups (e.g., carboxyl, hydroxyl, and epoxy groups) and surface destruction. Many efforts have recently been made on the therapeutic application of GO as an alternative to antibiotics in microbial infections, focusing on the antibacterial activity and mechanism.19-23 It has been demonstrated GO is a broad-spectrum antimicrobial agent against various species of microorganism, including Gram-negative (e.g., Escherichia coli, Pseudomonas aeruginosa) and


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positive bacteria (e.g., Staphylococcus aureus, Bacillus subtilis), drug-resistant bacteria (e.g. E. coli carrying blaNDM-1, E. coli carrying mcr-1, and methicillin-resistant S. aureus), and fungal pathogens (e.g., Fusarium graminearum, Fusarium oxysporum).20,

24, 25

Our previous work

showed GO could effectively kill the oral pathogenic bacteria growth, and inhibit Streptococcus mutans biofilm formation.26, 27 Three major hypotheses have been proposed to account for the excellent bactericidal capacity.20 Physical disruption of the microbial cell membrane resulted from GO’s sharp edges is a predominant mechanism.20-22 Knife-like GO can penetrate the microbial cellular membrane, induce the damage of the cellular membrane integrity and the leakage of cell content, and finally lead to the death of bacteria.21-23 Another mechanism of GO against the microorganism is oxidative stress. The generation of reactive oxygen species (ROS) mediated by GO can inactivate bacteria by destroying the bacterial membrane and disordering normal metabolism.28, 29 Besides, the instinctive flexibility and absorbability of GO enable it to coat the surface of bacteria and prevent the substances exchange between cells and environment, reducing the growth and proliferation of bacteria.30, 31 In order to further improve the antibacterial capacity of GO, more and more studies focus on the graphene oxide-silver nanoparticles nanocomposite (GO-AgNPs) in recent years.25, 32-35 Ag nanoparticles as an excellent antibacterial agent have potentially used in bacterial infection, but that it tends to aggregate due to high surface energy, which greatly weakens its antibacterial action.33 A recent study showed the aggregation of AgNPs made gram-negative bacterial become resistant to their toxic effects.36 It has also been reported that GO can provide the platform to anchor Ag nanoparticles and prevent its aggregation,25 therefore, GO-AgNPs showed enhanced antimicrobial capacity compared to pure GO and AgNPs.33, 34 However, these studies selected


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model microorganisms to examine the microbial toxicity of GO or GO-AgNPs under lab conditions. The impact of GMs on the clinical microbial communities has not been reported. In this study, we first studied the influence of GO and GO-AgNPs at different concentrations to the salivary microbial community in vitro. A clinical standard method was used to collect saliva from six healthy volunteers. MTT assay and flow cytometry were carried out to investigate the viability of bacteria in saliva samples after treatment with nanomaterials for 2 h. Confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) were carried out to observe the morphology of living and dead salivary bacteria. The 16S rDNA amplicon sequencing was performed to analyze the changes of bacterial community in saliva. This study will provide a promising antibacterial nanomedicine for oral disease therapeutics.

Experimental Section Preparation and characterization of GMs GO nanosheet was fabricated by our reported modified Hummers’ methods.22,

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The

concentration of 2 mg/mL GO solution was stored at 4 °C. GO-AgNPs was synthesized according to the proposal reported by Zhou et al.34 Briefly, the aqueous solution of 3.6 mM AgNO3 was added to 0.5 mg/mL GO solution with vigorous stirring. After 30 min of ultrasound treatment at room temperature, the GO solution with AgNO3 and sodium citrate was added slowly to fresh NaBH4 solution (0.12 M, 60 mL) under vigorous stirring. The pH value of the reaction system was adjusted to 11 by 0.5 M NaOH solution under magnetic stirring. The product was obtained by centrifugation, and then washed with deionized water three times and dialyzed for 3 days to remove residual ions.


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Transmission electron microscopy (TEM, Tecnai G2F20S-TWIN, FEI, USA) and atomic force microscopy (AFM, Nanoscope IIIa, USA) was used to characterize the morphology of GMs. The structure of nanomaterials was measured by UV-vis spectrophotometer (U-3010, Hitachi, Tokyo, Japan), and Raman spectroscopy (XPLORA INV). Collection and culture of saliva sample The study was approved by the Independent Ethics Committee of Ninth People’s Hospital affiliated to Shanghai Jiao Tong University, School of Medicine. Six volunteers who match inclusion and exclusion standard were selected for the study. They went to the laboratory for sampling and refrained from drinking or eating for 2 h. About 5 mL of whole unstimulated saliva (WUS) was collected from each volunteer. The saliva sample (100 μL) was added into BHI broth and cultivated at 37 °C for 48 h in an anaerobic system (80% N2; 10% H2; 10% CO2). Then the suspension of optimal concentration of cultured salivary bacteria was prepared for the subsequent experiment. The activity of salivary bacteria The cultured salivary bacteria (OD600=0.6, 200 uL) was cultured in brain heart infusion broth (BHI) with different concentrations of GMs. After 2 hours, MTT assay and flow cytometry were used to examine the cell viability treated with at all concentrations of GMs. Confocal laser scanning microscopy (CLSM, Leica TCS SP2, Germany) and transmission electron microscopy (TEM, PHILIP CM-120) were carried out the observation of bacterial morphology.37-39 16S rDNA amplicon sequencing


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After bacterial community exposure to GMs, PMA treatment was employed to filter DNA information of dead bacteria since PMA with high affinity for DNA is a cell membraneimpermeable dye.40 Total genome DNA from samples (live bacteria) was extracted using Sodium

Dodecyl

Sulfate

(SDS)

method.

Specific

primer

515F

(5’-

GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) with the barcode was carried out to amplify the distinct V4 region of 16S rDNA. Phusion® HighFidelity PCR Master Mix (New England Biolabs) was used for all PCR reactions. Libraries were sequenced on the Illumina HiSeq2500 platform (Illumina, San Diego, CA, USA), generating 250 bp paired-end reads. Statistical and bioinformatics analysis Statistical significance between groups was determined by Student’s t-test. If p < 0.05, the differences were considered to be statistically significant. Unweighted Unifrac Distance data were analyzed statistically using Wilcoxon Signed Rank Test. The minimum qualified sequence number of 18 genomic DNA samples were 55755. Uparse software (Uparse v7.0.1001, http://drive5.com/uparse/) was used for sequences analysis.

Results Preparation and Characterization of GMs A modified Hummers’ method was used for the fabrication of GO nanosheets.21, 22 Atomic force microscopy (AFM) showed the thickness of single-layer GO was about 1 nm (Figure 1A). As displayed by Raman spectra, the D, G and 2D peak of GO was at about 1353, 1590 and 2678


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cm-1 (Figure 1B), respectively, suggesting the successful preparation of the single-layer GO.30 On the basis of the structural defects and abundant oxygen-containing groups, GO-AgNPs nanocomposites were fabricated by reducing Ag ion to Ag nanoparticles on the surface of GO.34 The characteristic peak of GO performed by UV-vis spectroscopy was at 220 nm (Figure 1C), which was derived from π-π* transitions of aromatic C=C bonds. A novel peak appeared at 401 nm in the spectrum of GO-AgNPs, which resulted from the surface plasmon resonance (SPR) of ball-shaped Ag nanoparticles, revealing the successful anchor of AgNPs on the surface of GO.25, 41

TEM images confirmed the spherical shaped AgNPs (diameter ~12 nm) was coated on a

single-layer GO (Figure 1D-F). These results demonstrated GO-AgNPs was successfully synthesized.

Figure 1. Characterization of GO and GO-AgNPs nanocomposite. (A) AFM image and (B) Raman spectrum of GO. (C) UV-vis spectra of GO and GO-AgNPs. (D) TEM and (E) HRTEM image of GO-AgNPs. (F) Size distribution of AgNPs on GO.


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Inhibition of salivary bacteria by GMs Saliva samples were first collected following a clinical standard method from six healthy volunteers. To remove the biological molecular in saliva, such as ptyalase, DNA, and miRNA, the samples were cultured in an optimal medium for 24 h. Taxonomic profiling using 16S rDNA sequencing revealed no significant differences were observed on the species and abundance of bacteria at the genus level between experimental culture and direct salivary collection (Figure S1). Thus, the cultured salivary bacterial community as a substitute for the clinical sample was used for subsequent experiments. The defined optical density (OD600=0.6) of cultured salivary bacteria cells were expose to different concentrations of GO (0-160 µg/mL) and GO-AgNPs (0-40 µg/mL) dispersing in isotonic saline solution (0.9% NaCl), respectively. After 2 h, the salivary bacterial viabilities were detected by the MTT assay and flow cytometry. MTT is a sensitive indicator of cellular metabolic activity due to its reduction by succinate dehydrogenase of the cell.21 Flow cytometry was considered a fast and economic approach to study complex ecosystems, revealing the bacterial survival.42, 43 As shown in Figure 2, GO and GO-AgNPs both reduced the cell viability in a concentration-dependent manner, and GO-AgNPs displayed stronger toxicity to salivary bacteria than GO. MTT results showed the concentration of GO-AgNPs at 15 μg/mL decreased the viability to approximately 20% (Figure 2A). The concentration of GO at 160 μg/mL showed the comparable effect as GO-AgNPs at 15 μg/mL (Figure 2C). Although there were individual differences between samples exposed to the same concentration of GO or GO-AgNPs (except for GO 40 μg/mL), the similar tendencies of their inhibition activity were observed by flow cytometry in Figure 2B&D. Almost all bacteria in saliva were killed by 20 μg/mL GO-AgNPs. However, about 20% of the bacteria survived in the presence of GO at the concentration of 160


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μg/mL. The superior antibacterial capacities of GO-AgNPs may be attributed to the synergistic effect of GO and AgNPs.25, 35 Ag nanoparticles have been proved to inhibit the bacterial growth and disrupt the cell membrane,44 which has been extensively used for dental application.45, 46 The incorporation of AgNPs into GO confers additional antimicrobial activity. However, the fatal drawback of AgNPs is that it is very easy to aggregate.25 It is noteworthy that graphene nanosheets decrease AgNPs agglomeration and improve the contact probability of AgNPs with bacteria, resulting in the enhanced antibacterial properties of GO-AgNPs.33

Figure 2. Growth of salivary bacteria exposure to GMs. The viability of salivary bacteria treated with (A) GO-AgNPs and (C) GO. The number of live bacteria treated with (B) GOAgNPs and (D) GO. Salivary samples were cultured in the presence of different concentrations of GMs at 37 °C for 2 h. * means significant difference at p < 0.05 compared with control.


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Morphology changes of salivary bacteria by GMs The morphological changes in bacteria exposure to GMs were observed by CLSM and TEM. Confocal images and quantitative analysis were performed to investigate the salivary bacterial morphology. SYTO9 with green fluorescence is able to enter live cells, whereas red ﬂuorescent PI penetrates into cells with disrupted cytoplasmic membranes. As shown in Figure 3A, the control group was dominated by green fluorescence from SYTO9, suggesting nearly all bacteria were alive. Dead cells stained by red fluorescence of PI increased with GO and GO-AgNPs concentration, which suggested that GO and GO-AgNPs inhibited the salivary bacteria through damaging the cellular membrane of bacteria. The fluorescence intensity quantitatively confirmed the effective inhibition activity of GMs (Figure 3B&C). The morphologies of salivary bacteria were further observed by TEM (Figure 4). Untreated salivary bacteria showed the natural rodshaped and spherical morphologies, as well as the intact cell membranes. However, the morphological integrities of salivary bacteria were destroyed in the presence of 20 µg/mL of GO-AgNPs or 80 µg/mL of GO. The damaged cell walls and membranes, as well as a leakage of intracellular contents of the disrupted cell were observed (Figure 4, red arrow). This was in agreement with the result observed by confocal microscopy. Notably, some bacteria appeared to be coated by GO nanosheets (Figure 4, black arrow), which suggested that the bacteria were likely favored to interact with AgNPs on GO-AgNPs. Also, the sample preparation may promote the interaction between GO-AgNPs and the bacterial cells.47


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Figure 3. Confocal imaging of bacteria in saliva. (A) Confocal images of live and dead bacteria exposed to GMs of different concentrations for 2 h, with 0.9% NaCl treatment as the control. Live and dead bacteria were respectively labeled by SYTO9 (green) and PI (red). (B), (C) Quantitative evaluation of live and dead cells based on confocal images.


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Figure 4. TEM images of the morphology of salivary bacteria. Red and black arrows mark membrane disruption and nanomaterials, respectively.

Ecosystem composition of salivary bacteria exposure to GMs The landscape of the living salivary microbiome in all samples was examined by 16S rDNA amplicon sequencing. DNA information of dead bacteria was filtered by PMA treatment since its covalent linkage with PMA could not be amplified by PCR.40 A total of 1577508 unique reads and 4404 operational taxonomic units (OTUs) were obtained from 18 samples. The OTU numbers of control, GO and GO-AgNPs were 2872, 3081 and 3609, respectively. At a 3% dissimilarity level, the coverage estimations suggested 98.9% to 99.6% of bacterial species were present for all samples. The abundance of the most dominant phylum Firmicutes in samples treated with GO (81.5±12.0%) and GO-AgNPs (78.5±15.2%) both increased by about 10%, compared to untreated (68.6±21.0%) (Figure S2), but no significant difference was observed at the phylum level.


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To find out the change of microbial flora exposed to GMs, Figure 5A presented the 32 most abundant bacteria at the genus level in all samples with the relative abundance greater than 0.5%, that Veillonella and Streptococcus were enriched in all samples. The community diversity was examined by alpha diversity or within-sample diversity. Simpson diversity index of control, GO and GO-AgNPs group was 0.685, 0.676, 0.751, respectively (Figure 5B). Although GO-AgNPs possessed the highest level of bacterial diversity, there existed no significant difference when comparing the diversity of these groups. It revealed that GMs did not significantly impact the number of salivary bacterial species present, as well as the abundance of each species. The findings were confirmed by the Shannon diversity index and Chao 1, and no significant differences were reflected in the bacterial evenness and richness of three groups (Figure S3). Upon beta diversity, or between-sample diversity, with unweighted UniFrac, significant differences were observed in bacterial communities in Figure 5D (t-test, p < 0.05). These analyses revealed special genera may play a critical role in driving the bacterial communities. The relative abundance of Veillonella displayed no significant difference between bacterial communities in all samples. However, as shown in Figure 5E, the average relative abundance of Streptococcus in bacterial communities of GO or GO-AgNPs was significantly improved compared to the control group at the genus level (t-test, p < 0.05). Moreover, Streptococcus anginosus were more abundant in GO-AgNPs group than control at the species level (t-test, p < 0.05). The significant differences were analyzed by principal coordinate analysis (PCA), which showed that samples in control and GO groups clustered closed to each other, the samples of GO-AgNPs were clearly separated (Figure 5C). These findings demonstrated that the effect of GMs on community composition was less than that of microbial activity. They only significantly impact the abundance of one genus Streptococcus.


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Figure 5. Changes of salivary microbiome diversity after treatment with GO-base nanomaterials. (A) Stacked bar plot of phylogenetic composition of control, GO and GOAgNPs samples at the genus level (>0.5% abundance). Six samples were labeled with the number 1, 2, 3, 4, 5 and 6. (B) Simpson diversity scores of the microbiome in samples. (C) Principal coordinate analysis of salivary samples based on unweighted Unifrac distances. (D) Beta diversity scores of the microbiome in samples. (E) Comparison of the abundance of Streptococcus in three groups.


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Impact of GMs on Gram-negative and -positive bacteria The bacterial membrane disruption is the main cause of GMs against microorganism. On the basis of the different membrane or wall structure, bacteria are traditionally divided into Grampositive and Gram-negative bacteria. The percentage of two types of bacteria abundance (>0.9%) at genus level was assessed through high-throughput sequencing. Heat map revealed 17 genera were found, and 6 genera belonged to Gram-positive, 11 genera belonged to Gram-negative bacteria (Figure 6A). Streptococcus and Veillonella dominated the Gram-positive and Gramnegative bacteria, respectively. Gram-positive bacteria abundance was 15.7±18.5%, 50.6±27.2%, 31.2±7.9% in control, GO and GO-AgNPs groups, respectively, and the significant difference was observed (p ＜ 0.05) (Figure 6B). The results suggested that the toxicity of GMs to Gramnegative bacteria was higher than Gram-positive, probably resulting from their different cellular structures. Gram-positive bacteria own thicker cell walls composed of peptidoglycan, which may inhibit the direct interaction between GMs and phospholipids molecular of the membrane.48 However, graphene nanosheets could directly penetrate into the outer membrane of Gramnegative bacteria and extracted lots of phospholipids molecular, causing the damage of membrane and loss of cell integrity.23 Thus, Gram-positive bacteria might be difficult to be disrupted, showing the enhanced percentage in saliva microbiota.


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Figure 6. GMs impact the abundances of Gram-negative and -positive bacteria in saliva. (A) Heatmap of bacterial genera divided into Gram-positive and Gram-negative bacteria (>0.9% abundance) (B) The percentage of Gram-positive bacteria in saliva microbiota of three groups.

Discussion GMs are effective antimicrobial agents that inhibit the growth of a broad spectrum of bacterial species. However, these studies often employ the experimental model bacteria and evaluate the antibacterial properties of nanomaterials against the single microbe, which could not reflect the actual cases in clinical and environment. Actually, a primary survival strategy of bacterial is to form the homologous and heterologous ecosystem in nature. The microbial ecosystem balance plays an important role in the environment and human being health. Thus, an increasing amount of work begins to pay attention to the influence of GMs on microbial community, such as mouse gut, swine manure, and soil.49-51 Saliva is an important microbial community ecosystem, not only directly related to oral health but also can be used as a biomarker to predict systemic diseases. In our study, the influence of GMs on the human salivary microbial community was assessed. The results showed both GO and GO-AgNPs could effectively prevent the growth of the salivary microbial community, and GO exhibited the weaker inhibition activity than GO-AgNPs (Figure


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2). This is consistent with previously reported findings that the capacity of GO-AgNPs against bacteria is superior to GO.32, 34 Our previous work noted the minimal inhibition concentration (MIC) of GO-Ag against E. coli and S. aureus were 3 and 7 μg/mL, respectively, but the concentration of GO above 64 μg/mL could significantly prevent the bacterial growth.25 This because that the combination of GO and AgNPs synergistically enhanced the bactericidal efficacy of GO-AgNPs against the bacteria. It has been known that GO can threaten bacteria through two mechanisms, including physical damage and oxidative stress, causing the loss of membrane integrity.20 TEM images indicated that GO absorbed to the surface of bacteria, resulting in the damage of bacterial membrane and the leakage of intracellular substance (Figure 4). For GO-AgNPs, the higher affinity of GO for bacteria can promote the contact of AgNPs with bacteria.19 It is well known that AgNPs can react with proteins and DNA with sulfurcontaining groups in the cell, easily lead to the structural damage and function loss of these molecules, finally give rise to the damage of the cellular structure and the disturbance of normal metabolism, growth and proliferation.44 Additionally, GO as a platform can protect AgNPs from aggregating to retain strong bactericidal properties. GMs shows low cytotoxicity to mammalian cells (Figure S4), in agreement with previous reports. There, GMs provide a promising antimicrobial agent for dental applications.10-12 Compared to bacteria, the salivary bacterial community increased the tolerance to GMs, that GO concentration of 160 μg/mL can eradicate approximately 80% of the bacteria in saliva. However, more than 80% of oral bacteria such as S. mutans, Porphyromonas gingivalis, and Fusobacterium nucleatum was killed by only 80 μg/mL GO.26 The inhibition of salivary bacteria also need a higher concentration of GO-AgNPs (>20 μg/mL) compared to E. coli and S. aureus.25 The thick wall structure of Gram-positive might figure prominently in the tolerance of


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bacteria. High-throughput sequencing revealed the abundance of Gram-positive bacteria was significantly enhanced, especially Streptococcus. (Figure 6). Streptococcus widely inhabited various oral organs is also one of the richest bacteria in the salivary microbiome. The genus Streptococcus is comprised of many species of gram-positive, such as S. mutans, Streptococcus anginosus, Streptococcus salivarius, and Streptococcus mitis, and most of them are related to the oral and throat diseases.52 For example, S. mutans is considered to be a principal factor in the development of caries for years, and it plays a significant role in multispecies communities.53 However, Streptococcus sanguis inhibits the growth of some Gram-negative species such as Porphyromonas gingivalis through producing hydrogen peroxide and sanguicin, which has beneficial effects on periodontitis and halitosis.54,

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Our findings showed Streptococcus

anginosus were more abundant in GO-AgNPs group than control at the species level in Figure 5. Enhanced abundance may be due to the less susceptibility of S. anginosus to antimicrobial agents.56 However, it is not clear which species increase in the proportion of the genus Streptococcus after GO exposure. We found the effect of GMs on bacterial activity was more than their community ecosystem, while the cause of the phenomenon remains unclear. Further study based on these confusing issue will provide insights into the application of GMs for oral disease therapeutics.

Conclusions In summary, the influence of GMs on salivary microbiota was first investigated. Both GO and GO-AgNPs could effectively kill salivary bacteria and be concentration-dependent, and GOAgNPs was superior to GO due to the synergistic effect of GO and AgNPs. Interestingly, the


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salivary bacterial community showed more tolerance to GMs than homogenous bacteria. The effective bactericidal concentration of GMs is safe for mammalian cells. Additionally, GMs increased the relative abundance of Veillonella and Streptococcus confirmed by high-throughput sequencing, and that of Streptococcus showed a significant difference compared to the untreated sample. These findings suggested the impact of GMs on bacterial activity was related to the bacterial wall structure. Our study demonstrated the influence of GMs on bacterial activity was more than their community ecosystem, and thus GMs show a great application potential in oral disease therapy.

ASSOCIATED CONTENT Supporting Information Details of the experimental section; the bacterial diversity of saliva compared to that of the cultured saliva; the abundance of salivary bacteria at the phylum level; Shannon diversity scores and Chao 1 diversity scores of the microbiome in the samples; cytotoxicity of GMs.

AUTHOR INFORMATION Corresponding Author：[email protected]; [email protected] ‡These authors contributed equally. Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT


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This work was supported by National Key R&D Program of China (2017YFC0840100, 2017YFC0840110, 2016YFA0201200, 2016YFA0400900), The Open Large Infrastructure Research of Chinese Academy of Sciences, the National Natural Science Foundation of China (U1432116, 11675251, 81870749), and The Open Project of State Key Laboratory of Oral Diseases (SKLOD2018OF01).

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Impact of graphene exposure on microbial activity and community

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