Quaternized carbon quantum dots with broad-spectrum antibacterial activity for treatment of wounds infected with mixed bacteria

Quaternized carbon quantum dots (qCQDs) with broad-spectrum antibacterial activity were synthesized by a simple green “one-pot” method using dimethyl diallyl ammonium chloride and glucose as reaction precursors. The qCQDs showed satisfactory antibacterial activity against both gram-positive and gram-negative bacteria. In rat models of wounds infected with mixed bacteria, qCQDs obviously restored the weight of rats, signicantly reduced the death of rats from severe infection, and promoted the recovery and healing of infected wounds. Biosafety tests conrmed that qCQDs had no obvious toxic and side effects during the testing stage. The analysis of quantitative proteomics revealed that qCQDs mainly acted on the ribosomal proteins of gram-positive bacteria and signicantly down-regulated the metabolization-related proteins of gram-negative bacteria. Real-time quantitative PCR veried the expression levels of genes corresponding to the proteins with signicant differences expressed by the two species of bacteria after treated with qCQDs. The variation trend of the detected genes was consistent with the results of proteomics, meaning that qCQDs played the antibacterial effect on bacteria with a new antibacterial mechanism. and and the application of in the eld of the in this study provides a feasible strategy for studying the mechanism level. The proved pathway may also be extended as the new medicinal targets for development and the hemolysis rate was calculated. Normal saline and sterilized were used as negative and positive control, respectively.


Introduction
There is no doubt that bacterial infectious diseases have always posed a serious threat to human health and are always a sever challenge for medical workers. With the widespread and extensive use of antibiotics for the last 70 years or so, antimicrobial resistance (AMR) has become an imminent threat to the effective treatment of bacterial infections. The Review on Antimicrobial Resistance chaired by Jim O'Neill has predicted that the burden of deaths from AMR could balloon to 10 million lives each year by 2050, at a cumulative cost to global economic output of 100 trillion USD 1 . Thus, novel antimicrobial agents against severely resistant bacteria are urgently needed to address the problem. Otherwise, we may be entering a post-antibiotic era where advances in modern medicine would be jeopardized, resulting in an increase in serious infections 2 . In response to increasing antibiotic resistance in bacteria, alternatives different from traditional antibiotics are being investigated, including antibodies, probiotics, bacteriophages and antimicrobial peptides currently undergoing clinical trials, and advancements within arti cial intelligence, biotechnology, genetic engineering and synthetic chemistry have opened up new avenues towards the search for therapies that can substitute for antibiotics 3,4 . However, extensive use of these alternatives is limited by the cost of production and limited shelf life, and the potential biotoxicity and the occurrence of new bacterial resistance always puzzle the application of various new technologies in research and development of antimicrobial drug. Thus, the exploitation of novel antimicrobial agents can not only realize the speci c antibacteria of strains or species, but also achieve broad-spectrum antibacteria, so as to solve the bottleneck problem. Meanwhile, it is still an urgent task to clarify the molecular mechanism of antibacterial and avoid new bacterial resistance.
In more recent study, various nanomaterials have gradually emerged as excellent substitutes for antimicrobial agents, and have been widely explored for a broad range of bacterial infections via the photodynamic effect or/and photothermal ability [5][6][7] . Compared with reported nanomaterials, carbon nanomaterials (e.g. fullerenes, carbon nanotubes, graphenes, carbon quantum dots) with broad spectral coverage and other superior material properties have attracted wider attention in antibacterial and related applications in recent years [8][9][10] . Carbon quantum dots (CQDs), especially hetero-element doped CQDs with particle size of less than 10 nm, stand out in the eld of antibacterial research due to their advantageous properties including nontoxic nature, photostability, versatility in surface functionality for desired microbial adhesion and interactions, and their production from abundant and inexpensive precursors for extremely broad and low to ultralow cost applications [11][12][13][14][15][16] . At present, the mechanism of action of CQDs against bacteria mainly focuses on the three aspects of directly producing reactive oxygen species (ROS), such as superoxide (•O 2 − ) and hydroxyl radical (•OH) 17,18 , producing ROS assisted by photoactivation or other compound 6,[19][20][21] and damaging the cell membrane caused by the insertion of surface controlled CQDs through electrostatic interaction and/or hydrophobic interaction [22][23][24] . However, the above mechanism cannot further explain the signaling pathway of antibacterial CQDs against bacteria at the molecular level and there has been a lack of in-depth understanding of the interaction between such CQDs and bacteria. And the understanding of the molecular target of CQDs to bacteria will help the precision design of antibacterial CQDs for overcoming the need of AMR.
Quaternary ammonium compounds (QACs) with cationic groups can predominantly act on cytoplasmic (inner) membrane in bacteria or plasma membrane in yeasts to play antibacterial action 25 . Besides, combination of quaternary ammonium groups (QAGs) and nanomaterials can introduce new effects for improving antibacterial properties and safety [26][27][28] . In the other hand, quaternary ammonium carbon nanomaterials have become an important way to develop carbon nanomaterials with antibacterial activity 22,29,30 . For example, lauryl betaine (QACs) modi ed carbon dots prepared from 3-[2-(2aminoethylamino)ethylamino]propyl-trimethoxysilane and glycerol as the precursors could selectively kill gram-positive bacteria 29 ; a kind of quaternized carbon dots prepared from glycerol and quaternary ammonium-carrying organosilane could effectively disrupt the cell walls of gram-positive bacteria through the synergistic action of both electrostatic and hydrophobic interactions 22 ; and graphene oxidequaternary ammonium salt nanocomposite exhibited the synergistic antibacterial activity on Staphylococcus aureus and Escherichia coli on the basis of the combined action of mechanical membrane perturbation and oxidative stress induction 30 . The above antibacterial mechanisms of CQDs functionalized with QAGs mainly focused on electrostatic interaction, hydrophobic interaction and oxidative stress. However, the mechanism of QAGs functionalized carbon nanomaterials on bacteria has not been elucidated at the molecular level. Revealing and exploring the molecular antibacterial mechanism of carbon nanomaterial will promote the development of new antibacterial agents.
In this work, dimethyl diallyl ammonium chloride (DDA) and glucose as precursors were used to directly synthesize quaternized carbon quantum dots (qCQDs) with broad-spectrum antibacterial activity by a simple green "one-pot" method (Scheme 1). The qCQDs had the antibacterial activity against both grampositive and gram-negative bacteria, including Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis. In addition to the advantages of simple preparation, convenient synthesis and low cost, qCQDs had different antibacterial mechanisms from traditional quaternary ammonium compounds and other carbon quantum dots with antibacterial activities. The quantitative proteomics based tandem mass tags revealed that the targets of qCQDs on gram-positive and gram-negative bacteria were different. The qCQDs mainly interfered with the ribosomal proteins of gram-positive bacteria, but downregulated a variety of metabolized-related proteins for gram-negative bacteria, among which the citrate cycle was the most signi cant. At the same time, the effects of qCQDs on the corresponding genes of related proteins of gram-positive and gram-negative bacteria were veri ed by real-time quantitative PCR (RT-qPCR). The qCQDs were used to treat the wounds infected with mixed bacteria (Staphylococcus aureus and Pseudomonas aeruginosa), showing the similar therapeutic effect as positive control antibiotic and the promising prospect of qCQDs for the cure of complex bacterial infection.

Results
Characterization of qCQDs.
The size distribution of carbon nanomaterials is not only the simple physical change, but also is able to affect their antibacterial activity 31 . Transmission electron microscope (TEM) image showed that the average diameter of qCQDs was about 3 nm with homogeneous size distribution (Fig. 1a). Highresolution TEM (HRTEM) showed that the well-resolved lattice fringe with intercrystalline spacings of qCQDs was 0.22 nm (Fig. 1b), corresponding to the (100) facets of graphitic carbon 18, 32 . The particle size of qCQDs measured by dynamic light scattering (DLS) was centered at 4.5 nm with relatively concentrated distribution, suggesting the consistent and uniform morphology of qCQDs con rmed from TEM.
The optical property of qCQDs was evaluated. UV-vis absorption spectrum (Fig. 1d) of qCQDs showed π→π * transition of C=C (aromatic sp 2 domains) at 225 nm and n→π * transition of C=O and C-O at 283 nm 33 . The maximum emission wavelength of qCQDs uorescence increased with the red shift of excitation wavelength (Fig. 1e), suggesting the excitation-dependent luminescence behavior 32 . And qCQDs generated a uorescence spectrum with the maximum emission wavelength at 455 nm under the excitation wavelength of 355 nm (red arrows in Fig. 1e). Furthermore, Fig. 1f showed the typical mirror symmetry of maximum emission spectrum (black) and the maximum excitation spectrum (red). Using quinine sulfate in 0.5 mol/L H 2 SO 4 as standard reference 34 , the uorescent quantum yield of qCQDs was calculated to be 4.61% when the excitation wavelength was at 355 nm, suggesting the acceptable luminescence property.
The molecular functional groups of qCQDs was investigated by fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR), as shown in Fig. 2 and 1035 cm -1 in qCQDs were ascribed to alkyl aromatic ether, indicating that aromatic ether structure existed in qCQDs. The absorption peaks of qCQDs at 962 cm -1 and 879 cm -1 might be generated by the telescopic vibration of C-C and C-O, respectively. NMR was further applied to investigate glucose, DDA and qCQDs using D 2 O as solvent 27 . The 1 H NMR spectra in Fig. 2b revealed that the peaks of the proton signals of glucose at green area almost disappeared in qCQDs. Nevertheless, the proton peaks of C=C In vitro antibacterial activity of qCQDs.
The antibacterial activity of qCQDs on different species of bacteria was investigated (Fig. 3). Disk a, b and c on MHA plates contained 0.2 mg of qCQDs, 0.2 mg of glucose and 0.2 mg of DDA, respectively. Four species of gram-positive bacteria (S. aureus, MRSA, S. epidermidis and E. faecalis) and two species of gram-negative bacteria (E. coli and P. aeruginosa) were used as model bacteria to evaluate the antibacterial activity of qCQDs. Obvious inhibition zones appeared around disk a (qCQDs) on all the MHA plates with incubated bacteria, and the diameters of inhibition zones were more than 10 mm. In contrast, there was no inhibition zone around disk b (glucose) or c (DDA), indicating no antibacterial activity of two reaction substrates. Moreover, the minimum inhibitory concentration (MIC) of qCQDs on the above six species of bacteria was determined by broth dilution method, as shown in Supplementary Table 1. The MIC of qCQDs was 12.5 µg/mL for S. epidermidis, 25 µg/mL for S. aureus, MRSA and E. faecalis, and 50 µg/mL for both E. coli and P. aeruginosa. The above results con rmed that qCQDs exhibited antibacterial action on both gram-positive and gram-negative bacteria, implying the broad-spectrum antibacterial properties.
In order to better understand bactericidal and bacteriostatic effect of qCQDs, we investigated the number of colonies on nutrient agar plates after the six species of bacteria were exposed to different concentrations of qCQDs in different periods, as shown in Supplementary Fig. 1. When the concentration was up to 200 μg/mL, the changing tendency of the six species of bacteria was similar to that of 100 μg/mL, indicating that qCQDs can effectively inhibit and kill the gram-positive and gram-negative bacteria at 100 μg/mL. The detailed description for Supplementary Fig. 1

was provided in Supplementary
Information.
Antimicrobial mechanism of qCQDs against gram-positive bacteria and gram-negative bacteria.
The morphological changes of the six species of bacteria before and after the treatment of qCQDs were characterized by TEM, as shown in Fig. 4. Without the treatment of qCQDs, all of the bacteria exhibited normal cell structure, complete cell walls, and uniform density of substance inside the cells (Fig. 4a, 4c, 4e, 4g, 4i and 4k). In comparison, after the treatment of 100 μg/mL qCQDs for 12 h, bacterial cells were signi cantly changed with obviously broken cell wall and cell membrane (Fig. 4b, 4d, 4f, 4h, 4j and 4l). More speci cally, the bacterial cells treated with qCQDs were disintegrated to different degrees, the cytoplasmic density of bacterial cells obviously decreased, and even the phenomenon of cavities and substance agglomeration occurred in the bacterial cells. And bacterial cells lost their integrity, causing irreversible damage to bacteria cells and resulting in death of bacteria 40,41 , which was completely different from the untreated bacteria. The morphology of the treated bacteria indicated that qCQDs had the signi cant destructive effects on the above six species of bacterial cells, con rming the killing effect of qCQDs on both gram-positive and gram-negative bacteria. Furthermore, using S. aureus and E. coli as the representative bacteria, ow nanoanalyzer was applied to measure the changes of particle size of bacteria before and after the treatment of qCQDs, as shown in Supplementary Fig. 2. For untreated S. aureus and E. coli, particles larger than 1000 nm accounted for 78.3% and 92.2% of the total particles, respectively. After S. aureus and E. coli were treated with qCQDs, particles larger than 1000 nm were signi cantly reduced in both bacteria. In addition, with the increase of qCQDs concentration from 50 to 100 μg/mL and then to 200 μg/mL, particles larger than 1000 nm in the two species of bacteria decreased from 52%/64.2% to 16.8%/21.1% and then to 10.3%/1.0%, respectively, showing gradual decreasing trend. It is further con rmed that qCQDs could make bacterial cells lyse or disintegrate, resulting in death of the bacteria, which is consistent with the results observed by TEM. (ROS representative substance) was added, obvious uorescence at 520 nm appeared in the both bacterial suspension. However, for the direct interacting system of qCQDs and bacteria, there was hardly any uorescence at 520 nm in the both bacterial suspension containing DCFH-DA. Then, the above both bacterial suspension containing DCFH-DA and qCQDs showed obvious uorescence at 520 nm with the addition of H 2 O 2 . Therefore, it is preliminarily inferred that almost no ROS generated in the process of qCQDs combating bacteria.
In order to elucidate the antibacterial mechanism of qCQDs against bacteria, S. aureus and E. coli were used as the representative gram-positive and gram-negative bacteria, respectively. And then, tandem mass tag (TMT)-based quantitative proteomics was performed to analyze the protein changes of bacteria before and after the treatment of qCQDs 43,44 . Control group (C), low concentration group (L) and high concentration group (H) were set up in the test of quantitative proteomics. After S. aureus and E. coli were treated with qCQDs, the analysis of differential expressed proteins was conducted by comparing L with C and H with C. With the increase of qCQDs concentration, the amount of the differential proteins with both up-regulated and down-regulated expression increased in S. aureus and E. coli, respectively, and the differential expressed proteins in S. aureus were signi cantly more than those in E. coli ( Supplementary Fig. 4). After S. aureus and E. coli were treated with qCQDs, the identi ed proteins of the two bacteria were annotated in Cluster of Orthologous Groups of proteins (COG), showing that the functions of the proteins were mainly annotated in energy production and conversion, amino acid transport and metabolism, carbohydrate transport and metabolism, and translation, ribosomal structure and biogenesis ( Supplementary Fig. 5). The enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway revealed the proteins pathways with signi cant statistical differences. In the KEGG pathway for S. aureus, the differentially expressed proteins were mainly enriched in ribosome, RNA degradation, aminoacyl-tRNA biosynthesis and carbon xation in photosynthetic organisms by comparing L with C and H with C ( Supplementary Fig. 6). Ribosome, RNA degradation and aminoacyl-tRNA biosynthesis were mainly related to protein synthesis, among which the enrichment of ribosomal proteins showed the most signi cant difference (p=3.34×10 -5 ). The 46 differentially expressed proteins were enriched in ribosomes by further analysis of KEGG chord diagram ( Fig. 5a and Supplementary Table  2). The corresponding clustering heat map was drawn, and 33 ribosomal proteins were up-regulated and 13 were down-regulated by comparing H with C ( Fig. 5c). Correspondingly, for E. coli, the differentially expressed proteins in the KEGG pathway were mainly enriched in biosynthesis of antibiotics, microbial metabolism in diverse environments, citrate cycle, sulfur metabolism, metabolic pathways, biosynthesis of secondary metabolites, tryptophan metabolism, propanoate metabolism, carbon metabolism, lysine degradation, butanoate metabolism, benzoate degradation and nitrogen metabolism by comparing L with C and H with C ( Supplementary Fig. S8). Among them, further analysis of KEGG chord diagram showed that 10 differentially expressed proteins were enriched in the following six pathways ( Fig. 5b and Table  3), including biosynthesis of antibiotics, microbial metabolism in diverse environments, citrate cycle, metabolic pathways, biosynthesis of secondary metabolites and carbon metabolism. And these 10 proteins are essential enzymes for the citrate cycle (p=3.01×10 -5 ) ( Supplementary Fig. 8). The corresponding clustering heat map showed that the 10 differentially expressed proteins in citrate cycle were down-regulated by comparing H with C (Fig. 5d). After the differentially expressed proteins associated with ribosome in S. aureus and citrate cycle in E. coli were annotated in Gene Ontology (GO), the two types of proteins were classi ed according to cellular component, molecular function and biological processes, respectively. All the 46 ribosomal proteins belonged to intracellular cellular component which were mainly related to ribosomes, including large and small subunit, and involved in the biological process of protein translation, and their molecular functions were mainly focused on structural constituent of ribosome and RNA/rRNA binding ( Supplementary Fig. 9). In contrast, the molecular function of the 8 proteins associated with citrate cycle were mainly concentrated in the activity of various enzymes, including aconitate hydratase, isocitrate dehydrogenase (NADP + ), oxoglutarate dehydrogenase (succinyl-transferring), oxidoreductase and electron carrier, and the binding of thiamine pyrophosphate, metalion, cofactor and iron-sulfur cluster, and involved in the biological process including tricarboxylic acid cycle, metabolic process and oxidation-reduction process ( Supplementary Fig. 10).
Meanwhile, interaction analysis of differentially expressed proteins showed that 22 of the 46 ribosomal proteins were in the three protein functional interaction networks (Fig. 5e) and all the 10 proteins associated with citrate cycle were at the hub positions of the one protein functional interaction networks ( Fig. 5f).
In order to gain insight into the contributions of ribosome and citrate cycle to the antibacterial action of qCQDs against S. aureus and E. coli, gene set enrichment analysis (GSEA) was conducted to further analyze the genes of differentially expressed proteins in KEGG pathways, and real-time quantitative PCR (RT-qPCR) was used to verify the expression of related protein genes. GSEA showed that there were positive and negative correlations in the function set of ribosomal proteins (Fig. 6a), but all the proteins in the function set of citrate cycle were negatively correlated (Fig. 6b), which were consistent with the differentially expressed proteins in Fig. 5c and 5d, respectively. At the same time, GSEA gave the sequence of the corresponding genes of the proteins in the function set. According to the rank in gene list, six proteins in S. aureus and E. coli were selected from the corresponding differentially expressed proteins, respectively, and the information of selected proteins was shown in Supplementary Table 5. The proteomics analysis showed that three ribosomal proteins were down-regulated and the other three were up-regulated after S. aureus was treated with qCQDs ( Supplementary Fig. 11A). Fig. 6c showed the RT-qPCR results of the corresponding genes of six ribosomal proteins in S. aureus. The gene expression of the six ribosomal proteins had no signi cant difference at the low-concentration of qCQDs. When S. aureus was treated with high-concentration of qCQDs, the gene expression of down-regulated and upregulated ribosomal proteins changed signi cantly, which was consistent with the variation trend in the proteomics. The proteomics analysis showed that all six proteins associated with citrate cycle in E. coli were down-regulated ( Supplementary Fig. 11B). The RT-qPCR results in Fig. 6d showed that the gene expression of these six proteins had no signi cant difference at the low-concentration of qCQDs, while the gene expression of these six proteins signi cantly down-regulated at the high-concentration of qCQDs, corresponding to the variation trend of the six proteins in the proteomics.
In vivo antibacterial activity of qCQDs.
When the skin is compromised, bacteria from environment and skin surface are able to in ltrate into the subcutaneous tissue that is physiologically optimum for colonization and growth, eventually leading to wound infection. The impact of wound infections on health care is enormous, including burns, surgicalsite infections, and nonhealing diabetic foot ulcers. S. aureus and P. aeruginosa among the most common organisms isolated from both acute and chronic wounds of various etiologies have been attributed to causing infections of penetrating trauma and burn wounds in surgical site infections as well as in the military setting 45 . Double infections with S. aureus and P. aeruginosa is more virulent and/or results in worse patient outcomes than single infections, and both species are notorious for their resistance to antimicrobials 46 . Therefore, we established the rat trauma model with mixed infection of S. aureus and P. aeruginosa to evaluate the in vivo antibacterial activity of qCQDs. The experimental process was shown in Fig. 7a. aeruginosa were about 3.0×10 8 CFU/mL in the mixed bacterial suspension. After 48 h, the massive abscess appeared on the wounds. The qCQDs in the experimental group, levo oxacin in the positive control group, and normal saline in the negative control group were employed to treat the infected skin wounds, respectively, investigating the in vivo antibacterial activity of qCQDs. After treatment for 3 days, compared with the negative control group, the obvious scab was observed on the infected skin wounds with decreased exudate in the experimental group and the positive control group. After treatment for 5 days, although exudate appeared in all three groups, purulent lesions and the number of bacteria in the experimental group and the positive control group were signi cantly lower than that in the negative control group. After treatment for 7 days, exudate, purulent lesions and the number of bacteria signi cantly reduced in both the experimental group and the positive control group. After treatment for 10 days, the wound area in the experimental group and the positive control group signi cantly decreased, but the wound size in the negative control group was similar to that of the other two groups on the seventh day and there was still a large amount of exudate and numerous bacteria. After treatment for 14 days, the wounds in the two treated groups completely had no exudate and basically healed without detectable bacteria, but the wound size of the negative control group was the same as that of the two treated groups on the tenth days with the still present bacteria colonies. The above results indicated that qCQDs could promote the recovery of the wound infected with mixed bacteria, which had the same therapeutic effect as levo oxacin at a certain concentration. Fig. 8a showed that three groups of SD rats lost weight after the wounds were infected with mixed bacteria for 48 hours, and the weight of the rats in the experimental group and the positive control group increased gradually after intervention. The increasing trend continued within 14 days. Meanwhile, the weight of the negative control group increased slowly in the rst 6 days intervention, and always were lower than that of the experimental group and the positive control group. Within the whole course of intervention, the dead rats appeared in the negative control group on 2 nd , 3 rd , 5 th , 6 th and 9 th day, as shown in Fig. 8b, with a nal survival rate of 56.4%. All the rats of experimental group and positive control group survived in the treating process. The above results indicated that qCQDs could effectively combat the infection with mixed bacteria of S. aureus and P. aeruginosa, greatly contribute to the weight recovery of experimental rats, and signi cantly reduce the death rate of the rats.
H&E staining was carried out on the pathological sections of the wound tissues after 48 hours infection and 14 days treatment to evaluate the recovery of the infected wounds in different groups, as shown in Fig. 8c. For the infected wounds, a large number of neutrophils appeared in the wound tissues, with enlarged blood vessels and increased blood cells (black arrow in Fig. 8c), which con rmed the successfully infected wounds with S. aureus and P. aeruginosa. After intervention for 14 days, capillaries in the wound tissues of the experimental group and the positive control group were signi cantly reduced, neutrophils basically disappeared, and obvious connective tissue formed on the surface of the wound (red arrow in Fig. 8c). In the negative control group, there were still a large number of neutrophils in wound tissues and many blood cells in the enlarged capillaries accompanying with the uncontrolled in ammation in the wounds after intervention for 14 days. The above results indicated that qCQDs were bene cial for the abatement of in ammation in the infected wound tissues and had similar therapeutic effect to levo oxacin in the wounds infected with mixed bacteria.
Biosafety evaluation of qCQDs.
At rst, the possible induced drug resistance of qCQDs to bacteria was evaluated to assess the biosafety ascribed to the serious and common problem of bacterial antibiotic resistance. Supplementary Fig. 13 showed that S. aureus, E. coli and MRSA did not develop resistance to qCQDs after induced resistance for 30 days at sub-MIC level, meaning that bacteria could not easily develop new or secondary resistance to qCQDs. It may be due to the fact that the qCQDs can play an antimicrobial role via new signaling pathways or effect targets that is different from the acting targets of traditional antibiotics. In vitro cytotoxicity of qCQDs was further investigated by MTT test based on the effects of qCQDs at different concentrations on macrophages and HepG2 cells at different times. As shown in Supplementary Fig. 14, when the concentration of qCQDs was 150 μg/mL, the survival rates of macrophages ( Supplementary  Fig. 14a) and HepG2 (Supplementary Fig. 14b) were above 80% within 6 to 12 hours, and 67.3% and 70.7% after exposed for 24 h, respectively. When the concentration reached 200 μg/mL, the survival rates of the two kinds of cells were somewhat reduced with 63.2% and 42.9% after exposed for 24 h. The above results showed that certain cytotoxic effects of qCQDs on eukaryotic cells would also occur in vitro when the concentration reached a high level. The reason might be the poor selectivity of qCQDs for target ascribed to the broad-spectrum antibacterial activity. In vitro hemolysis test was also conducted to verify whether qCQDs could cause erythrolysis. As can be seen from Supplementary Fig. 14c, when red blood cells were added to the sterilized water, the solution changed from colorless to red after centrifugation, meaning that red blood cells were ruptured due to different osmotic pressures inside and outside the cells. Red blood cells were added to normal saline containing different concentrations of qCQDs, and subjected to centrifugation. Red blood cells were deposited at the bottom of the centrifuge tube, and the solutions were still transparent and colorless. Even if the concentration of qCQDs reached 400 μg/mL, compared with the negative control, the hemolysis rate is only 1.9% (<5%), as shown in Supplementary   Fig. 14d, indicating that qCQDs would not cause red blood cells rupture and hemolysis in physiological conditions and has good blood compatibility.
The in vivo toxicity of qCQDs to the main organs (including heart, liver, spleen, lung and kidney) of rats was evaluated by pathomorphology, as shown in Supplementary Fig. 15. Rats in the experimental group were given with 4 mg/mL of qCQDs solution by continuous gavage at the dose of 5 mL/day for 7 days, and the control group was given with the same dose of sterilized normal saline by gavage. Within 7 days, the rats in the two groups showed normal activity, diet and mental state. H&E-stained pathological sections of heart, liver, spleen, lung and kidney showed no signi cant difference in the tissue structure between the two groups, meaning that qCQDs would not cause pathological damage to main organs of rats by intragastric administration for 7 days. It may be the reason that qCQDs does not generate toxicity and side effects on the main organs of the body, or qCQDs can be converted into non-toxic substances for the main organs in the body. Thus, it is preliminarily concluded that qCQDs may be a kind of carbon nanomaterial without detectable in vivo toxicity for in vivo antibacterial applications.

Discussion
The qCQDs with the particle size of 3~4.5 nm showed the broad-spectrum antibacterial activity against gram-positive and gram-negative bacteria. It was con rmed by TEM that qCQDs could destroy the integrity of bacterial structure, make the substances inside the cell agglomerate and leak, and result in the death of bacteria. It is well known that the generation of ROS is the main combating mechanism of antibacterial nanomaterials with broad-spectrum antibacterial activity 47 . According to the results of ROS detection, we could clearly know that qCQDs did not generate ROS in the process of interaction with bacteria. Proteomics is an attractive ~omics technique that allows the identi cation of secreted active compounds, the selection of drug-targets and the elucidation of action mechanisms of drug candidates 48 . According to the comprehensive analysis of the quantitative proteomics, we further con rmed that qCQDs had different targets and pathways for generating antibacterial activity against S. aureus (gram-positive) and E. coli (gram-negative). The qCQDs mainly acted on ribosomal proteins in gram-positive bacteria to interfere with and disrupt the synthesis of bacterial protein, thus playing an antibacterial effects (Fig. 9a). For gram-negative bacteria, qCQDs mainly interfered with its cellular respiration by down-regulating the essential proteins in citrate cycle to further disrupt multiple relevant metabolic pathways in bacterial cells, thus producing antibacterial effects (Fig. 9b). RT-qPCR successfully veri ed the gene expression of six representative differentially expressed proteins screened from S. aureus and E. coli by interaction analysis of proteins and GSEA, respectively. Therefore, it is further con rmed that qCQDs against gram-positive and gram-negative bacteria had different target pathways and different antibacterial mechanisms to produce irreversible damage to bacteria and make bacteria rupture or lyse, leading to the death of bacteria. The antibacterial mechanisms of qCQDs on bacteria is obviously different from the common mechanism of ROS generation, electrostatic interaction, surface adsorption or peroxidase-like activity of antibacterial agents reported in the literature 23, 30, 33, 42, 49-51 .
Compared with the reported representative antibacterial carbon nanomaterials (Supplementary Table 6) 6, 24, 30, 33, 42, 52-55 , the mechanism of antibacterial action of the as-synthesized CQDs in this work provided a new way of thinking for studying the antibacterial mechanism of CQDs with broad-spectrum antibacterial activities. This study provides a feasible basis for the research and development of new antibiotics and the application of CQDs in the eld of antibiotics. In addition, the application of quantitative proteomics combined with RT-qPCR in this study provides a feasible strategy for studying the antibacterial mechanism of antimicrobial CQDs at molecular level. The proved pathway of qCQDs on bacteria may also be extended as the new medicinal targets for the development of bactericidal agents.
In this work, biocompatible qCQDs functionalized by quaternary ammonium group without detectable resistance evolution was synthesized with DDA and glucose by a simple "one-pot" method. According to the analysis of quantitative proteomics, it was found that qCQDs exerted the antibacterial effect against gram-positive and gram-negative bacteria through different targets. The qCQDs exhibited effective therapeutic effects on the wounds infected with mixed bacteria in SD rats, and signi cantly reduced the death of rats caused by infection. This work provides a new feasible way to prepare carbon quantum dots with broad-spectrum antibacterial activity and study the antibacterial mechanism of carbon quantum dots on bacteria, and expends a new development direction for the preparation of novel carbon quantum dots with low risk of AMR evolution.

Synthesis of qCQDs.
Glucose and DDA were used as the reaction precursor to synthesize qCQDs. At rst, glucose (0.5 g) was placed and heated at 150 ℃ for 20 min in a 50 mL of round-bottomed ask, and then 1 mL of DDA was added into the ask and continually stirred for 150 min at 150 ℃. After that, it was cooled to room temperature to get the dark brown solid substance. After the addition of 10 mL of deionized water dissolved the solid substance, the solution was ltered with needle cartridge lter, and then was dialyzed in a cellulose dialysis bag with deionized water for 24 h. After dialysis, the solid qCQDs were obtained via freeze-drying. A stock solution of qCQDs with accurately controlled concentration by deionized water was prepared for the subsequent experiments.
In vitro antibacterial activity test of qCQDs.
Bacterial culture and preparation of bacterial suspension were shown in Supplementary Information S1.1.
Disk diffusion test: the bacterial suspension with a concentration of 1.5×10 8 CFU/mL was smeared evenly with sterile medical swabs on the MH agar medium, and then the disks containing the tested substances (including qCQDs (a), glucose (b) and DDA (c)) were placed onto the above medium. The diameter of MH agar medium was 90 mm. The center distance of all the disks was at least 24 mm. After incubation at 35 ℃ for 18 h, the diameter of inhibition zone around each disk was measured. Each disk diffusion test was carried out three times independently.
Broth dilution test: thirteen sterile test tubes (12×75 mm) were placed in a row and numbered successively, 1.6 mL of MH broth was added into Tube No.  g in the experiment. A total of 18 SD rats were randomly divided into three groups, i.e. experimental group (treated with qCQDs), positive control group (treated with levo oxacin), and negative control group (treated with sterilized saline). After the SD rats were anesthetized with 10% chloral hydrate solution (3 mL/kg) by intraperitoneal injection, the dorsal hair of the SD rats was shaved with a hair clipper and a nummular full-thickness skin was resected with a scalpel [15]. The diameter of all the wound was about 1.8 cm, about the size of a dime, as shown in Supplementary Fig. 16. The operation was performed under aseptic conditions before the infection. The sterile wound dressing added with 2 mL of bacterial suspension was adhered to the nummular wound and was fastened with medical reticular elastic bandage. The bacterial suspension contained S. aureus and P. aeruginosa, both of which had concentrations of 1.5×10 8 CFU/mL. Then, all the experimental SD rats were separately raised in cages at a standardized temperature. After 48 h, the nummular wounds were infected with S. aureus and P. aeruginosa and the wound dressing with bacteria was discarded, obtaining the wound model infected with mixed bacteria. The wounds were observed and photographed before and after infection.
Treatment of wounds infected with mixed bacteria.
The infected wounds were treated with local application in this work. Experimental group and positive control group were treated with sterile wound dressing containing 2 mL of qCQDs solution (2.5 mg/mL) and the same volume of levo oxacin (0.25 mg/mL), respectively. Meanwhile, the negative control group was treated with the sterile wound dressing containing 2 mL of sterilized normal saline. The wound dressings were changed once a day in each group in the period of 14 days. On the 3 rd , 5 th , 7 th , 10 th and day, all the experimental rats were sacri ced by euthanasia, and the tissues of wounds were harvested for bacterial culture of wound tissue and H&E staining of histopathological sections. Within 14 days of treatment, the weight of rats in each group and the number of rats that died from infection were recorded, and the weight change curve and survival curve were plotted, respectively.
In vitro cytotoxicity test of qCQDs.
Mouse mononuclear macrophages (macrophages, RAW264.7) and human hepatoma cells (HepG2 cells) were selected to evaluate the toxicity of qCQDs by MTT assay. The 300 µL of cell suspension (2×10 4 cells/mL) was added into 96-well plates, and made the cell counts about 6000 cells/well. After incubation at 37 ℃ for 24 h, the cells stuck to wall and the culture medium was discarded. The 300 µL of culture medium containing different concentration of qCQDs were added into the preset wells, and the concentrations of qCQDs were 25, 50, 100, 150 and 200 µg/mL, respectively. After incubation at 37 ℃ for 6, 12 and 24 h, respectively, the culture medium was discarded, and 30 µL of 0.5% MTT solution and 70 µL of serum-free culture medium were added to each well, and then incubated at 37 ℃ for 4 h. After that, the MTT solution in each well was discarded and 300 µL of DMSO was added. The absorbance of each well was measured by a microplate analyzer at a wavelength of 490 nm after the 96-well plates were shaken at a low speed for 15 min on the oscillator. According to the measured absorbance value, the survival rate of cells was calculated.
Hemolysis test of qCQDs.
Human blood samples for hemolysis test were obtained from First A liated Hospital of Fujian Medical University with the consent of healthy volunteers. Firstly, a certain volume of blood was centrifuged at 1500 rpm for 15 min to collect red blood cells. And then, the red blood cells were cleaned with normal saline three time with the assist of centrifugation. The 50 µL of erythrocyte suspension was dispersed in 1 mL of normal saline containing different concentration of qCQDs, and the concentrations of qCQDs were 5, 10, 50, 100, 200 and 400 µg/mL, respectively. After incubation at 37 ℃ for 3 h, the erythrocyte suspension was centrifuged at 12000 rpm for 15 min. The absorbance of the supernatants at 540 nm was measured, and the hemolysis rate was calculated. Normal saline and sterilized water were used as negative and positive control, respectively.
Preparation of bacteria samples for quantitative proteomics and RT-qPCR.
S. aureus and E. coli as models were used for the study of quantitative proteomics and RT-qPCR. After  (H1, H2, H3). After incubation at 37 ℃ for 12 h, the above bacterial solutions were centrifuged at 5000 rpm at 4 ℃ for 5 min, and the bacterial precipitations were collected. After cleaning with pre-cooled PBS three times, the bacterial precipitates were stored at -80 ℃ when not in use.
Detection of quantitative proteomics labeled with Tandem Mass Tags (TMT) was detailed in Supplementary Information S1.6. Real-time quantitative PCR (RT-qPCR) of bacteria treated with qCQDs was detailed in Supplementary Information S1.7, and primer sequences for RT-qPCR were listed in Supplementary Table 7        Antibacterial activity on MHA plates. qCQDs (a), glucose (b) and DDA (c). Six species of bacteria is S. aureus, MRSA, S. epidermidis, E. faecalis, E. coli and P. aeruginosa, respectively. The histogram is derived from the diameter of the inhibition zone around disk a.

Figure 3
Antibacterial activity on MHA plates. qCQDs (a), glucose (b) and DDA (c). Six species of bacteria is S. aureus, MRSA, S. epidermidis, E. faecalis, E. coli and P. aeruginosa, respectively. The histogram is derived from the diameter of the inhibition zone around disk a.    The circles represent ribosomal proteins and citrate cycle related proteins, and the diamonds represent other proteins. Red indicates up-regulated expression, while green indicates down-regulated expression. Ribosomal proteins (c) and citrate cycle related proteins (d) in clustering heat map were detailed in Supplementary Table 2 and 4, respectively.

Figure 5
Page 35/46 Differentially expressed proteins associated with ribosome and citrate cycle. KEGG chord diagram between differentially expressed proteins and enriched pathway in S. aureus (a) and E. coli (b), (more information was described in Supporting Information); Clustering heat map of differentially expressed proteins associated with ribosome in S. aureus (c) and citrate cycle in E. coli (d). Protein-protein interaction network of differentially expressed proteins associated with ribosome (e) and citrate cycle (f).
The circles represent ribosomal proteins and citrate cycle related proteins, and the diamonds represent other proteins. Red indicates up-regulated expression, while green indicates down-regulated expression. Ribosomal proteins (c) and citrate cycle related proteins (d) in clustering heat map were detailed in Supplementary Table 2 and 4, respectively.