Zinc Imidazole Framework‐8 Nanoparticles Disperse MRSA Biofilm by Inhibiting Arginine Biosynthesis and Down‐Regulating Adhesion‐Related Proteins

Nanomaterials, including ZIF‐8 nanoparticles (NPs), are shown to be effective antimicrobial agents against Methicillin‐resistant Staphylococcus aureus(MRSA). However, the antibiofilm properties and mechanisms of ZIF‐8 NPs remain uncertain. In this study, ZIF‐8 NPs are prepared using the room temperature solution reaction method and characterized. Biofilm formation inhibition test and biofilm eradication test are performed and the results show that ZIF‐8 NPs can inhibit the formation of MRSA biofilm as well as disperse established MRSA biofilm. Proteomics and real‐time fluorescence quantitative polymerase chain reaction (PCR) are conducted to prove that ZIF‐8 NPs reduce the expression of adhesion‐related proteins, namely the fibronectin‐binding proteins A and B (fnbA/fnbB), fibrinogen binding protein caking factors A and B (clfA/clfB), elastin binding protein (ebps), and fibrin binding protein (eno). ZIF‐8 NPs also inhibit the arginine biosynthesis pathway by affecting the activities of argininosuccinate lyase, ornithine carbamyl transferase, Glutamate dehydrogenase, carbamate kinase, and arginine deiminase. A conclusion can be drawn from the above results that ZIF‐8 NPs can inhibit bacterial adhesion and kill bacteria directly, ultimately destroying MRSA biofilm. This study provides a molecular basis for the treatment of MRSA biofilm with ZIF‐8 NPs.


Introduction
[3] MRSA can live not only in planktonic form but also in a consortium of different or the same species, called biofilm. [4,5]Biofilm is a structure formed of bacteria and biofilm matrix, which is less sensitive to antibiotics than planktic bacteria due to their tolerance and resistance mechanisms. [6]he biofilm development of MRSA can be described as five stages: attachment, multiplication, exodus, maturation, and dispersal. [7]During the attachment stage, MRSA can be attached to inorganic surfaces through electrostatic and hydrophobic interactions, [8] while it attaches to the host cell by cell wall-anchored proteins (CWA proteins) and secreted proteins. [9]o survive in harsh environments, MRSA produces an extracellular matrix (ECM) composed of proteins, carbohydrates, and/or extracellular DNA (eDNA), [10] forming a thick matrix to block outside damage.These stages, together with the survival of individual bacteria, affect biofilm formation and dispersion.
It has been proved that one of the important causes of biofilmassociated infection is biofilm colonization on the implant surface. [11]Staphylococcus aureus biofilms have been found to colonize various human implant surfaces, such as pacemaker lead, [12] titanium locking plates, [13] biomaterials commonly used in dentistry (titanium alloy, zirconium alloy, zirconia, and cobaltchromium alloy). [14,15]Coating the implant surface with antibiotics is a common way to prevent Staphylococcus aureus from attaching, but this approach carries the risk of screening for drugresistant bacteria, a case in point is MRSA. [16]Therefore, we need to take a different approach to deal with the risk of Staphylococcus aureus and MRSA biofilm-associated infections, and antibacterial nanomaterials are potential agents.
At present, antimicrobial and anti-biofilm composite nanomaterials are important roles to fight against bacterial resistance as many research results have been achieved.[19][20][21][22] The mechanism of antimicrobial nanomaterials enables them to eliminate resistant pathogenic microorganisms in environmental and healthcare applications, while down regulating their drug resistance genes and avoiding targeted resistance of bacteria, which is of great significance for controlling nosocomial infections and protecting public health. [23]26][27] MOFs materials can also be used as capacitors, sensors, adsorption materials, catalytic materials, drug slow-release carriers, etc. [28] Zinc imidazole framework-8 nanoparticles (ZIF-8 NPs) belongs to the MOFs' family, with 3D dodecahedral structure, can be synthesized with zinc ions and 2-methylimidazole.It has been discovered that ZIF-8 NPs has many application, such as antibacterial agent, nano-drug delivery system, [29,30] electrochemical sensor, [31] and adsorption agent. [32]In previous studies, we have successfully synthesized ZIF-8 NPs and demonstrated that it has antibacterial effect as well as can promote the healing of infected wounds, and we analyzed its mechanism utilizing transcriptomics and metabolomics. [33]][36][37][38] However, no studies on the anti-biofilm mechanism of ZIF-8 NPs as an independent anti-biofilm agent have been reported so far.
Therefore, in this study, we further investigated the anti-MRSA biofilm performance of ZIF-8 NPs.The anti-biofilm effect of ZIF-8 NPs, namely MRSA biofilm formation hindering and MRSA biofilm dispersing effect was assessed.Using proteomic methods, we tested and analyzed the differential proteins of ZIF-8 NPs treated MRSA biofilm, to elucidate the molecular mechanism of ZIF-8 NPs against biofilm formation.Our proteomic results suggest that ZIF-8 NPs may disrupt bacterial biofilms by inhibiting the arginine biosynthesis pathway and reducing the expression of adhesion-related proteins.

Preparation and Characterization of ZIF-8 NPs
Using the room-temperature solution reaction method, we successfully synthesized ZIF-8 NPs. [39]SEM and TEM images showed that the ZIF-8 synthesized in this study has a dodecahedral structure with a diameter of ≈66.58 nm (Figure 1a,b).The average hydrated particle size was 113.4 nm, with the Polydispersity index (PdI) of 0.141 (Figure S1, Supporting Information).Due to the agglomeration of ZIF-8 NPs in water, their average hydrated particle size was larger than the size measured in TEM images.Zeta Potential of ZIF-8 NPs was 26.6 mV, which means the ZIF-8 NPs are more likely to attach to the surface of bacteria carrying negative charge (Figure S2, Supporting Information).The crystallinity of the material was determined by XRD, and it was confirmed that the synthesized substance was ZIF-8 and the specific compound name was catena-(bis(μ 2 -2-Methylimidazolato-N, N')-zinc(ii) (Figure 1c).C, N, O, and Zn signals could be observed in the XPS result, confirming the successful synthesis of ZIF-8 NPs (Figure 1d).The FTIR spectrum (Figure 1e) shows crystallization peaks, which are consistent with previous studies. [39]

ZIF-8 Hinders MRSA Biofilm Formation
Our study has proved that MRSA could form a stable bacterial biofilm at 24 h (Figure S3, Supporting Information).Therefore, in the following study, we chose 24 h as incubation time for the MRSA biofilm formation.The results of the Crystal Violet staining solution showed that compared to the control group, MRSA biofilm was significantly reduced in the ZIF-8 NPs treatment group (Figure 2a).The formation of biofilm was evaluated after crystal violet was dissolved.When the dose of ZIF-8 NPs is 200 μg mL −1 , it can inhibit biofilm formation by >80% (Figure 2b).
At the same time, the MRSA biofilm was observed by a laser confocal microscope, and a 3D image of the biofilm was obtained.A strong green fluorescence signal can be seen in the control group, indicating that MRSA forms a biofilm (Figure 2c).The higher the concentration of the ZIF-8 NPs treatment group, the weaker the green fluorescence, indicating that the formation of MRSA biofilm is hindered, and the correlation is dosedependent.The fluorescence intensity of MRSA biofilm was analyzed (Figure 2d).After 25 μg mL −1 ZIF-8 NPs treatment, the relative activity of MRSA biofilm was weakened, and decreased by 18% compared with the control group, with an extremely significant difference (p < 0.01).When the concentration of ZIF-8 NPs was higher than 25 μg mL −1 , the fluorescence intensity decreased by >38%, and the difference was extremely significant (p < 0.001).In conclusion, ZIF-8 NPs with a concentration > 50 μg mL −1 can significantly inhibit the formation of MRSA biofilm.
Additionally, the effect of ZIF-8 NPs on the formation of MRSA biofilm was observed by scanning electron microscopy.The biofilm structure of the control group was complete and compact, with clear bacterial morphology and complete structure, no obvious abnormal changes were observed (Figure 2e,f).After treatment with ZIF-8 NPs, the bacteria in the biofilm were scattered, along with cell membranes breaking and necrosis (Figure 2g,h).Some bacterial morphology was relatively complete.

ZIF-8 Disperses Established MRSA Biofilms
Extracellular polymeric substance (EPS) is a vital component of the biofilm matrix, it helps the bacteria to survive against harsh environmental conditions. [40]Due to the protective effect of EPS in bacteria biofilms, complete removal of established biofilms is often more difficult than suppression of bacteria isolates, which poses a challenge for the treatment of biofilm-associated infections.Previously, we confirmed that ZIF-8 NPs significantly inhibited the formation of MRSA biofilm, and we further explored the eradication ability of ZIF-8 NPs on MRSA biofilm.To analyze the eradication ability of ZIF-8 NPs on MRSA biofilm, the crystal violet staining method was first used for detection.MRSA biofilm without ZIF-8 NPs treatment turned purple after staining, while in the ZIF-8 NPs treatment group, Except for 25 μg mL −1 of ZIF-8 NPs, the crystal violet color of the other concentrations of ZIF-8 NPs became lighter and showed a significant dose-dependent relationship (Figure 3a).The results showed that a high concentration of ZIF-8 NPs had an obvious destructive ability to MRSA biofilm.Quantitative analysis of crystal violet showed that compared with the control group when the concentration of ZIF-8 NPs reached 100 μg mL −1 , the inhibition rate of bacterial biofilm reached 63.62% (Figure 3b).In conclusion, when the concentration of ZIF-8 NPs reaches 100 μg mL −1 , it has a significant eradication effect on MRSA biofilm.
To more directly observe the eradication effect of ZIF-8 NPs on MRSA biofilm, a laser confocal microscope 3D imaging system was used.A large area of green fluorescence was observed in the control group, while in the ZIF-8 NPs treatment group, the higher the concentration, the weaker the bacterial fluorescence intensity, indicating that the MRSA biofilm content was reduced in a dose-dependent manner (Figure 3c).The fluorescence intensity of MRSA biofilm was analyzed (Figure 3d).After 50 μg mL −1 ZIF-8 NPs treatment, the relative activity of MRSA biofilm decreased by ≈43% compared with the control group, with a highly significant difference (p < 0.001).When the concentration of ZIF-8 NPs was 100 and 200 μg mL −1 , the relative fluorescence intensity of MRSA biofilm was reduced respectively by ≈63% and 84%.These results indicate that ZIF-8 NPs can effectively eradicate MRSA biofilm.
The morphology of MRSA biofilm treated with ZIF-8 NPs was observed by scanning electron microscope.The MRSA biofilm in the control group had a complete and compact structure, clear bacterial morphology, and complete structure, and a large number of EPS could be seen among the bacteria (Figure 3e,f).After treatment of MRSA biofilm with ZIF-8 NPs, bacterial biofilm was damaged, only part of the biofilm remained, and some bacteria on the biofilm were deformed and necrotic (Figure 3g,h).These results indicate that ZIF-8 NPs can significantly eradicate MRSA biofilm.
In the previous study, we confirmed that ZIF-8 NPs have good bactericidal effects on MRSA, but it is unclear whether they also have eradication effects on MRSA biofilms. [27]At present, there is no literature reporting the anti-biofilm properties of ZIF-8 NPs, and ZIF-8 NPs only function as a carrier.Peng et al. developed ZIF-8 NPs coated mesoporous dopamine core-shell nanoparticles, loaded with PES, and formed MPDA@ZIF-8 /PES has good antibacterial effects and treats bacterial biofilm infections. [41]Our results confirm that ZIF-8 NPs have an eradication effect on MRSA biofilm and can inhibit the formation of MRSA biofilm.Therefore, ZIF-8 NPs play an important role in antibacterial drug species, as well as serving as carriers themselves.

Proteomics Analysis of ZIF-8 NPs Anti-MRSA Biofilm
The previous experimental results have confirmed that ZIF-8 NPs can damage the MRSA biofilm.To further understand the specific mechanism of ZIF-8 NPs damaging the MRSA biofilm, proteomics was used for research and analysis.The experiment set up a control group and a ZIF-8 NPs treatment group.
Correlation analysis and PCA analysis were conducted on the samples based on the expression of proteins between different samples to evaluate the differences between the groups (Figures S4 and S5, Supporting Information).
According to the analysis of the detection results, a total of 22 791 peptides and 2672 proteins were identified.The detected proteins in the two groups were screened utilizing Student ttest, combined with Fold change, FC, and the ratio of average expression between the two groups.Both p-value<0.05and FC >1.2 or <1/1.2 are considered differential proteins.According to this screening method, 383 differential proteins were screened (Figures S6-S8, Supporting Information).The horizontal coordinate was the logarithmic transformation of FC, and the vertical coordinate was the negative logarithmic transformation of the pvalue.Each point represented a protein, and the red dot represented a significantly high-expression protein.Green dots are a significantly low expression of protein; The gray dots are nondifferentiated proteins, of which 208 proteins are up-regulated and 175 proteins are down-regulated.
GO analysis was performed on differential proteins, the top 10 significant enrichment items of differential proteins under the three branches of biological process (BP), molecular function (MF), and cellular component (CC), respectively (Figure 4a).The horizontal axis represents the negative logarithmic transformation of p-value enrichment significance, while the vertical axis represents the GO term.Each circle represents a term, and the size of the circle represents the count of differential proteins.The three major branches are represented in different colors.In biological processes, differential proteins are mainly concentrated in the processes of ornithine metabolism, cellular amino acid decomposition, and organic nitrogen compound decomposition; In terms of molecular function, differential protein enrichment mainly occurs in metal ion binding, ATPase coupled cationic transmembrane transport protein activity, and ornithine carbamyltransferase activity.
We conducted KEGG analysis to gain a more systematic and comprehensive understanding of the impact of ZIF-8 NPs on the biological processes, disease mechanisms, drug action mechanisms, and other most direct and necessary pathways of MRSA biofilm.The signal pathways that downregulate the significance of differential protein enrichment, including arginine biosynthesis, alanine, Asparagus cochinchinensis and glutamate metabolism, Amino sugar and nucleotide sugar metabolism Phosphotransferase system (PTS), and nitrogen metabolism (Figure 4b).The signal pathways that regulate the significance of differential protein enrichment, include Staphylococcus aureus infection, Glycolysis/Gluconeogenesis, and RNA degradation (Figure 4c).The KEGG analysis results indicate that arginine biosynthesis, phosphotransferase system, and nitrogen metabolism signaling pathways are likely involved in the process of ZIF-8 NPs disrupting MRSA biofilm.

ZIF-8 NPs Affect Arginine Biosynthesis, QS Signaling, and Adhesion in MRSA Biofilm
It can be seen from the KEGG analysis results of proteomics that the arginine biosynthesis signal pathway is significantly inhibited, including argininosuccinate lyase, ornithine carbamoyltransferase, Glutamate dehydrogenase, carbamate kinase The activity of these five enzymes, arginine deiminase, was significantly inhibited (Figure 5a).The above results indicate that ZIF-8 NPs may play an anti-MRSA effect by targeting to inhibit the main protein activities of arginine biosynthesis (argininosuccinate lyase, ornithine carbamyl transferase, Glutamate dehydro-genase, carbamate kinase, arginine deiminase), thus playing an anti-MRSA biofilm effect.
To further demonstrate that ZIF-8 NPs disrupt bacterial biofilm by affecting the activity of five MRSA enzymes, we detected the gene expression levels of these five proteins through real-time fluorescence quantitative PCR.All qPCR primer sequences employed in this study have been shown in Table S1 (Supporting Information).ArcA, arcB, arcC, argH, and gudB are argininosuccinate lyase, ornithine carbamyltransferase, Glutamate dehydrogenase, carbamate kinase, and arginine deiminase genes, respectively.The mRNA expression of arcA, arcA, arcA, argH, and gudB in the ZIF-8 NPs group was significantly downregulated compared with the control group (Figure 5b).The results above indicate that ZIF-8 NPs can disrupt the formation of biofilms by inhibiting the activity of key enzymes involved in arginine biosynthesis.
Staphylococcus aureus can express a variety of microbial surfactants that recognize adhesion matrix molecules, such as fibronectin binding proteins a and B (fnbA/fnbB), fibrinogen binding protein caking factors A and B (clfA/clfB), biofilm-related proteins (bap), elastic binding proteins (ebps), collagen binding proteins (can), laminin-binding proteins (eno), fibrinogen binding proteins (fib), etc. [40,42] The results of proteomics showed that the fibronectin-binding proteins a and B (fnbA/fnbB), fibrinogen binding protein caking factors A and B (clfA/clfB), elastin binding protein (ebps), and fibrin binding protein (eno) of the biofilm in the ZIF-8 NPs treatment group were significantly lower than those in the control group (Figure 5c).The results indicate that ZIF-8 NPs affect the adhesion of biofilms by inhibiting the expression of adhesion matrix molecules.
The mRNA changes of biofilm adhesion matrix molecules were detected by fluorescence quantitative PCR.Compared with the control group, the expressions of clfA, clfB, ebps, eno, fnbA, and fnbB were down-regulated, and the differences were significant (Figure 5d).These results indicate that ZIF-8 NPs can inhibit the expression of biofilm adhesion matrix molecular gene, and then inhibit the expression of its protein, leading to the damage of biofilm matrix.

Conclusion
[45] ZIF-8 is used as a common carrier of composite nanomaterials to treat bacteria and biofilms in research studies.The researches on the antibiofilm mechanism of composite nanomaterials containing ZIF-8 is usually based on the loaded material or drug, and ZIF-8 itself is rarely studied as an independent antibiofilm drug.Previous studies have shown that ZIF-8 NPs can inhibit biofilm formation, but no studies have explored the mechanism of the antibacterial biofilm effect of ZIF-8 NPs. [46,47]t has been shown that arginine is an essential amino acid for bacteria and its consumption directly leads to bacterial death. [48]e have demonstrated that ZIF-8 can induce the death of plankton bacteria by inhibiting arginine biosynthesis in previous studies, in which we discussed how arginine biosynthesis inhibition could cause ROS generation. [33]Arginine is an essential amino acid for humans and animals, can catalyze the ornithine cycle, promote the formation of urea, and make ammonia in the body into non-toxic urea. [49]The synthesis of arginine begins with Lglutamic acid.The precursors involved include glutamine, proline, and ornithine, and the key enzymes are glutamine transpeptidase, arginase, arginine succinate synthetase, and arginine succinate lyase. [50]Our results mentioned above indicated that arginine biosynthesis inhibition also plays an essential part in the anti-biofilm mechanisms of ZIF-8 NPs.
In addition, the enrichment of CWA proteins in the differential protein KEGG signaling pathway was significantly reduced.CWA proteins are critical for MRSA to attach to and multiply on injured tissue, the most common of which is the MSCRAMMs family. [51]In many cases, MSCRAMMs are covalently anchored to cell wall Peptidoglycan.Fibronectin binding proteins A and B (fnbpA/fnbpB), fibrinogen binding protein caking factors A and B (clfA/clfB), elastic binding proteins (Ebps), and fibrin binding proteins (eno) all belong to this family.Proteomics results in the present study showed that fibronectin-binding proteins A and B (fnbA/fnbB), fibrinogen binding protein caking factors A and B (clfA/clfB), elastic binding proteins (Ebps), and fibroin binding proteins (eno) were significantly down-regulated.Therefore, it can be concluded that ZIF-8 NPs can induce the down-regulation of MSCRAMMs-related protein expression in biofilms, thereby inhibiting bacterial adhesion and ultimately damaging the biofilm.These novel anti-biofilm mechanisms of ZIF-8 NPs can provide new ideas for exploring synthetic anti-biofilm materials using ZIF-8 NPs as a carrier or component in the future.
In conclusion, the ZIF-8 NPs synthesized in this study can effectively inhibit the formation of MRSA biofilm and eradicate the established biofilm.ZIF-8 NPs reduced the expression of adhesion-related proteins (fnbA, fnbB, clfA, clfB, ebps, eno) as well as inhibited the synthesis of five enzymes related to arginine biosynthesis pathway (argininosuccinate lyase, ornithine carbamyl transferase, Glutamate dehydrogenase, carbamate kinase, arginine deiminase).To summarize, the anti-biofilm effect of ZIF-8 NPs is reflected in restraining bacterial adhesion to damage the biofilm matrix component and blocking the arginine biosynthesis pathway to kill bacteria directly.

Experimental Section
Materials: Zinc nitrate (Zn (NO 3 ) 2 •6H 2 O) was obtained from the Tianjin Chemical Reagent 3 Plant Co., Ltd.(Tianjin, China).2methylimidazolate was supplied by Beijing J&K Chemicals Co., Ltd.(Beijing, China).The LIVE/DEAD Bac Light Bacterial Viability Kit was purchased from Invitrogen (Waltham, USA).The MRSA ATCC 33 591 isolates were gifted by the College of Veterinary, China Agricultural University.
Synthesis of ZIF-8 NPs: ZIF-8 NPs was prepared by room temperature solution reaction method.The specific synthesis method can be summarized as follows: weighed 0.325 g of 2-methylimidazole and dissolved in 10 mL methanol (solution A), then weighed 0.147 g of zinc nitrate and dissolved in 10 mL methanol (solution B).Slowly added solution A into solution B, stirred at 500 RPM for 1 h at 25 °C, then the reaction product contains ZIF -8 NPs was obtained.The reaction product was centrifuged for 10 min (rotational speed was 10 000 RPM, the precipitation was dissolved with methanol for ultrasonic treatment (2 min) until completely dissolved.Repeat the above centrifugation and ultrasonic dissolution process twice, and the product of the third methanol dissolution was the methanol solution of ZIF-8 NPs.
Preparation of ZIF-8 NPs for Biological Assays: Centrifuged at 10 000 revolutions at 4 °C for 5 min, discarded the supernatant, and replaced it with the required volume of pure water.The ZIF-8 aqueous solution was sonicated for 10 min to disperse completely.
Bacterial Cultivation: MRSA isolates were cultured on LB AGAR medium.Individual bacterial colonies were inoculated in brain-heart infusion (BHI) medium and cultured at 37 °C(200 rpm) to OD600 of 0.6.After the culture was centrifuged, the suspensions were washed three times with PBS and diluted to 10 6 CFU mL −1 for use.
Measurement of MRSA Biofilm Growth over Time: After overnight cultivation of MRSA, adjusted the bacterial concentration to 10 6 CFU mL −1 and added 200 μL to the 96 well plate bacterial suspension, incubated at 37 °C for 4, 8, 12, 24, and 48 h, washed with PBS for three times, and then added with 150 μL 0.1% Crystal violet, dyed for 20 min, washed with PBS for three times, after naturaldrying, added 200 μL 33% acetic acid solution per hole, stood for 3 min, then an enzyme-linked immunosorbent assay was used to measure the absorbance value at 562 nm, with 3 replicates for each group.
ZIF-8 Hinders MRSA Biofilm Formation: ZIF-8 NPs solution (200, 100, 50, 25 μg mL −1 ) was used to resuspend bacteria with a CFU of 10 6 mL −1 , and 200 μL of bacterial suspension was added to a 96 well plate.After 24 h of constant temperature cultivation at 37 °C,biofilm was washed with PBS for 3 times and added 150 μL 0.1% Crystal violet, dyed for 20 min, washed with PBS for three times, after natural drying, added 200 μL 33% acetic acid solution per hole, stood for 3 min, then an enzyme-linked immunosorbent assay was used to measure the absorbance value at 562 nm, with three replicates for each group.
Characterization of ZIF-8 NPs: Transmission electron spectroscope (JEOL JEM-F200 (Japan)) and Scanning electron microscope (ZEISS Gem-iniSEM 300 (German))were used to investigate the morphology of ZIF-8.Diameters of ZIF-8 and zeta potentials of all the nanoparticles in water were measured by Malvern Zetasizer Nano ZS90 (UK).Powder Xray diffraction (XRD) patterns were recorded on D8 Advance X-ray diffractometer (Rigaku SmartLab SE (Japan)).X-ray photoelectron spectroscopy (XPS) was used to detect the molecular structure and valence state of ZIF-8 (Thermo Scientific K-Alpha (USA)).FTIR spectra were collected on a FTIR spectrometer (Thermo Scientific Nicolet iS20 (USA)).
ZIF-8 NPs Disperses Established MRSA Biofilms: Prepared bacterial suspension (10 6 CFU mL −1 ) and added in 96 well plates, then incubated at 37 °C for 24 h.After the formation of the biofilm, washed with PBS for three times, then added the ZIF-8 NPs solution in each well to make final concentration reach200, 100, 50, 25, 0 μg mL −1 in each group, co-incubated for 24 h at 37 °C.After co-incubation, washed with PBS for three times, then added 150 μL 0.1% Crystal violet, dyed for 20 min, washed with PBS for three times, after natural drying, added 200 μL 33% acetic acid solution to each hole, stood for 3 min, then used an enzyme-linked immunosorbent assay to measure the absorbance value at 562 nm, with three replicates for each group.
Laser Confocal Observation of MRSA Biofilm: The pretreatment method was the same as above (ZIF-8 Disperses Established MRSA Biofilms).The bactericidal effect of ZIF-8 NPs against E. coli biofilm was detected using the LIVE/DEAD Bac Light Bacterial Viability Kit (ThermoFisher, Shanghai, China).After pretreatment, green fluorescent dye (SYTO9) was added to the 96-well plate and dyed in darkness for 30 min.After three times of PBS washing, the biofilm was observed under a confocal laser scanning microscope with the same parameters.And obtain 3D images of bacterial biofilms.
Proteomics Study of ZIF-8 NPs anti-MRSA Biofilm: According to the method described in ZIF-8 NPs Hinders MRSA Biofilm Formation, after the MRSA biofilm was established, washed with PBS for three times, then a fresh culture medium containing ZIF-8 NPs was added to the 24 well plate.Continued to cultivate at 37 °C for 24 h, removed the culture medium and suspended bacteria, collected bacterial biofilm, ground the sample liquid hydrogen, then added 80% of the sample separately μL lysate was incubated at 95 °C for 3 min, broken by ultrasound for 2 min, and centrifuged for 20 min (4 °C, 16 000 g).The supernatant was taken and protein quantification was performed using the BCA method.After enzymatic hydrolysis of the sample, TMT peptide labeling and peptide grading were performed.
Fluorescence Quantitative PCR Detection: According to the results of Proteomics analysis, the arginine synthesis, LuxS, and adhesion-related genes were verified by RT qPCR.After bacteria extract RNA, reverse transcription was performed, with a reverse transcription procedure of 50 °C for 15 min; and 85 °C for 5 s.After completion of reverse transcription, stored cDNA at 4 °C for backup.The sequences of related genes and primers are shown in Table S1 (Supporting Information).
Statistical Analysis: All data were expressed as mean ± SD unless otherwise specified.One-way analysis of variance (ANOVA) and GraphPad Prism 9.0 Software (CA, USA) were used to further perform Tukey's multiple comparison post hoc tests.p < 0.05 was considered statistically significant.All experiments in this study were conducted at least three times.

Figure 1 .
Figure 1.a) The SEM image of ZIF-8 NPs.The average size of ZIF-8 measured with ImageJ is 66.58 nm (Five times of measurements, SD = 2.472).b) The TEM image of ZIF-8 NPs.c) The XRD diffraction pattern of ZIF-8 NPs.d) The XPS spectroscopy of ZIF-8 NPs.e) The FTIR spectrum of ZIF-8.

Figure 4 .
Figure 4. GO analysis and KEGG enrichment analysis of differential proteins.a) Bubble map of differential protein enrichment in the ZIF-8 NPs versus control.b) KEGG pathway enrichment diagram of down-regulated proteins in ZIF-8 versus control group.c) KEGG pathway enrichment diagram of up-regulated proteins in ZIF-8 versus control group.

Figure 5 .
Figure 5. ZIF-8 NPs affect arginine biosynthesis and adhesion of MRSA biofilm.a) Changes of proteins involved in the arginine biosynthesis in MRSA biofilm.b) Changes of proteins involved in the bacterial adhesion in MRSA biofilm post ZIF-8 NPs treatment.c) RT-qPCR determined the relative expression levels of fnbA, fnbB, clfA, clfB, ebps, and eno in MRSA biofilm.d) RT-qPCR determined the relative expression levels of arcA, arcB, arc, argH, and gudB in MRSA biofilm.Each group had three replicates.Error bars stand for standard errors.Statistics were achieved by a two-tailed t-test.Asterisks indicate values that are significantly different as follows: ns, not significant; *p < 0.05; **p < 0.01.