Targeting of Uropathogenic Escherichia coli papG gene using CRISPR-dot nanocomplex reduced virulence of UPEC

Urinary tract infections (UTI) are the most common infectious diseases in the world. It is becoming increasingly tough to treat because of emergence of antibiotic resistance. So, there is an exigency to develop novel anti-virulence therapeutics to combat multi-drug resistance pathogenic strains. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) discovery has revolutionized the gene editing technology for targeted approach. The greatest obstacle for CRISPR/Cas9 is cargo delivery systems and both viral and plasmid methods have disadvantages. Here, we report a highly efficient novel CRISPR based gene editing strategy, CRISPR-dots for targeting virulence factor Fimbrial Adhesion (papG gene), the bacterial adhesion molecule. Carbon quantum dots (CQD) were used as a delivery vehicle for Cas9 and gRNA into CFT073, a UPEC strain. CQDs were covalently conjugated to cas9 and papG-targeted guide RNA (gRNA) forming a nanocomplex CRISPR-dots (Cri-dots) as confirmed by DLS and transmission electron microscopy. Cri-dots-papG significantly targeted papG as demonstrated by decrease in the expression of papG.Further papG deficient UPEC had significantly reduced adherence ability and biofilm forming ability as demonstrated by fluorescence microscopy and scanning electron microscopy. Also, papG deficient UPEC had reduced virulence as shown by significantly increased survival of Caenorhabditis elegans (C. elegans) worms compared to UPEC. Our findings suggest that targeting of papG gene using Cri-dots nanocomplexes significantly reduced the pathogenicity of UPEC. Thus, Cri-dots nanocomplex offer a novel anti-bacterial strategy against multi-drug resistant UPEC.

www.nature.com/scientificreports/ in women 18 . The global emergence of antimicrobial resistance (AMR) in uropathogens during the last decade has complicated the UTI treatment 19,20 . Hence, there is an urgency to develop an alternate treatment strategy. Anti-virulence therapeutic strategies can selectively target uropathogens sparing the commensal bacteria 21,22 . CRISPR-Cas9 system has emerged as one of the foremost technology for programmable genome-engineering tool for targeting and manipulation of genomic sequences in pathogens and mammals [23][24][25] . CRISPR-Cas9 system was programmed to resensitize the bacteria by targeting AMR-encoding plasmids genes involved in biofilm formation and virulence [26][27][28] . However, the major hurdle for the application of CRISPR-Cas systems as antimicrobial agents is the lack of an effective and specific delivery method 29 . Although viral methods are the most common CRISPR/Cas9 delivery vehicle, but they have several drawbacks such as potential off-target effects and risk of immunogenicity 30 . Recently Nanocomplexes have been successfully used for delivery of CRISPR-Cas in both bacteria 31 and humans 32 .
In this study, we have exploited carbon quantum dots (CQDs) as a potential delivery vehicle for CRISPR-Cas gene editing system in UPEC. CQDs are nanoparticles with a size less than 10 nm and have special optical properties due to quantum confinement 33 . Because of its excellent biocompatibility, CQDs have emerged as a potential candidate for theranostic application 34 . Also, carbon dots are proved to be potential alternative for fluorescencebased cell-labelling assays as they are non-toxic and cell internalization occurs quickly 35 . We applied the Cri-dots editing strategy to target virulence factor, papG in UPEC, important for adhesion to host. The establishment and application of Cri-dots editing strategy would improve the genetic engineering in bacteria and provide insights for the development of anti-virulence strategy in other bacterial pathogens.

Results
Carbon quantum dots (CQDs) were not cytotoxic to human cell lines. Quantum dots have gained a lot of interest in nano-theranostics applications such as sensors, drug delivery and biomedical imaging. For therapeutics purpose, CQDs should be biocompatible and show low toxicity 36 . MTT proliferation assays were performed to evaluate the cytotoxicity of the CQDs on HeLa and THP-1 cells. The viability of both HeLa and THP-1 cells remained above 95% at CQD concentrations, ranging from 12.5 to 200 μg/ml post 24 h-and 48 h-of treatment ( Fig. 1a,b). The results showed that CQDs have no considerable cytotoxicity in the human cells lines used.
CQDs display no antibacterial properties and no haemolytic ability. CQDs toxicity was evaluated on E. coli. i.e., CFT073 (uropathogenic strain) and K12 (non-pathogenic strain) by performing MTT assay. CQDs did not show any observable effect on the viability of bacteria. The cell viability of CFT073 and K12 bacteria remained above 98% after 24 h and 48 h of treatment, with CQD concentrations ranging from 12.5 to 200 μg/ml (Fig. 1c,d). Agar well diffusion method is used to evaluate the antimicrobial activity. Supplementary  Fig. S1a and S1b shows the photographic images of agar plates with bored wells inoculated with CFT073 and K12. Overall, the data indicated no inhibitory activity of CQDs against CFT073 and K12 strains.
Characterization of CQD and CQD-cas9 using TEM and DLS. The synthesis of CQD and CQDs-cas9 was analysed using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Size measurements were performed before and after conjugation to check the increase in the hydrodynamic diameter of CQD-cas9 complex. Average size distribution data of the TEM demonstrated that size of CQDs was 8.23 ± 2.41 nm and size of CQDs-cas9 was 88.01 ± 7.68 nm (Fig. 2a,b). Figure 2c,d represents frequency size distribution bar graph for corresponding TEM images. DLS showed an increase in size of CQDS (10.86 ± 1.05 nm) to CQD-cas9 (89.31 ± 15.16 nm) (Fig. 2e). DLS plot of CQD-cas9 sample showed another small peak at 330 ± 30.64 nm, which implies the presence of some aggregates within the sample. The polydispersity index (PDI) of CQD and CQDs-cas9 conjugate is calculated to be 0.3 and 0.6, respectively. The values of PDI suggest the higher monodisperse nature of CQDs and increase in PDI of Cri-dots qualifies the complex formation with varying disperse nature.
papG targeting reduced adherence of UPEC. P-fimbriae are fundamental for bacterial adherence to the uroepithelium through the Galα1-4Gal-restricting papG adhesin 37 . Therefore, a qualitative analysis of adherence potential of control and papG targeted CFT073 strain to HeLa cells was investigated with the help of light microscopy (Fig. 3). The rod-shaped CFT073 were observed adhered to HeLa cells in the typical diffuse manner, (see arrow, Fig. 3b). Whereas, significantly fewer bacteria were visibly adhered to HeLa cells, infected with Cridots-papG (Fig. 3c). This showed a perceptible reduction in adherent ability of bacteria after CRISPR targeting of papG gene. Also, we observed Cri-dots-papG CFT073 had no significant effect on the morphology of HeLa cells as compared to control CFT073 ( Supplementary Fig. S3). www.nature.com/scientificreports/ In addition, we quantified the adherence potential of control and Cri-dots-papG CFT073 strain to HeLa cells by flow cytometry. Total number of events acquired in 12µL of sample, for both unstained and Syto9 stained cells obtained after adherence to HeLa cells, were calculated. There was a significant decrease in the number of the Cri-dots-papG CFT073 as compared to control CFT073 (p < 0.001). The percentage of unstained Cri-dots-papG strain was 2.157 ± 0.2069 as compared to adhered control strain obtained, which was 98.20 ± 1.556 ( Fig. 3d-f). Similar results were obtained when bacterial cells were stained with SYTO9 ( Fig. 3g-i). A drastic reduction in adherence property of Cri-dots-papG CFT073 strain (100.1 ± 4.115% Cri-dots-papG vs 4.270 ± 0.2096% control) was observed. Supplementary Fig. S4 represents the flow cytometric gating strategy of the unstained CFT073. The results were consistent in T24 urinary bladder epithelial cell lines both checked by giemsa staining and flow cytometry showing more than 90% reduction in adherence ( Supplementary Fig. S5).
papG targeted CFT073 showed mannose sensitive haemagglutination (MSHA). The expression of the PapG II adhesin was compared in the CFT073, Cri-dots-papG, and K12.As established PapG II adhesin exhibit mannose-resistant hemagglutination (MRHA) while fimH adhesion show mannose-sensitive hemagglutination (MSHA) to human blood type OP1 17 . As expected CFT073 agglutinated human blood type OP1 both in presence and absence of mannose indicating MRHA whereas Cri-dots-papG did not agglutinate erythrocytes in presence of mannose which showed MSHA. These results were consistent with the K12 strains. The results confirmed the absence of papG in Cri-dots-papG targeted CFT073. In order to estimate the effect of mannose on www.nature.com/scientificreports/ adherence, we tested bacterial adherence to T24 cell line with untreated(-) and mannose pre-treated( +) Control and Cri-dots-papG targated strains (Fig. S6). In concurrence with our observations from the haemagglutination assay, Control CFT073 demonstrated adherance (64.61 ± 1.002 N%) in presence of 1% mannose aa compared in absence of mannose, but there was only a slight change in adherence of Cri-dots-papG (0.9610 ± 0.1068%) in presence and absence of mannose as adherence is already reduced to significant levels after papG targeting.
Cri-dots mediated editing of papG reduced the biofilm forming ability of CFT073. Expression of papG has been reported to be prevalent in strong biofilm producers 38,39 . We estimated whether Cri-dots mediated papG gene editing was affecting the biofilm forming ability of CFT073. A quantitative analysis of biofilm formation ability of the Cri-dots-papG CFT073 was performed by crystal violet assay (CV) (Fig. 5a). The observed CV value of Cri-dots-papG CFT073 was significantly lower as compared to control CFT073 (0.570 ± 0.019, Cridots-papG vs 1.072 ± 0.043, control) (p < 0.0001). These data showed that Cri-dots mediated editing of papG gene in CFT073 significantly reduced their biofilm forming ability. Moreover, there was no effect seen on biofilm forming ability of K12 treated with Cri-dots-papG as K12 does not have papG gene ( Supplementary Fig. S6). This further proves the specificity of cri-dots-papG. Quantitative measurements of CV assay were further supported by florescence microscopy (Fig. 5c,d). The difference in intensity, because of SYTO9 staining of bacterial cells, showed the noticeable reduction in the biofilm forming tendency of CFT073 after Cri-dots mediated papG gene targeting. Similar results were observed from a quantitative analysis of the florescence images (Fig. 5b), where quenching was observed in Cri-dots-papG CFT073 as compared to control CFT073 (0.310 ± 0.1504, Cri-dots-papG vs 24.125 ± 1.2996, control) (p < 0.0001). These data confirm that the Cri-dots mediated editing of papG reduced the biofilm forming ability of CFT073.  www.nature.com/scientificreports/ SEM confirmed a reduced biofilm forming capability in papG-targeted CFT073. Bacterial adhesins (Flagella and pili) are known to be directly involved in the attachment to abiotic surfaces in Gramnegative bacteria 40 . The effects of papG targeting on CFT073 biofilms forming ability and adhesion capacity were further investigated using SEM. The bacteria in the control CFT073 were observed to be encased within a self-produced exopolymeric matrix forming strong biofilm as comparison to the Cri-dots-papG CFT073, which showed a significant reduction in their number (Fig. 5e,f). In addition, control CFT073 had a higher number of adhered cells, while Cri-dots-papG CFT073 had individual bacterial cells.
Decreased Virulence of CFT073 after targeting papG using Cri-dots. We further evaluated the virulence of CFT073 and Cri-dots-papG with an in vivo C. elegans infection model. This was done to assess the cumulative importance of our results on the virulence of CFT073 after using Cri-dots targeting papG. We found that Cri-dots-papG significantly increased the survival of C. elegans worms compared to CFT073 ( Fig. 6 and Fig. S7).

Discussion
UTIs are widespread around the globe and around 150 million people develop UTI every year, resulting in high social costs 41,42 . It is estimated that 11% of women report at least once UTI in their lifetime, and the prevalence in women over 65 years of age is approximately 20% 43,44 . The introduction of the antibiotic therapy had contributed significantly to the UTIs management, but antibiotic resistance in the UPEC causing UTIs has further worsened the treatment options. Because of the global emergence of multi-drug resistant uropathogens, it is imperative to embark on parallel strategies that target specific virulence systems of bacteria to combat pathogenesis. However, www.nature.com/scientificreports/ some potential drug targets of uropathogens such as adhesins, toxins, capsule, urease, iron metabolism and motility have been explored, but clinical studies are still in progress 22 . Bacterial adhesion to urothelial cells is a key step in pathogenesis of UTIs. One large family of adhesive organelles are pili assembled by the chaperone-usher pathway (CUP) pili. The most studied of the CUP pili of UPEC are the Type 1, P, and S pili that mediate microbial attachment to host tissues and biofilm formation 45 . P fimbriae are the second most common virulence factor of UPEC, which plays an important role in the pathogenesis of ascending UTIs and pyelonephritis in humans (Bien et al., 2012). P-fimbriae are encoded by the pap (pyelonephritis-associated pili) operon and encoded by protein subunits such as PapA, PapD, PapE, PapF, and PapG 10 . Previous studies had investigated the efficacy of vaccine containing PapA subunit, but failed due to poor generation of adherence-inhibition antibodies in-vivo 46,47 . Purified PapDG vaccine protected cynomolgus monkeys from pyelonephritis, however no further studies were conducted 23,48,49 . Here, we designed a novel non-viral CRISPR-cas9 gene editing strategy by Cri-dots for targeting virulence factor, papG in UPEC.
Recent work on the CRISPR adaptive bacterial immune system has led to the identification of new RNAguided DNA-binding platform that can be reprogrammed to target transcription of many genes. It only requires a single protein, and a customized sgRNA designed with a complementary region to the gene of interest 50 . Many studies have used Cas9-directed cleavage at the targeted genomic site for manipulating bacterial systems 25,51 .
Several approaches have been used, including plasmid, viral, bacteriophage and nanoparticle for delivery of CRISPR-cas9. Genetically encoded phage genomes were used to deliver CRISPR-cas9 antibacterial into bacteria, but challenges arise because of the varying size and structure of different phages 52 . Although viral delivery methods are very promising, potential off-target effects and risk of immunogenicity arise due to long-term exposure in vivo 53 . Plasmid methods have been reported for bacterial gene editing 54 , but limitations lie in off-target effects and delivery time 55 . The advantage of nonviral delivery of the Cas9 protein and sgRNA into mammalian and bacterial cells is the widely studied strategy in recent years because of its specificity, minor stimulation of immune response, and minimal exposure to nucleases 32,56 . Also, covalent conjugation of cas9 and delivery vehicle gives an advantage over non-covalent conjugation 31 .
In order to address these challenges, we developed a non-viral delivery method, Cri-dots. We used carbon quantum dots (CQDs) as a potential delivery vehicle for gene editing because of their biocompatibility and small sizes. CQDs were found to be non-toxic to both bacterial and mammalian cells. CQDs were further covalently conjugated with cas9 using EDC/NHS chemistry. Further sgRNA was also complexed with cas9-CQD conjugate to form Cri-dots nanocomplexes (Fig. 7a). Cri-dots are being reported for the first time as a targeted anti-bacterial therapy. Cri-dots could be an easy and efficient gene targeting strategy as compared to conventional plasmid and bacteriophage-based methods.
Previous studies have shown that papG mutant failed to colonize or cause inflammation in kidney and are crucial for microbial adherence 57,58 . P fimbriae is strongly associated with pathogenic strains, with at least 70-90% of acute pyelonephritis in contrast to another type 1 fimbriae, which can be expressed by both commensal and uropathogenic E. coli 14,59 . Thus, we have chosen papG for CRISPR-cas9 targeting using this novel strategy involving Cri-dots for direct delivery of cas9 protein and sgRNA into the bacteria. Before proceeding with the targeting strategy, we checked the cytotoxicity of of CQDs for mammalian and bacterial cells. CQDs showed excellent biocompatibility with both mammalian and bacterial cells (Fig. 1). Further, CQDs were successfully conjugated with cas9 protein using EDC/NHS chemistry (Fig. 2). The targeting efficiency of Cri-dots was evaluated by studying the gene expression levels of papG gene in control and Cri-dots targeted cells. We observed drastic reduction in mRNA expression of papG in Cri-dots-papG targeted CFT073 strain, compared to control CFT073 (Fig. 3a). PapG has been found to be indispensable for the adherence of UPEC to epithelial cells for establishing early growth in urinary tract 16 . Hence we evaluated the adherence of papG targeted CFT073 to human cells. As expected, we have observed 96% reduction in adherence of papG targeted CFT073 as compared to control (Fig. 3). Moreover, papG targeted CFT073 showed mannose sensitive hemagglutination (MSHA) while control CFT073 showed Mannose resistance hemagglutination(MRHA) (Fig. 4). P fimbriae are involved in the adhesion of bacteria to mucosal epithelial through TLR4 and hence activation of immune response causing inflammation and pain 60 . Our results showed that Cri-dots targeting papG gene quashed the adherence ability of bacteria to a supreme extent. Thus Cri-dots-papG might reduce the virulence of UPEC to a greater extent. Interestingly, Cri-dot-papG targeted bacteria showed significant reduction in biofilm formation. Bacteria were seen scattered in papG targeted CFT073 as compared to control where dense layer in typical diffused pattern was observed (Fig. 5). Furthermore, in order to comprehend how the respective virulence alteration induced by papG targeting contributes to the virulence of CFT073, an in vivo C. elegans infection model was used. We found that Cri-dots papG targeted bacteria did not significantly alter the survival of C. elegans worms as compared to control CFT073 (Fig. 6). These results not only validate the Cri-dots as novel vehicle for targeting CRISPR-cas gene editing strategy but also propose papG as a good candidate for anti-virulence therapy against UPEC involving ascending UTIs and pyelonephritis.
Bacterial relapses and recurrent infections are related to the ability of UPEC to form biofilms. Biofilm formation enables bacteria to persist in vagina and bladder, raising the risk of recurrent UTI 61 . Bacterial antibiotic resistance in the biofilm communities imparts to the chronic infections, which leads to the spread of chronic drug resistance bad bugs 62 . Conventional antimicrobials are not effective enough to control biofilm infections and the number of novel therapeutic strategies are still in development 63 . Hence, there is a need for innovative strategies with combination of both anti-biofilm and anti-virulence effects 64 . papG has been found to be more prevalent in strong biofilm producer strains 38,39,65 . Also, papG is essential for E.coli causing recurrent UTI in women 18 . We observed that Cri-dots mediated papG targeting significantly diminished the biofilm forming ability of UPEC. Large cellular aggregates of control strain are embedded in exopolymeric matrix, whereas individual cells with small clumps were observed for papG targeted strain, which further confirms the reduced adherence and biofilm www.nature.com/scientificreports/ forming potential of papG targeted strain (Fig. 7b). Therefore, papG was verified as an important target for developing combination strategy with both anti-adhesive and anti-biofilm effect against UPEC.
In conclusion, this is the first report suggesting use of biocompatible and non-cytotoxic CQDs for CRISPR mediating gene editing in bacteria where Cri-dots can be used as a delivery system for targeting various virulence genes. We showed that the Cri-dots targeting papG gene can be successfully delivered into bacteria. A direct role of papG, in both adherence and biofilm formation is showed. Therefore, papG could be a probable candidate for anti-virulence therapy in drug resistant UPEC. This study might open a new strategy of antimicrobials that can specifically target pathogenic strain without affecting commensal strain. However, further research is required to validate the in vivo efficacy of Cri-dots as a successful therapeutic strategy for UPEC mediated UTIs.

Materials and methods
Materials. UPEC      Haemolysis analysis. Haemolysis assay was performed as described by Black et al. 69 . Fresh human blood (5 ml) was taken from a healthy volunteer and stabilized with EDTA. The plasma was removed from blood by centrifugation at 3000 rpm for 10 min. Then the RBCs were washed thrice with freshly prepared 150 mM NaCl. Thereafter, the RBCs were diluted by 10% (w/v) with 100 mM sodium phosphate buffer (PBS) (pH 7.4). Diluted RBCs (0.3 ml) were then incubated with different concentrations of CQDs (0, 12.5, 25, 50, 100 and 200 μg/ml) for 3 h at 37 °C. 1% TritonX-100 and 1X PBS served as positive control (PC) and negative control (NC), respectively. After incubation, samples were centrifuged at 3000 rpm for 10 min and 100 ml of supernatant was collected. Absorbance was measured at 541 nm using a UV-vis spectrophotometer. The haemolysis % was calculated according to this formula: Haemolysis % = [(sample absorbance − NC)/ (PC − NC)] × 100, and the average value was obtained from five parallel samples and results were represented using GraphPad's Prism5 software.
Synthesis of Cri-dots (CQD-cas9) conjugates. The standard EDC/NHS conjugation protocol was applied to covalently conjugate carboxyl CQDs to amine group containing cas9 protein. EDC and NHS were mixed in 10 mM (pH 5.5) HEPES buffer at a concentration of 30 and 36 mg/mL, respectively. 10 μl of CQDs (20 μg/ml) were mixed with EDC/NHS mix solution and incubated for 30 min at room temperature to sufficiently activate the carboxyl groups. The carboxyl activated CQD were vortexed in 1 ml of PBST and centrifuged at 6500 g for 30 min. After centrifugation, most of the supernatant was removed. Then, 10 µl of cas9 (8 µg) was added to the mixture and incubated for 4 h at room temperature with mixing. Again, the mixture was thoroughly vortexed in 1 ml of PBST and centrifuged at 6500 g for 30 min. Finally, most of the supernatant was removed, and the solution was dissolved in 50 µl PBS. The conjugate was stored at 4 °C. Synthesis of single-guide RNA (sgRNA). Designing of gRNA. The gRNA sequences were designed using 'CHOPCHOP' webtool 70 . Target site on papG gene was 20 nucleotides in length with adjacent PAM sequence (5′-AGG-3′). The gRNA sequence was designed from the negative strand of targeted gene (Supplementary Fig. S8). On-target gRNA activity was predicted using the CHOPCHOP software analysis, and detailed BLAST searches of E. coli genome were conducted to predict any off-target binding of gRNA.
In vitro synthesis of gRNA (papG). In vitro synthesis of gRNA was performed using the GeneArt™ Precision gRNA Synthesis Kit. Oligonucleotide primers for synthesizing gRNAwere purchased from Sigma, with the forward primer containing a T7 promoter sequence. papG gene Specific primers were designed as shown in Supplementary S1 Table. The DNA template for in vitro sgRNA transcription was prepared by assembly PCR using Phusion™ High-Fidelity PCR Master Mix, Tracr fragment, T7 primer mix and 0.3 μM Target F1/R1 oligonucleotide mix according to kit cycling parameters. The DNA template was then in vitro transcribed and purified using gRNA clean up kit. The purified gRNA was quantified using a Nanodrop (ND-1000) and stored at − 80 °C.
Delivery of Cri-dots-papG nanocomplex to CFT073. CFT073 was grown overnight in LB medium in a shaking incubator at 200 rpm and 37 °C. For the experiment, the overnight cultures in log phase at OD600 between 0.6-0.8 (4.8-6.4 × 108 cells/mL) was used for the experiment. Bacterial culture was washed with 1X PBS and 1 ml of culture was incubated overnight with Cri-dots-papG nanocomplex in a 1.5 ml Eppendorf on a rotor spinner (20 rpm). After the treatment, cells were examined for gene targeting efficacy.
Quantitative real time PCR (qRT-PCR) and mRNA quantification. RNA isolation and cDNA synthesis. Total RNA was extracted from bacterial cultures Cri-dots-papG immediately after targeting and control CFT073 using TRI reagent according to manufacturer's instructions. After quantification using Nanodrop, 1000 ng of total RNA was subjected to cDNA synthesis using cDNA synthesis kit (RevertAid) as per manufacturer's protocol. www.nature.com/scientificreports/ and flow rate was set at low (12 µL / min). The data were analysed using Flowing software 2.5.1 67,72-74 and quantitative analysis was done using Prism software (GraphPad) .
Haemagglutination assay. Hemagglutinating activity was used for the determination of expression of the papG II and Type 1 fimbrial adhesins. For this, CFT073, Cri-dots-papG and K12 were grown on Luria Bertani plates at 37 °C for 24 h, suspended and serially diluted in PBS (10 10 bacteria/ml). A suspension of 1% fresh human group O Rh positive erythrocytes from healthy volunteer was mixed with bacterial suspension and added to slide. Haemagglutination was observed both in presence and absence of 1% D-mannose after 2 min. Wells containing only the suspension of erythrocytes were utilized as negative control. Slides were viewed under a microscope (Motorized Inverted Microscope. Ii2; Nikon).
Biofilm analysis. Crystal violet (CV) assay. Biofilm formation was estimated by CV assay as described by Schiebel et al. with some modifications 75 . The procedure was followed as described in the supplementary file for antibiofilm activity determination of CQDs (see supplementary Note S1). Each experiment was performed in triplicate with three independent experiments and analysed using Prism software (GraphPad).
Florescence microscopy. For microscopic visualization, biofilms were made as described in the antibiofilm activity determination of CQDs section. After removing planktonic growth from a 96 well plate, 3 µM SYTO 9 green fluorescent nucleic acid stain was added to each well and incubated for 15 min in dark at room temperature. Finally, plate was washed again with PBS (1X) and viewed under a fluorescence microscope (Motorized Inverted Microscope. Ii2; Nikon) using fluorescence setting for FITC (green/SYTO 9). Quantitative analysis of florescence images was carried out using ImageJ software (NIH) 76  Statistical analyses. Statistical analyses were conducted using GraphPad's Prism5 software. A Student's t test was conducted for two-sample analyses and a one-way analysis of variance (ANOVA) with post-hoc Tukey's honest significant difference was conducted for multiple sample analyses.

Data availability
Data available within the manuscript.