Mitigation of acyl-homoserine lactone (AHL) based bacterial quorum sensing, virulence functions, and biofilm formation by yttrium oxide core/shell nanospheres: Novel approach to combat drug resistance

The present study evaluated the efficacy of Y2O3:Tb (core) and Y2O3:Tb@SiO2 nanospheres (core/shell NSs) against virulence functions regulated by quorum sensing (QS) and biofilm formation in pathogenic bacteria. Scanning electron microscope (SEM) images were used to study the size, shape, and morphology. The images clearly displayed spherical shaped, mono-dispersed particles with narrow size distribution and an average grain size of 110–130 nm. The chemical composition of the samples was determined by using energy dispersive X-ray (EDX) and X-ray photoelectron spectroscopy (XPS). We determined the impact of core and core/shell NSs on QS using sensor strains of Chromobacterium violaceum CVO26 and Pseudomonas aeruginosa PAO1 in a comparative study. Sub-MICs of core and core/shell NSs substantially suppressed QS-controlled violacein production in C. violaceum. Similar concentration-dependent effect of sub-MICs of synthesized core and core/shell NSs was observed in the QS-regulated virulence functions (elastase, total protease, pyocyanin production, swarming motility, and exopolysaccharide production) in PAO1. A concentration-dependent decrease (14–60%) was recorded in the biofilm forming capability of PAO1, upon treatment with core and core/shell NSs. Moreover, core/shell NSs were more effective in inhibiting biofilm at higher tested concentrations as compared to core-NSs. The synthesized NSs demonstrated significantly impaired attachment of cells to the microtiter plate indicating that NSs target biofilm inhibition at the attachment stage. Based on these results, we predict that core and core/shell NSs may be an alternative to combat the threat of drug-resistant pathogenic bacteria.

Violacein inhibition assay. Overnight grown Chromobacterium violaceum CV026 (OD 600 nm = 0.1) was inoculated in Erlenmeyer flasks containing Luria broth (LB). LB supplemented with C6-HSL (10 µM/L) and the core and core/shell NSs and incubated under shaking for 24 h 25 . Violacein production by Chromobacterium violaceum (CVO26) in presence of core-NSs and core/shell NSs was studied using the method described by Husain et al. 25 .
Effect on virulence factors production. LasB elastolytic activity assay. The elastolytic activity was determined as described 26 . Briefly, treated and untreated PAO1 was incubated for 16 h at 37 °C. Culture supernatant (100 μL) was added to ECR buffer (900 μL) and left at shaking for 3 h at 37 °C. Centrifugation was performed to remove insoluble ECR, and absorption was measured at 495 nm.
Azocasein-degrading proteolytic activity. Proteolytic activity in cell-free supernatants of PAO1 was determined using the assay described by Kessler et al. 27 . Briefly, culture supernatants (150 μL) from NSs treated and untreated cultures were added to 1 mL of 0.3% azocasein (Sigma, USA) and incubated at 37 °C for 15 min. Trichloroacetic acid was added to stop the reaction. Absorbance was read at 400 nm.
Pyocyanin assay. Pyocyanin was measured by extracting 5 mL culture supernatant of PAO1 with 3 mL of chloroform and then re-extracted in 1 mL of 0.2 M HCl, to get a pink to deep-red colored solution. The absorbance of the extracted solution was measured at 520 nm 28 . Swarming motility assay. Swarming motility was determined by point inoculation overnight culture of P. aeruginosa (PAO1) at the center of the plate medium consisting of LB broth supplemented with 0.3% agar with or without various sub-inhibitory concentrations (25-200 µg/mL) of core and core/shell NSs 29 . Plates were incubated at 37 °C for 24 h and diameter of swarm was measured.
Extraction and quantification of exopolysaccharide (EPS). EPS produced in treated and untreated PAO1 was extracted by centrifugation, and the resultant supernatant was filtered. EPS was precipitated by adding ethanol (100%) to the supernatant and incubating at 4 °C 30 . Method described by Dubois et al. 31 was used to quantify the EPS.
Assay for biofilm inhibition. Microtiter plate (MTP) assay was employed to assess the effect of the synthesized NSs on biofilm formed by PAO1 32 . Briefly, cells with biofilms grown for 24 h in MTP were stained with crystal violet and quantified by measuring absorbance at 470 nm. cell attachment assay. Core and core/shell NSs (100 μL) were added to 96-well microtiter plates, and culture (100 μL) was added into each well. The MTPs were incubated at 37 °C for 8 h without shaking to allow cell attachment and biofilm development. Following incubation, the modified crystal violet assay was performed to assess biofilm biomass 33 . Analysis of lasB and pqsA transcriptional activity in E. coli. The effect of NSs on the lasB and pqsA transcriptional activity in reporter strains, E. coli MG4/pKDT17 and E. coli pEAL08-2 was measured using the β-galactosidase assay 32,33 . Briefly, quorum-sensing signal molecules (AHLs) from overnight grown PAO1 culture supernatant were extracted with ethyl acetate. Subsequently, 2 mL of reporter strains and 0.5 mL of the extracted supernatant were incubated in a rotatory water bath for 5 h at 30 °C. Further, the mixture was centrifuged, resultant pellet was resuspended in Z buffer (Na 2 HPO 4 . Upon appearance of yellow color, the reaction was stopped by addition of 1 mL of 1 M Na 2 CO 3 . Absorbance was read at 420 and 550 nm. Units of β -galactosidase were calculated as 1000 × OD 420 nm-(1.75 × OD 550nm )/ time × volume × OD 600nm .

Statistical analysis.
All experiments were performed in triplicates and data obtained from the experiments were presented as mean values. The differences between control and test were analyzed using Student's t test.

Results and Discussion
Morphological characterization. Scanning electron microscopy was performed to analyze the morphology of the prepared samples. Figure 1(a-c) demonstrates that the formed core/shell particles were well separated, spherical shaped, uniform, with a rough surface, narrow size distribution, and average grain size of 110-130 nm. SEM micrographs displayed that the particles are interconnected to each other because of the hydrophilic surface. This was expected owing to the grafting of silica layer over the surface of yttrium oxide NSs. The surface was covered with abundant silanol (Si-OH) groups, which easily form a colloidal solution in aqueous media by hydrogen bonding. Due to the presence of hydrogen bonding, they were connected to each other and slightly aggregated. Energy dispersive X-ray analysis was used to monitor the chemical composition of the designed core/shell NSs (Fig. 1d). Figure 1d, shows all elements in the spectrum, including silica (Si), oxygen (O), yttrium (Y), and terbium (Tb), which correspond to the core/shell NSs. This verified the core/shell nanostructure of the sample.

Minimum inhibitory concentration (Mic).
The MIC of core-NSs and core/shell NSs was tested against all test pathogens. The MICs (µg/ mL) of core-NSs were found to be 512, 512 and 1024 against CV12472, CVO26, and PAO1, respectively. Similarly, the MIC of core/shell NSs against all three test bacteria was 256 µg/mL. Sub-MICs were used to assess the QS and biofilm inhibitory potential of the NSs. Our finding is in accordance with those reported against pathogenic bacteria using various nanomaterials 40-44 . Anti-QS assay (violacein inhibition). Potential quorum sensing inhibitory potential of the synthesized NSs was screened using the biosensor strain Chromobacterium violaceum CV12472. Production of a violet-colored pigment called violacein in this strain is regulated by CviIR-dependent quorum sensing system. Therefore, pigment inhibition is indicative of interference with AHL-regulated QS 45 . Violacein levels were quantified by extracting violacein from biosensor strain Chromobacterium violaceum CVO26 at sub-MICs of NSs. Colorimetric analysis revealed a substantial drop in violacein production by CVO26 at all tested concentrations of NSs. Core-NSs at sub-MICs of 25, 50, 100 and 200 µg/mL demonstrated 18%, 43%, 56% and 75% reduction in violacein, respectively (Fig. 3a). Similarly, core/shell NSs also interfered with AHL-regulated violacein production in a concentration-dependent manner and 38-84% reduction was recorded (Fig. 3a). The effect of the core-NSs and core/shell NSs (200 µg/mL) on the growth of CVO26 was also examined and no significant difference in cell density was recorded (Fig. 3b). These findings demonstrate that the inhibition of violacein by NSs in CVO26 is growth independent i.e. the growth is not affected. Thus, it is anticipated that the NSs (core & core/shell NS) interfere with AHL-regulated QS signaling. Our findings are validated by reports on anti-QS properties of NSs. Silver nanowires demonstrated 80% decrease in violacein production in CVO26 at 4 mg/mL concentration 46  inhibition of QS-regulated virulence factors by core and core/shell nSs. Growth kinetics studies of PAO1 revealed no significant change in cell density after treatment with 200 µg/mL concentration of core and core/shell NSs as compared to untreated control (Fig. 4a). Thus, the synthesized NSs do not inhibit the growth of the pathogen. The effect of sub-MIC of synthesized core and core/shell NSs on various QS-regulated virulence functions in PAO1 was also assessed. Core-NSs demonstrated the reduction of all virulence factors in a concentration-dependent manner (25-200 µg/mL): elastase (18-57%), total protease (21-61%), pyocyanin (06-55%) (Fig. 4b). Core/shell NS was found to be more potent in inhibiting the studied virulence factors over www.nature.com/scientificreports www.nature.com/scientificreports/ the same concentration range. The core/shell NS treated PAO1 cells displayed substantially reduced azocasein degrading protease activity (84%), elastase activity (81%), and pyocyanin production (89%) at 200 µg/mL concentration as compared to untreated control (Fig. 4c).  www.nature.com/scientificreports www.nature.com/scientificreports/ Protease and elastase enzymes play a major role in the pathogenesis of P. aeruginosa, as they are responsible for host tissue degradation. Production of pyocyanin in P. aeruginosa is also QS-regulated and this secondary metabolite along with its precursor causes severe toxic effects. In the present study, the production of these virulence traits was reduced significantly at sub-MICs. These findings also support our previous observations on ZnO nanoparticles that reduced the production of elastase and pyocyanin in clinical strains of P. aeruginosa 48 . The effect of synthesized NSs on swarming motility and EPS production by PAO1 was also examined. The addition of sub-inhibitory concentrations (25-200 µg/mL) of core-NSs and core/shell NSs demonstrated a statistically significant reduction of swarming motility and EPS production. The reduction in swarming motility by core-NSs ranged from 12-62% while EPS production decreased by 19-59% over untreated control (Fig. 4b). The core/ shell NSs demonstrated a significant reduction in swarming migration and EPS at all tested concentrations. The motility of PAO1 was reduced by 32-90% and EPS production was impaired by 41-89% (Fig. 4c). These findings are significant as these two virulence traits play an important part in biofilm formation by P. aeruginosa. Motility and EPS are responsible for the successful attachment, development, and maturation of biofilm. Interference with either of the two leads to impaired biofilm formation thereby reducing the resistance of sessile cells to drugs and host immune system. Singh et al. 47 demonstrated reduced migration diameter of PAO1 upon treatment with 25 µg/mL of biogenic silver NSs. This was probably the first report on NS targeting motility and EPS production in PAO1.
Effect on β-galactosidase activity. The effect of 25-200 µg/mL concentration of core and core/shell NSs on lasB and pqsA transcriptional activity was examined using β-galactosidase assay. Core and core/shell NSs reduced the lasB transcriptional activity by 41% and 65% respectively. Moreover, 55% and 67% downregulation in pqsA was also recorded at 200 µg/mL concentration of core and core/shell NSs (Table 1). Pearson et al. 49 reported a positive correlation between AHL concentration and lasB-lacZ expression. Our findings correlate well with the above observations, as reduced β-galactosidase activity is indicative of reduced AHL levels and, therefore, reduced expression of the lasB gene. Similarly, core and core/shell NSs reduce the transcriptional activation of pqsA and inhibit the pqs system that controls the production of virulence factors like pyocyanin. These results www.nature.com/scientificreports www.nature.com/scientificreports/ indicate that the synthesized core and core/shell NSs have broad-spectrum anti-QS activity and are active against both las and pqs system of P. aeruginosa.
Inhibition of biofilm formation. Biofilm inhibition by core-NSs and core/shell NSs was tested using microtiter plate assay. Biofilm formation was reduced by 14-60% by core-NSs in a concentration-dependent manner (Fig. 5a). Core/shell NSs were more effective in inhibiting biofilm at higher tested concentrations as compared to core-NSs. At 25, 50, 100 and 200 µg/mL concentrations, core/shell NSs impaired the biofilm formation by 15%, 34%, 58%, and 75% over untreated control (Fig. 5a). CLSM images confirmed the results of light microscopy displaying major disruption in the architecture of biofilms of PAO1 (Fig. 5b-d). A biofilm is a complex congregation of micro-colonies that is resistant to drugs and plays a key role in the pathogenesis of opportunistic bacteria such as P. aeruginosa 50 . Biofilms are therefore interesting drug targets in fight against drug resistance and persistent infections. We recorded a significant reduction in biofilm biomass upon treatment with the core and Core/shell NSs. Our study is in accordance with previous reports that showed the reduction of biofilms in P. aeruginosa PAO1 by nanoparticles of zinc oxide 14,51 , tin oxide 52 and iron oxides 53 , silica nanoparticles 54 antimicrobials like doxycycline and ceftazidime 29,55 .
Effect on cell attachment. Cell attachment is the first step in biofilm formation and it has been established by previous reports that inhibition of cell attachment to a substrate is easier to achieve than inhibition of the growth of an already established biofilm 56 . Therefore, NSs were examined for anti-attachment property against PAO1. The synthesized core-NSs showed significant inhibition of cell attachment to the microtiter plate. Core and  www.nature.com/scientificreports www.nature.com/scientificreports/ core/shell NSs showed maximum activity of 45% and 67% at 200 µg/mL concentrations, respectively. Core-NSs at concentrations 25, 50, and 100 µg/mL showed 19%, 31%, and 40% inhibition of cell attachment over control. Similarly, 16%, 41%, and 54% inhibition was recorded at lower tested concentrations (25, 50, 100 µg/mL), respectively (Fig. 6). These results indicate that the synthesized core/shell NSs inhibit cell attachment and hence prevent biofilm formation. These core/shell NSs may be used as a coating on surfaces to prevent biofilm formation.

conclusion
To conclude, spherical shaped monodispersed core and core/shell NSs were successfully produced by homogeneous urea-based co-precipitation process. The morphology, size, and elemental composition of the samples were characterized by scanning electron microscopy, EDX, and XPS analysis. The present findings highlight the QS and biofilm inhibitory potential of chemically synthesized NSs (Core and Core/shell NSs), which may be exploited to treat drug-resistant infections of P. aeruginosa in future. The anti-biofilm activity results clearly indicate that core and core/shell NSs may be helpful in preventing and fighting drug-resistant infections.