Five bacterial strains, Escherichia coli MTCC 1304, P. aeruginosa MTCC 741, K. pneumoniae MTCC 109, S. aureus MTCC 96, and Chromobacterium violaceum CV026, were used in the study. All bacterial strains except C. violaceum were maintained in nutrient agar (NA) slant at 4°C, and C. violaceum was maintained in trypticasein soy (TS) broth slant at 4°C. The culture was inactivated prior to any test at 37°C and 27°C.

All the chemicals used for different experiments and resazurin dye (7-hydroxy-10-oxidophenoxazin-10-ium-3-one, sodium) were purchased from HiMedia Chemicals Ltd, Mumbai, India.

Biofilm of Gram-negative (E. coli MTCC 1304, P. aeruginosa MTCC 741, and K. pneumoniae MTCC 109), Gram-positive (S. aureus MTCC 96), and the mixed strain was formed on petri plates filled with glass wool. One percent of the overnight-grown culture of each bacterium was used as an inoculum, added to 10 ml of fresh media, inoculated on 5-g glass wool, and kept undisturbed for 2 days at 37°C. The biofilm grown on glass wool was visually observed after 48 h. For mixed biofilm, 25% volume of each fresh bacterial culture was added on glass wool and kept for 5 days at 37°C under static conditions. The mixed biofilm formed on glass wool was visually observed, and EPS extraction from the cultured media was performed.

Biofilm inhibition and degradation tests were performed in 96-well microtiter plates as described by Kalia et al. (2015), with minor modifications, and crystal violet (CV) dye was used for the quantification of the biofilm. In a 96-well plate, 100 µl of the freshly prepared bacterial suspension (0.5 McFarland’s standard) and 100 µl of 2 mg/ml stock of the plant extract was added and incubated for 48 h at 37°C. After the incubation medium was removed, wells were washed with distilled water to remove planktonic cells. Further 200 µl of 0.4% CV dye was added and incubated for 20 min to stain the attached biofilm followed by washing the wells with distilled water to remove the extra dye, and then plates were dried. The biofilm was re-solubilized with dimethyl sulfoxide (DMSO) (99.5%) and then read with a spectrophotometer at 620 nm in an ELISA reader. The percentage of biofilm inhibited was calculated by the following formula.

Biofilm degradation assay was performed as explained by Perumal and Mahmud (2013), with minor modifications. The 24-h-old biofilm was formed in 96-well plates, and after incubation, the medium was removed. Adherent biofilm was treated with 100 µl of plant extract (2 mg/ml of stock solution) and incubated for 24 h at 37°C. The CV dye was added after washing the wells with distilled water and was kept for 20 min for staining purposes. After staining, the extra dye was removed by washing, and the plate was dried. Further DMSO was added to resolubilize the biofilm, and plates were read at 620 nm in an ELISA plate reader. All the experiments were performed in triplicate. The degradation percentage was calculated by the following formula:

For EPS production in mixed and individual bacterial biofilm, an inoculum of bacterial strains, S. aureus, P. aeruginosa, E. coli, and K. pneumoniae, was prepared in 15 ml of nutrient broth in petri-plates with 0.5-g glass wool in it. The treatment of biofilm was done with plant protein after 48 h. The plates were incubated at 37°C for 5 days under static conditions. The steps involved in the extraction of EPS from 5-day-old culture medium were performed as described by Gong et al. (2009) and are depicted as follows, Various microbes’ inoculated culture was incubated for 5 days at 37°C and then heat-inactivated at 100°C for 10 min. The culture was centrifuged at 10,000 rpm for 10 min, and the supernatant was precipitated with cold ethanol (double volume). After overnight incubation at 4°C, the mixture was centrifuged at 10,000 rpm for 25 min in a cooling centrifuge, and the obtained pellet was dissolved in distilled water. Total carbohydrate, reducing sugars, and the total soluble protein were estimated by various analytical methods.

Various plants ( Table SI1 ) were collected freshly from the Institute of Engineering and Technology Campus, Sitapur Road, Lucknow (26.9143°N, 80.9410°E), and the local market of Alambagh, Lucknow (Uttar Pradesh), India (26.8128°N, 80.9014°E). The plant parts were collected fresh, washed, shade dried, and powdered. Each plant sample measuring 2 g was dissolved in 20 ml of methanol and 20 ml of aqueous solvent (1:10 w/v distilled water) and kept at a rotary shaker overnight. Flask was kept in a stable position for 1 h, and then extract was filtered through Whatman No. 2 filter paper, was concentrated to dry crude, and stored in a sterile Eppendorf tube at 4°C. The methanolic and aqueous plant extracts were used to test the existence of various phytochemicals like flavonoids, glycoside, phenol, amino acids, saponins, tannins, quinone, coumarins, carbohydrate, alkaloids, and terpenoids using the standard protocols (Goyal et al., 2016; Sánchez et al., 2016; Santhi and Sengottuvel, 2016) ( Table SI2 ).

The protein content present in the aqueous medium of the plant extract was isolated according to the steps mentioned by Gangwar et al. (2016), with minor modifications.

Liquid chromatography–tandem mass spectrometry (LC-MS) analysis of the sample was done with a C-18 250 × 4.6 mm column (Waters Instrument, Milford, MA, USA).

To analyze the sample, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed using Mini-PROTEAN apparatus (Bio-Rad Laboratories Inc., Hercules, CA, USA) following the protocol of Laemmli et al. (1970). The gel was made with urea and other solutions as done with SDS–PAGE. The sample solution was prepared with Bio-Rad AG501-X6 mixed bed resin. Methyl green was used as a tracking dye, and peptide markers were used. The gel was run at 110 V for 2–3 h. The gel was stained with a staining solution containing Coomassie Brilliant Blue (CBB) 250 dye overnight and was destained with a destaining solution.

The 24-h-old bacterial cultures were inoculated in fresh nutrient broth media. Bacterial culture measuring 5 ml was dispensed in petri dishes and then incubated at 37°C for 24 h. Biofilms were prepared on sterilized coverslips kept in petri dishes. After incubation, planktonic cells were discarded by pipetting out the media, and coverslips were rinsed with 1× phosphate-buffered saline (PBS) twice. Coverslips were stained with 0.4% CV dye for 15 min. Coverslips were rinsed twice for extra dye removal. Then coverslip was kept on a glass slide and observed under a light microscope at 100× for visualization of biofilms.

The anti-quorum-sensing activity (QQ) of plant extracts was evaluated by using biosensor bioassay with C. violaceum strain by using the agar well diffusion method. A lawn of bacteria was formed on an agar plate by using freshly prepared culture in an LB medium. The wells were inoculated with 100 µl of stock solution of 2 mg/ml of aqueous and methanolic plant extract. Plates were incubated at 27°C for 48 h. Methanol (99%) and distilled water were used as a negative control. The anti-quorum-sensing activity was determined by the zone of inhibition (measured in mm) formed around the wells.

All the experiments were performed in triplicate, and the mean value of the data was presented. The statistical significance of the data was analyzed through one-way ANOVA by setting the significance level of p-value <0.05.

The dialyzed sample was subjected to DEAE cellulose ion-exchange chromatography, and the fractions collected from the column were examined for the presence of protein by Lowry assay. The maximum protein content was eluted with 0.1 M of NaCl. All the fractions were subjected to anti-quorum-sensing activity, and the fractions showing positive activity were subjected to phytochemical activity. The results of phytochemical activities suggest that except quinone and coumarins, all the tested phytochemicals were present in the aqueous extract of the plant leaves. However, with the progress in the purification steps, their number reduces, and finally, in the dialyzed sample, only phenol, terpenoid, protein, saponin, and reducing sugar were present ( Table 1 ). LC-MS analysis revealed that at retention times of 19.88 and 20.96, basic peaks were observed, further molecular mass of compounds was determined, and 312.5 m/z and 445 m/z were found to be present in maximum concentration ( Figure 1 ). Further analysis suggested the presence of amino acids like aspartic acid, phenylalanine, and glutamic acid.

The plant protein extract was tested for having maximum anti-quorum-sensing activity at different physiochemical conditions like temperature, pH, solvents, metal ions, and surfactants. Under examined incubation temperatures (4°C–60°C), the protein retains the maximum anti-quorum-sensing activity between 4°C and 10°C. However, 80% of its activity is retained up to 40°C, which is in accordance with some previous reports that suggest plant proteins are stable up to 50°C temperature. Similarly, pH 7 (phosphate buffer) was found most suitable for protein stability. Metal ions like Mg²⁺ and surfactant EDTA were showing a positive effect on the isolated protein fraction. Plant sample dissolved in aqueous solvent was more active than in other solvents like methanol, ethanol, and DMSO; this suggests that the solvents lead to denaturation of the isolated protein resulting in the decrease of activity ( Figure 2 ).

The plant extract of C. carandas was showing significant inhibition and degradation percentage, and the results are presented in Table 3 . The plant extract was found to show 80% inhibition against Gram-positive strains while 60% inhibition against Gram-negative strains. The degradation percentage of biofilm by plant extract was found at 70% and 60% against Gram-positive and Gram-negative strains, respectively. Ampicillin (D2) and cefotaxime (D3), each measuring 2 mg/ml, were also taken as standard.

Biofilm inhibition and degradation analysis of the selected plant extract (C) were done by using a 96-well microtiter plate and CV dye. The inhibition percentage of 48-h-old biofilm in the presence of selected plant extract was compared with the control adherent biofilm without treatment. Antibiotics like ampicillin (D2) and cefotaxime (D3), each measuring 2 mg/ml, were also taken as standard. The percentage inhibition and degradation are tabulated in Table 3 . All the values were examined by one-way ANOVA using GraphPad Prism 7 software, and p-value >0.5 was considered significant.

Through the present study, it can be concluded that the selected plant compound was capable of inhibiting biofilm formed by Gram-positive microbes up to 80% while by Gram-negative microbes up to 60% at 100 μl of concentration. The selected plant extract has the potential to degrade biofilms formed by Gram-positive microbe up to 70% and by Gram-negative microbes up to 60% at 100 μl of concentration.

Biofilm on coverslips was made with E. coli, P. aeruginosa, S. aureus, K. pneumoniae, and C. violaceum bacterial strains and was visualized under bright-field microscopy. By examining the microbial colonies under a microscope, it was observed that the microbial colonies that were treated after 24 h had deformed and had fewer colonies as compared to non-treated ones ( Figure 4 ). This endorses that the plant extract has microbial colony-degrading capability. The biofilm has a network-like appearance when seen under the microscope, while in the bacterial samples where the plant extract was applied, the degraded network was observed with fewer colonies in number.

EPS of the biofilm was extracted by ethanol precipitation method and analyzed according to the carbohydrates, proteins, and lipid staining using iodine, ninhydrin, and Sudan black B dye, respectively. The slides were visualized under 100× oil immersion in a light microscope, the presence of the mentioned components was observed ( Figure 5 ). Further, in the quantitative analysis, the control biomass (10.85 g) and EPS (765 mg) were found to decrease nearly 40% after the treatment with the protein extract of the C. carandas leaves ( Table 4 ).

Methanolic and aqueous extracts of C. carandas leaves were tested for their anti-quorum-sensing activity against C. violaceum. The methanolic and aqueous extracts showed anti-quorum sensing with the zone of inhibition of 10 and 20 mm, respectively. The aqueous extract showed better activity than methanolic extract and suggests the proteinaceous nature of the compound, which was further confirmed by the protein assay. The results were further validated by biochemical activity at each step of protein purification.