Wing characterization
Dragonflies (Pantala flavescens) were collected from Botanical gardens in Coimbatore, India. The wings of dragonflies were rinsed with milliQ water (18.2 MΩ cm resistivity), dried in the laminar hood and stored for experiments. The wings were sputter coated using Emitech SC7620 Mini Sputter Coater and imaged using FEI Quanta 250 Field Emission Scanning electron microscope (FESEM). Water contact angle of the wing was measured using KRUSS-Drop Shape Analyzer DSA 25E. Chemical characterization was performed using Energy Dispersive X-ray spectroscopy (Bruker) and Fourier Transformed Infrared Radiation (Perkin Elmer FTIR model C96836).
Bacterial culture preparation
Pseudomonas aeruginosa strain ATCC 9027 (MTCC 1688) was procured from Microbial Type Culture Collection, Chandigargh, India and, P. aeruginosa PAO1 was procured from National Collection of Industrial Microorganisms, National Chemical Laboratory, Pune, India. P. aeruginosa clinical isolates (named 1570, 1589 and 1595) were obtained from PSG Institute of Medical Sciences & Research, India.
Single colony of P. aeruginosa PAO1 and ATCC 9027 were separately inoculated in 5 mL of nutrient broth and cultured overnight at 37 °C shaking at 120 rpm. The cultures were centrifuged, and the pellets were resuspended in Phosphate Buffered Saline (PBS) pH 7.4 to adjust OD600nm to 1.0. The above inoculums were diluted ten times with PBS. PBS does not support bacterial growth and hence control over the cell count could be achieved. Coverslip and wing were cut to 10 mm diameter. Glass coverslip was used as control as reported earlier (Ivanova et al. 2013). In 48-well plate, 1 mL culture was added to each well containing coverslip/wing and incubated for discrete time periods. The above procedure was followed for all the tests unless otherwise mentioned.
Bacterial viability
Quantitative analysis of bacterial viability
Bacterial viability was analyzed using flow cytometer (BD FACS verse). 1 mL of P. aeruginosa cultures were incubated separately with coverslip/wing for 30 min, 1 h, 2 h, 7 h, 24 h and 48 h under static condition in 48-well plate. After incubation time, 1 mL of the cell suspensions were transferred to fresh 2 mL centrifuge tubes. Coverslip and wing were sonicated (Lab companion ultra Sonic cleaner UCP-02) to detach the cells from coverslip/wing in 0.5 mL PBS. Post-sonication, coverslip/wing was discarded and the contents were pooled to their respective 1 mL cell suspensions that were collected earlier. Cells attached to the coverslip/wing were pooled to the suspension to get cell concentration of 107, which is the minimal cell count required for analyses using flow cytometry. To this pooled suspension, 10 µM propidium iodide (MP Biomedicals 195458) was added, mixed and incubated in dark for 10 min. Propidium iodide stains the dead cells red. The viable cells remain unstained. The suspensions were loaded onto the BD Facs Verse flow cytometer and medium speed was selected to analyze the cells. 10,000 events were studied. The results were obtained using BD FACSuite software application. The flow cytometry data obtained are presented in percentage and not in log scale, as it likely eliminates the low-intensity data due to signal compensation (Herzenberg et al. 2006).
Qualitative analysis of bacterial viability
Pseudomonas aeruginosa strains were incubated with coverslip/wing in 48-well plate for 24 h at 37 °C. After incubation, coverslip/wing were rinsed gently and transferred to fresh 1.5 mL centrifuge tube containing 1 mL 0.85% saline (PBS affects the efficiency of fluorescent staining, hence saline was used). Coverslip/wing in saline was sonicated to detach the cells. After sonication, coverslip/wing was discarded. The cells were stained with BacLight Live/dead bacterial fluorescent staining kit (Invitrogen L7012) as per manufacturer’s instructions. Nucleic acid of live cells were stained green using SYTO9 and dead cells red using propidium iodide (Dickson et al. 2015). 5 µL of the suspension was transferred to glass slide and imaged under fluorescent microscope (Nikon Ti eclipse, 100× oil immersion). Images were captured using NIS-Elements BR version 4.50.00 version.
Quantification of DNA in suspension
It is presumed that cells whose cell walls were damaged on the nanopillars release the cellular DNA into the suspension. The DNA released into the cell suspension was quantified without disturbing the cells with intact cell walls. After incubating the P. aeruginosa with coverslip/wing for discrete time periods, 1 mL of cell suspension was collected in fresh 2 mL centrifuge tubes. Equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) were added, mixed well and incubated for 10 min at room temperature. The contents were centrifuged at 12,000×g for 15 min at 4 °C. The top aqueous layer was transferred to a fresh 2 mL centrifuge tube. 2.5 volume of ice cold isopropanol was added to precipitate the DNA. The contents were mixed and allowed to stand for 10 min in ice. The tubes were centrifuged at 12,000×g for 15 min at 4 °C to pellet the DNA. Supernatant was discarded and the DNA pellet was washed with 75% ethanol and centrifuged again. The DNA pellet was suspended in 1× Tris EDTA buffer. Using 1× TE buffer as blank, the concentrations of the DNA obtained were read using nanospectrophotometer (Quickdrop Spectramax–Micro volume spectrophotometer).
Bacterial attachment
Pseudomonas aeruginosa strains were separately incubated with coverslip/wing for discrete time periods in 48-well plate. After incubation time, the coverslip and wing were rinsed gently and sonicated in 1 mL PBS. The coverslip/wing was discarded post-sonication. The cells were stained and enumerated under light microscope using hemocytometer. Triplicates were used.
Scanning electron microscopic imaging
Bacterial attachment on the coverslip/wing was studied using scanning electron microscopy. P. aeruginosa strains were incubated with coverslip/wing for 2 h, 7 h and 24 h. After incubation, the cell suspension was discarded and coverslip/wing was rinsed with PBS gently. The cells on the coverslip/wing were fixed with 2.5% gluteraldehyde for 15 min. The gluteraldehyde was discarded and coverslip/wing was rinsed with PBS thrice. The samples were dehydrated gradually with increasing percentages of ethanol 30%, 50%, 70%, 90%, 100% and 100% replacing each after every 10 min (Tang et al. 2014). The samples were then gold coated using Emitech SC7620 Mini Sputter Coater and imaged in FEI Quanta 250 Scanning electron microscope.
Adhesion force measurement
Adhesion force measurements were studied using Scanning Probe Microscope (NT-MDT). Overnight grown P. aeruginosa were centrifuged and the pellet was resuspended in PBS and the OD600nm was adjusted to 1.0. 1 µL of the culture suspended in PBS was added to the cantilever (CSG10 series, NT-MDT) and allowed to dry at room temperature. Bacterial attachment to cantilever is carried out according to the protocol specified by Touhami et al. (2006). The cantilever with bacteria attached was mounted onto the scanning probe microscope. Meanwhile, glass coverslip and wing were cut into 1 × 1 cm2 and fixed onto two separate stubs. Using contact mode, the adhesion forces were measured at 25 random points on the coverslip/wing. The cantilever with the bacteria was allowed to rest on coverslip/wing for 1 s, and retracted. For each point, force-distance curves were constructed using Origin 8.5 software. The difference between the cantilever approach and retract value is the height (h). The ‘h’ value denotes the adhesion. The spring constant of the cantilever used was 0.11 N/m.
The ‘h’ values obtained were substituted in the Hooke’s law to get the adhesion force.
By Hooke’s law,
$${\text{Adhesion}}\;{\text{force}},{\text{ F}}\, = \,{\text{k }} \times \, \Delta {\text{h}}$$
where, k, the spring constant is 0.11 N/m.
Number of points to which the P. aeruginosa strain shows adhesion was studied. In addition, the adhesion forces exerted on each point were also calculated. The adhesion forces were plotted as a Box–Whisker graph using Origin 8.5 software.
Biofilm forming assay
The biofilm forming ability of the P. aeruginosa strains were tested using the standard crystal violet assay in 96-well plate (O’Toole 2011). The absorbance values are proportional to the biofilm formed. Depending upon the absorbance values, Hassan et al. (2011) reported that bacteria can be classified as non-biofilm former (OD of test ≤ 2× OD of control), weak-moderate biofilm former (OD of test > 2× to ≤ 4× OD of control) and, potent biofilm former (OD of test > 4× OD of control).
Real-time PCR
The PAO1 and ATCC 9027 cultures were diluted to OD600 of 0.1 using PBS. The cultures were incubated with wings and coverslip separately in 48-well plate at 37 °C under static condition. After 30 min, 1 h, 2 h, 7 h, 24 h and 48 h, total RNA was extracted from the cells using Tri Reagent (Sigma Aldrich Catalogue No. T9424). The total RNA was used to synthesize cDNA using ThermoFisher kit (K1622). The synthesized ss cDNA was used in Bio-rad tubes (TCS 0801, TCS 0803) with Bio-rad SYBR mix (170-8880AP) for real-time PCR as per the manufacturer’s instructions in the CFX96 Bio-rad RT-PCR instrument. The primers of the genes fliE, fleS, pelA, gcbA, rsmZ and mreB were synthesized commercially using the sequences provided by Petrova et al. (2014).