Behavior of Pseudomonas aeruginosa strains on the nanopillar topography of dragonfly (Pantala flavescens) wing under flow conditions

Studies that reported the bactericidal activity of nanopillars were mostly experimented under static conditions.2121. J. Yayan, B. Ghebremedhin, and K. Rasche, PLoS One 10, e0139836 (2015). https://doi.org/10.1371/journal.pone.0139836 However, in nature, bacteria are subjected to hydrodynamic conditions. Understanding the behavior of bacteria under hydrodynamic conditions would provide better insights into employing nanopillars for medical applications.Among the methods employed to study bacteria-substrate interactions under hydrodynamic conditions, the flow cell is considered the better option than the rotary shaker, as the cells in the latter would be carried away with the swirl.2828. H. J. Busscher and H. C. van der Mei, Clin. Microbiol. Rev. 19, 127 (2006). https://doi.org/10.1128/CMR.19.1.127-141.2006 When fluid flows through a channel, the flow exerts a shear along the walls. The shear rate is higher on the walls and decreases gradually toward the center. Shear rates trap bacteria and enhance bacteria-substrate interactions.2929. R. Rusconi, J. S. Guasto, and R. Stocker, Nat. Phys. 10, 212 (2014). https://doi.org/10.1038/nphys2883 Flow conditions used in bacteria-substrate interactions are expressed as shear rates than flow rates, as the same flow rate may have different shear rates with variations in the tube diameter.2828. H. J. Busscher and H. C. van der Mei, Clin. Microbiol. Rev. 19, 127 (2006). https://doi.org/10.1128/CMR.19.1.127-141.2006The shear rates were calculated as reported earlier.2828. H. J. Busscher and H. C. van der Mei, Clin. Microbiol. Rev. 19, 127 (2006). https://doi.org/10.1128/CMR.19.1.127-141.2006 The shear rates corresponding to the flow rates of 250, 450, and 600 μl min−1 were found to be 1.0, 1.8, and 2.4 s−1, respectively.

A. Surface topography of the dragonfly wing

The surface topography of the dragonfly wing characterized using SEM (Fig. 2) indicated the presence of nanopillars that had an average height of 188 nm and spaced 88–157 nm apart.figure

B. Bacterial viability under flow condition

The viability of P. aeruginosa strains postinteraction with the coverslip and wing and subjected to three different shear rates for periodical intervals were tested quantitatively using flow cytometry and qualitatively analyzed by fluorescent staining of live/dead cells.

1. Analysis of bacterial viability

The viability of P. aeruginosa strains after interaction with the coverslip and wing at shear rates of 1.0, 1.8, and 2.4 s−1 are shown in Figs. 3(a)3(c). The results in Fig. 3 indicate that P. aeruginosa ATCC 9027 postinteraction with the coverslip showed average viabilities of 58%, 50%, and 50% at shear rates of 1.0, 1.8, and 2.4 s−1, respectively. P. aeruginosa ATCC 9027 postinteraction with the wing showed a viability of 50% at all three shear rates. On the other hand, PAO1 postinteraction with the coverslip showed average viabilities of 46%, 52%, and 74%, while with the wing, the viabilities were 32%, 40%, and 65% at shear rates of 1.0, 1.8, and 2.4 s−1, respectively. Statistically significant difference in the mean values was observed between ATCC 9027 and PAO1 at a shear rate of 2.4 s−1 (p = 0.021).figureThese results suggested that the viability of P. aeruginosa ATCC 9027 was less likely affected by the shear rates tested, while the increase in shear rates aided the viability of PAO1 postinteraction with the wing surface. Flow cytometry data are represented as percentage rather than the log scale, as the log scale likely eliminates low intensity data.3030. L. A. Herzenberg, J. Tung, W. A. Moore, L. A. Herzenberg, and D. R. Parks, Nat. Immunol. 7, 681 (2006). https://doi.org/10.1038/ni0706-681

2. Fluorescent imaging of live/dead cells

Qualitative analysis of bacterial viability postinteraction with the coverslip and wing at three different shear rates by fluorescent staining of nucleic acids is presented in Table I. The live cells appear green by taking up SYTO9, and dead cells appear red by taking up propidium iodide. The number of green cells was higher in PAO1 postinteraction with the wing surface. The live/dead staining results corroborated with the results in Fig. 3.Table icon

TABLE I. Fluorescent stained images of P. aeruginosa ATCC 9027 and PAO1 on coverslip and wing after 24 h of exposure to shear rates. Live cells stained with SYTO9 appear green, and dead cells stained with propidium iodide appear red. Bar represents 2 μm.

Shear rate (s−1)1.01.82.4ATCC 9027 on coverslipATCC 9027 on wingPAO1 on coverslipPAO1 on wing

C. Bacterial attachment under flow condition

Bacterial attachment under flow conditions is usually tested by mounting the flow cell on the fluorescent microscopic stage and continuous imaging. The bacterial cells would be fluorescently labeled or stained for imaging. However, fluorescent staining significantly affects the properties of cell surface and their adhesion. Yet another disadvantage with continuous imaging of cells is that, cells that are carried in the flow would also be captured.2828. H. J. Busscher and H. C. van der Mei, Clin. Microbiol. Rev. 19, 127 (2006). https://doi.org/10.1128/CMR.19.1.127-141.2006 Differentiating the moving cells from the attached cells is critical and crucial. This study used the method of simple staining and enumerating under an optical light microscope.

1. Staining and enumeration of attached bacteria

Enumeration of bacterial attachment on the coverslip and wing is presented in Fig. 4. The results indicated that the number of P. aeruginosa ATCC 9027 attached to 1 × 1 cm2 of coverslip increased from 11.87 × 104 to 12.68 × 104 cells, and on the wing from 9.89 × 104 to 11.56 × 104 cells with increase in the shear rate. An increase in the shear rate aided the attachment of ATCC 9027 on the coverslip and wing with the wing showing approximately 1.5 × 104 cells lower than that on the coverslip.figureThe number of PAO1 attached to the coverslip increased from 8.25 × 104 to 11.80 × 104 cells, and the number of wings attached on PAO1 increased with the increase in the shear rate, showing maximal attachment of 12.5 × 104 cells at a shear rate of 1.8 s−1. The number of cells attached to the substrate increased with the increase in the shear rate from 1.0 to 2.4 s−1. Similar observation of gradual increase in the attachment of P. aeruginosa cells with increase in shear rate from 1 to 10 s−1 was reported.2929. R. Rusconi, J. S. Guasto, and R. Stocker, Nat. Phys. 10, 212 (2014). https://doi.org/10.1038/nphys2883 A statistically significant difference in the mean values was observed between ATCC 9027 and PAO1 at a shear rate of 1.0 s−1 (p = 0.034).

Under flow conditions, the number of ATCC 9027 attached to the coverslip was nearly 2 × 104 cells higher and 1 × 104 cells lower on the wing surface than PAO1.

2. Scanning electron microscopic imaging

P. aeruginosa strains attached to the coverslip and wing were imaged using SEM. The results (Table II) indicated that P. aeruginosa ATCC 9027 was attached in higher numbers to the coverslip than the wing. On the contrary, PAO1 was attached in higher numbers to the wing than the coverslip. This is in line with the staining and enumeration results presented in Fig. 4. From the SEM images, in addition to the cellular attachment, EPS deposited on the surface was also observed.Table icon

TABLE II. Scanning electron microscopic images of P. aeruginosa ATCC 9027 and PAO1 attached to the surface of glass coverslip and wing after 24h of interaction under flow conditions with shear rates of 1.0, 1.8, and 2.4s−1. Images were captured at 12000× and the bar represents 5μm.

Shear rate (s−1)1.01.82.4ATCC 9027 on coverslipATCC 9027 on wingPAO1 on coverslipPAO1 on wingThe results of bacterial viability (Fig. 3) and attachment (Fig. 4) indicated that P. aeruginosa ATCC 9027 attached to the wing was lesser by 1 × 104 cells than PAO1 but was killed in relatively higher numbers than PAO1. Table II showed the bacterial EPS deposition. Bacteria secrete EPS for aiding the initial attachment and for biofilm formation.3131. V. Carniello, B. W. Peterson, H. C. van der Mei, and H. J. Busscher, Adv. Colloid Interface Sci. 261, 1 (2018). https://doi.org/10.1016/j.cis.2018.10.005 EPS secretion in bacteria was also observed to increase with stress. Bacteria attached to the nanopillars experience more stress than bacteria attached to the relatively flat surface.3232. C. D. Bandara, S. Singh, I. O. Afara, A. Wolff, T. Tesfamichael, K. Ostrikov, and A. Oloyede, ACS Appl. Mater. Interfaces 9, 6746 (2017). https://doi.org/10.1021/acsami.6b13666 Bacterial attachment to the nanopillars create a high local pressure in the bacterial cell wall enhancing EPS secretion.3333. K. Myszka and K. Czaczyk, Curr. Microbiol. 58, 541 (2009). https://doi.org/10.1007/s00284-009-9365-3In addition to the nanopillar surface topography, the shear rate also creates an additional stress for bacterial attachment. Nanopillars on the wing surface were less conducive for the bacterial attachment, which was indicated by the presence of relatively more EPS on the surface of the wing than on the coverslip (Table III). A study reported that bacterial EPS deposited on the nanopillars avoided the direct exposure of the bacterial cell wall to the nanopillars.3232. C. D. Bandara, S. Singh, I. O. Afara, A. Wolff, T. Tesfamichael, K. Ostrikov, and A. Oloyede, ACS Appl. Mater. Interfaces 9, 6746 (2017). https://doi.org/10.1021/acsami.6b13666Table icon

TABLE III. Scanning electron microscopic images showing the EPS secreted by P. aeruginosa strains that are left behind. Bars represent 5 μm.

Shear rate (s−1)1.01.82.4P. aeruginosa on the coverslipP. aeruginosa on the wing

D. Imaging and estimation of EPS

The SEM image of P. aeruginosa in Fig. 5 showed the secreted EPS surrounding the bacterial cell.figureThe nanospace between the bacterial membrane and nanopillars was filled by EPS secretion. Excess EPS secretion on the nanopillars, though aided bacterial attachment, eventually hampered the bactericidal efficiency of nanopillars under static conditions.3232. C. D. Bandara, S. Singh, I. O. Afara, A. Wolff, T. Tesfamichael, K. Ostrikov, and A. Oloyede, ACS Appl. Mater. Interfaces 9, 6746 (2017). https://doi.org/10.1021/acsami.6b13666 Among the three shear rates studied, 1.0 s−1 had a relatively higher bactericidal efficiency of killing 6% ATCC 9027 cells and 16% PAO1 cells when the flow setup was run for 24 h.Excess EPS secreted by P. aeruginosa was observed to be left behind on the coverslip and the wing (Table III). A similar observation was reported in P. aeruginosa in the flow cell.3434. C. Gómez-Suárez, J. Pasma, A. J. van der Borden, J. Wingender, H. Flemming, H. J. Busscher, and H. C. Van der Mei, Microbiology 148, 1161 (2002). https://doi.org/10.1099/00221287-148-4-1161 Flow rates stimulate the secretion of EPS. EPS secreted by P. aeruginosa strains masked the nanopillar topography, which could be the plausible reason for the higher viability of PAO1 on the wing surface.Figure 6 indicated that EPS secreted by ATCC 9027 increased with the increase in the shear rate both on the coverslip and the wing. On the contrary, EPS secreted by PAO1 was observed to decrease slightly with the increase in the shear rate. However, EPS produced by PAO1 was higher than ATCC 9027 till 1.8 s−1. The difference observed between PAO1 and ATCC 9027 at the shear rate of 2.4 s−1 was found to be statistically significant with a p value of 0.028. The results suggest that under flow conditions, P. aeruginosa that is able to produce more EPS would likely escape the bactericidal effect of nanopillars, as EPS masks the tips of the nanopillars.figureBacteria tend to form robust biofilms under nutrient-limited conditions than in nutrient-rich environments.3535. O. E. Petrova and K. Sauer, J. Bacteriol. 194, 2413 (2012). https://doi.org/10.1128/JB.00003-12 Bacterial biofilms formed under flow conditions attach robustly to the substrate.3636. A. K. Epstein, T. Wong, R. A. Belisle, E. M. Boggs, and J. Aizenberg, Proc. Natl. Acad. Sci. U.S.A. 109, 13182 (2012). https://doi.org/10.1073/pnas.1201973109 Higher flow rate stimulates the formation of thick and strong biofilms,3737. P. A. Araújo, J. Malheiro, I. Machado, F. Mergulhão, L. Melo, and M. Simões, J. Environ. Eng. 142, 04016031 (2016). https://doi.org/10.1061/(ASCE)EE.1943-7870.0001068 as it increases the secretion of EPS and bacterial attachment. Non-EPS producing P. aeruginosa was reported to show attachment similar to EPS producing P. aeruginosa over time under repeated running cycles in a parallel-plate flow chamber. In addition, EPS deposited by the previously run bacteria discouraged attachment of forthcoming bacteria in the flow condition. Non-EPS producing bacteria perhaps secreted EPS for adaptation to stress for attachment. EPS deposited on the substrate discourages the adhesion of itself or the competitors.3434. C. Gómez-Suárez, J. Pasma, A. J. van der Borden, J. Wingender, H. Flemming, H. J. Busscher, and H. C. Van der Mei, Microbiology 148, 1161 (2002). https://doi.org/10.1099/00221287-148-4-1161Under static conditions, P. aeruginosa PAO1 restrained itself from attaching to the wing surface and showed higher viability than ATCC 9027.2121. J. Yayan, B. Ghebremedhin, and K. Rasche, PLoS One 10, e0139836 (2015). https://doi.org/10.1371/journal.pone.0139836 Under flow conditions, P. aeruginosa adopted a different mechanism of secreting more EPS and masking the nanopillar tips to avoid the bactericidal effect. P. aeruginosa PAO1 though attached in higher numbers to the wing surface were killed in lower numbers, and this could be attributed to excess EPS secretion or its virulence trait.

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