Dynamics of Pseudomonas aeruginosa association with anionic hydrogel surfaces in the presence of aqueous divalent-cation salts

Abstract

Binding of bacteria to solid surfaces is complex with many aspects incompletely understood. We investigate Pseudomonas aeruginosa uptake kinetics onto hydrogel surfaces representative of soft-contact lenses made of nonionic poly(2-hydroxyethylmethacrylate) (p-HEMA), anionic poly(methacrylic acid) (p-MAA), and anionic poly(acrylic acid) (p-AA). Using a parallel-plate flow cell under phase-contrast microscopy, we document a kinetic "burst" at the anionic hydrogel surface: dilute aqueous P. aeruginosa first rapidly accumulates and then rapidly depletes. Upon continuing flow, divalent cations in the suspending solution sorb into the hydrogel network causing the previously surface-accumulated bacteria to desorb. The number of bacteria eventually bound to the surface is low compared to the nonionic p-HEMA hydrogel. We propose that the kinetic burst is due to reversible divalent-cation bridging between the anionic bacteria and the negatively charged hydrogel surface. The number of surface bridging sites diminishes as divalent cations impregnate into and collapse the gel. P. aeruginosa association with the surface then falls. Low eventual binding of P. aeruginosa to the anionic hydrogel is ascribed to increased surface hydrophilicity compared to the counterpart nonionic p-HEMA hydrogel.

Fig. 1

Advancing contact angles for surfaces: p-HEMA pretreated with PBS (pH = 7.4), p-MAA pretreated with PBS (pH = 7.4) with 100-mM MgCl₂, clean glass, and silanated glass each immersed in aqueous PBS (pH = 7.4) (*denotes statistically significantly different data with comparison to p-HEMA with a p-value <0.05).

Fig. 2

Volume swelling/shrinking kinetics of 4-μL p-MAA beads: (a) initially soaked in distilled/deionized water (pH = 6.2) and immersed in PBS (pH 7.4) (b) initially soaked in PBS (pH = 7.4) and immersed in PBS (pH 7.4) with 100-mM NaCl.

Fig. 3

Surface uptake kinetics of PAK suspended at 5 × 10⁶ cfu/mL in Mian’s medium accumulating on a p-HEMA surface pretreated in PBS (pH = 7.4). Open circles correspond to total bacterial accumulation. Filled circles reflect bound bacteria.

Fig. 4

Surface uptake kinetics of PAK at 5 × 10⁶ cfu/mL suspended in Mian’s medium accumulating on a 70% p-HEMA/30% p-MAA surface pretreated in PBS (pH = 7.4). Open circles correspond to total bacterial accumulation. Filled circles reflect bound bacteria.

Fig. 5

Total surface uptake kinetics of PAK at 5 × 10⁶ cfu/mL suspended in Mian’s medium accumulating on anionic-polycarboxylate homopolymers p-MAA (filled circles) and p-AA (open circles) each pretreated in PBS (pH = 7.4).

Fig. 6

Total surface uptake kinetics for PAK at 5 × 10⁶ cfu/mL suspended in Mian’s medium accumulating on p-MAA membranes with differing pretreatments: aqueous 100-mM NaCl in PBS solution (pH = 7.4) (filled circles) or distilled/deionized water (pH = 6.2) (open triangles).

Fig. 7

Volume shrinking kinetics of 4-μL p-MAA beads initially soaked in PBS (pH = 7.4) followed by immersion in PBS (pH 7.4) with 10-mM MgCl₂.

Fig. 8

Total surface uptake kinetics of PAK at 5 × 10⁶ cfu/mL accumulating on p-MAA membranes pretreated in Mian’s medium (open triangles), in 10-mM MgCl₂ aqueous PBS solution (pH = 7.4) (open squares), and in 100-mM MgCl₂ aqueous PBS solution (pH = 7.4) (closed squares). Open triangles are partially hidden.

Fig. 9

Burst initiation with divalent cations. Total surface uptake kinetics of PAK at 5 × 10⁶ cfu/mL on a p-AA membrane pretreated in PBS (pH = 7.4). (a) Initially the injected suspension medium is Mian’s without MgSO₄ (pH = 7.4). After 1 h, 10-mM MgCl₂ was added to the flowing bacterial suspension. (b) Initially, the injected suspension medium is 100-mM KCl and 10-mM CH₃COONa at pH = 7.4. After 1 h, 5-mM CaCl₂ was added to the flowing bacterial suspension.

Fig. 10

Volume swelling kinetics of 4-μL p-MAA beads upon exposure to EDTA. p-MAA was initially soaked in PBS (pH = 7.4) followed by immersion in PBS (pH 7.4) with 10-mM MgCl₂ (see Fig. 7). At the end of shrinkage in Fig. 7, 10-mM EDTA was added to the MgCl₂/PBS solution.

Fig. 11

Magnesium burst recovery with EDTA. Total surface uptake kinetics of PAK at 5 × 10⁶ cfu/mL in Mian’s medium (pH = 7.4) for p-AA membranes pretreated in PBS (pH = 7.4). After 1 h, 10-mM EDTA is added to the suspending solution. After about an additional 30 min, 10-mM MgCl₂ is added to the suspending solution.

Fig. 12

Calcium burst recovery with EDTA. Total surface uptake kinetics of PAK at 5 × 10⁶ cfu/mL in aqueous 100-mM KCl, 10-mM CH₃COONa, and 5-mM CaCl₂ (pH = 7.4) for p-AA membranes pretreated in 100-mM KCl and 10-mM CH₃COONa (pH = 7.4).

Fig. 13

Schematic of proposed bridging/collapse mechanism for PAK bursting. Initially, the hydrogel is expanded allowing divalent-cation bridging between bacteria and substrate surface. Later, the hydrogel collapses destroying surface bridging sites and releasing accumulated bacteria.

Fig. 14

PAK total uptake rates at 5 × 10⁶ cfu/mL on p-HEMA pretreated with PBS (pH = 7.4), p-MAA pretreated with PBS (pH = 7.4) and 100-mM MgCl₂, glass, and silanated glass. (*denotes statistically significantly different data with comparison to p-HEMA with a p-value <0.05).