the threat of implant infection. Fu et al. emphasized that the ideal coating to prevent implant infection both reduces bacterial adhesion and kills bacteria that adhere.8 To study the bactericidal effects of NO release on adhered P. aeruginosa, a BacLight LIVE/DEAD
Table 2.1. P. aeruginosa adhesion (percent surface coverage) under static conditions. av/v, balance BTMOS. 1 h 2 h % AHAP3a Control NO- releasing ANOVA P-value Control NO- releasing ANOVA P-Value 10 35 ± 10 23 ± 7 4.42 x 10-5 41 ± 5 33 ± 6 9.64 x 10-5 20 34 ± 9 19 ± 4 2.95 x 10-6 42 ± 7 25 ± 9 3.66 x 10-7 30 35 ± 8 13 ± 4 2.17 x 10-12 39 ± 6 18 ± 6 1.12 x 10-13 40 34 ± 4 9 ± 3 1.16 x 10-20 43 ± 7 9 ± 3 1.28 x 10-18
Table 2.2. P. aeruginosa adhesion (percent surface coverage) in parallel plate flow
chamber.a
aFlow rate = 0.2 mL/min.
bVolume percentage of AHAP3 (balance BTMOS).
cControl xerogels consist of the same silane compositions without NO-release capabilities.
Adhesion to controls at both time points was invariant of composition. For clarity, these values represent the average of adhesion to all controls.
Xerogel
Compositionb 1 h ANOVA P-Value With Control 2 h ANOVA P-Value With Control Controlc 40 ± 7 -- 40 ± 6 -- 10 32 ± 3 1.44 x 10-3 39 ± 4 4.50 x 10-1 20 28 ± 4 2.54 x 10-5 32 ± 3 1.19 x 10-3 30 21 ± 4 2.46 x 10-7 25 ±3 7.01 x 10-10 40 9 ± 1 1.56 x 10-17 14 ± 4 4.40 x 10-17
fluorescent viability kit was used to distinguish live and dead cells. After exposure to the fluorescent nucleic acid stains, live bacteria fluoresce green (Syto 9, λmax ≈ 533 nm) while
red fluorescence (propidium iodide, λmax ≈ 622 nm) indicates dead cells. Bright field and
fluorescent (Syto 9 and propidium iodide) images of bacteria adhered to control 40% AHAP3 xerogels are shown in Figure 2.6. Separate samples were stained either immediately after the 2 h adhesion procedure (Fig. 2.6 A, B, C) or after additional incubation of the surface- adhered bacteria for 7 h in PBS (Fig. 2.6 D, E, F). The Syto 9 fluorescent images (Fig. 2.6 B, E) indicate that at each time point, the vast majority of bacteria adhered to control surfaces were alive. Propidium iodide fluorescent images of the same fields (Fig. 2.6 C, F) were dark (i.e., no fluorescence), indicating that none of the adhered bacteria were dead either immediately after adhesion or after 7 h incubation. Thus, control xerogel surfaces were not bactericidal to adhered P. aeruginosa.
Similar experiments were conducted with NO-releasing 40% AHAP3 xerogels (Fig. 2.7). Syto 9 fluorescent images (Fig. 2.7 B) of adhered bacteria after the 2 h adhesion procedure indicate that the majority of bacteria were viable. In contrast, Syto 9 fluorescence was not observed for adhered cells at 7 h (Fig. 2.7 E). Images of the same field using the propidium iodide filter (Fig. 2.7 F) showed strong fluorescence, indicating that the adhered bacteria were killed by 7 h. Similar fluorescence viability studies indicated that after 5 h incubation, the adhered bacteria were still viable (data not shown), suggesting that the dose of NO necessary to kill adhered P. aeruginosa is between 375 nmol·cm-2 and 425 nmol·cm-2 (the total amount of NO released from 40% AHAP3 xerogels after 5 h and 7 h, respectively). Further examination revealed that only those bacteria cells adhered directly to the NO- releasing xerogel were killed by the NO. P. aeruginosa cells adhered to the bare glass side
Figure 2.6. Bright field (A, D), Syto 9 fluorescent (B, E), and propidium iodide fluorescent
(C, F) micrographs (20x magnification) of P. aeruginosa adhered to control (non-NO- releasing) 40% AHAP3 (v/v) xerogels (balance BTMOS). Images were acquired immediately (A, B, C) and 7 h after (D, E, F) initial bacterial adhesion.
D E F B A C D E F B A C
Figure 2.7. Bright field (A, D), Syto 9 fluorescent (B, E), and propidium iodide fluorescent
(C, F) micrographs (20x magnification) of P. aeruginosa adhered to NO-releasing 40% AHAP3 (v/v) xerogels (balance BTMOS). Images were acquired immediately (A, B, C) and 7 h after (D, E, F) initial bacterial adhesion.
D E F B A C D E F B A C D E F B A C D E F B A C
opposite the NO-releasing xerogel-coated side remained viable at 7 h (Fig. 2.8). Cells adhered directly to the glass substrate exhibited strong Syto 9 fluorescence, while no fluorescence was observed from propidium iodide. Thus, only bacterial cells in direct contact with the surface that released the NO were killed at 7 h, while cells adhered farther from the source of NO remained viable. Collectively, these results suggest that NO-releasing xerogels may mitigate biofilm formation even when bacteria manage to adhere.
Previous studies have shown that NO-releasing xerogels coated with a PVC overlayer maintained their ability to reduce bacterial adhesion despite a ~20% reduction in the 24 h NO flux.23 As such, the antibacterial efficacy of such materials was attributed solely to NO and not the xerogel matrix, for example. To further isolate the effect of NO on bacteria, we evaluated the viability of bacteria adhered to PVC-coated xerogels. Of note, the thickness of the PVC overlayer used for these studies was ~10 µm. As expected, a significant reduction in bacterial adhesion was observed after only 1 h at PVC-coated NO-releasing xerogels compared to PVC-coated controls (8 ± 3% vs. 25 ±4% surface coverage, respectively; ANOVA P-value = 3.9 x 10-6). Moreover, the NO release was cytotoxic to adhered P. aeruginosa. The propidium iodide fluorescence observed from P. aeruginosa cells adhered to PVC-coated NO-releasing xerogels for 7 h was intense, indicating cell death (data not shown). In contrast, bacteria adhered to PVC-coated controls remained viable through 7 h as evidenced by Syto 9 fluorescence. In addition to further corroborating NO’s role as an antibacterial agent, the data indicate that NO release may prove effective at rendering a range of polymers antibacterial.
To further characterize the antibacterial properties of NO-releasing xerogels, the viability of adhered P. aeruginosa was examined as a function of the total amount of NO
Figure 2.8. Bright field (A, D), Syto 9 fluorescent (B, E), and propidium iodide fluorescent
(C, F) images of P. aeruginosa adhered to the xerogel-coated side (A, B, C) and the glass side (D, E, F) of a glass microscope slide (20x magnification). Images were acquired after 7 h incubation in PBS. The cells adhered to the glass remain viable, while those adhered to the NO-releasing xerogel were killed after 7 h. Xerogel coating is 40% AHAP3 (v/v, balance BTMOS). D E F B A C D E F B A C
released. Due to both blurring during the exposure times required to obtain fluorescent images of adhered bacteria and the widely variable fluorescent intensities of Syto 9 and propidium iodide, the threshold/image analysis procedure used to determine percent bacterial surface coverage was not a valid method for obtaining quantitative viability information as a function of total NO release. Such information was instead obtained by removing adhered bacteria from the xerogel surfaces via sonication, and determining the extent of bacterial survival with a reproductive viability assay.11 Identical surface coverage values were obtained with the parallel plate flow cells by exposing each xerogel to the flowing bacterial suspension for the time required to achieve 20% coverage (verified by optical microscopy). The flowing bacterial suspension was then replaced with sterile PBS to facilitate the continued release of NO from the xerogels for 15 h. Over this period, the total NO release from 10, 20, 30, and 40% AHAP3 xerogels was 25, 53, 170, and 750 nmol·cm-2, respectively. After the 15 h incubation, the bacteria were removed from the substrate surface by sonication, serially diluted (10-fold dilutions), and plated on nutrient agar plates. The number of colonies counted on each plate after overnight incubation were then used to calculate the number of viable cells removed from the xerogel surface. As shown in Figure 2.9, a dramatic decrease in viability was observed with increasing total NO release. The number of viable P. aeruginosa cells removed from the surface of 10% AHAP3 xerogels was 6.9 x 106 while only 2.7 x 105 viable cells were removed from the 40% AHAP3 xerogels, representing a 96% decrease in viability upon increasing the total amount of NO released from 25 nmol·cm-2 to 750 nmol·cm-2 over 15 h.
Figure 2.9. Viable P. aeruginosa adhered to AHAP3 xerogels with varying NO release
capabilities removed by sonication after 15 h incubation in sterile PBS. Initial levels of P. aeruginosa adhered to each substrate were identical (20% surface coverage). Values represent average ± standard error of the mean.
0 100 200 300 400 500 600 700 800 900 1000 Vi abl e Ba cte ria Remove d F ro m Surf ace ( x 10 4 CFU )
Xerogel Composition (% AHAP3)