Scott P. Nichols and
Mark H. Schoenfisch*
Department of Chemistry, Caudill Laboratories, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. E-mail: schoenfisch@unc.edu
First published on 17th July 2013
Nitric oxide (NO) is an endogenous antibacterial agent produced by immune cells in response to pathogens. Herein, the NO fluxes necessary to reduce bacterial adhesion of different bacteria (S. aureus, methicillin-resistant S. aureus, S. epidermidis, E. faecalis, E. coli, and P. aeruginosa) were investigated to ascertain the sensitivity of these bacteria to NO. S-Nitrosothiol NO donor-modified xerogels were selected as a model NO-release surface due to their extended NO-release kinetics relative to other NO donor systems. The xerogels were coated with poly(vinyl chloride) (PVC) to achieve consistent surface energy between NO-releasing and control substrates. Fibrinogen was pre-adsorbed to these materials to more accurately mimic conditions encountered in blood and promote bacteria adhesion. Nitric oxide fluxes ranging from 20–50 pmol cm−2 s−1 universally inhibited the bacterial adhesion by >80% for each strain studied. Maximum bacteria killing activity (reduced viability by 85–98%) was observed at the greatest NO payload (1700 nmol cm−2).
Implant-associated infections are the result of protein adsorption, bacterial adhesion, and subsequent colonization.4–6 The colonized bacteria excrete an extracellular matrix offering protection from the immune response and external treatment (e.g., antibiotics) in the form of a biofilm. Eradication of biofilm bacteria requires significantly greater antibiotic concentrations than killing of planktonic bacteria.7 Without complete eradication, bacteria can proliferate and recolonize the material.8 With the emergence of antibiotic-resistant strains, device removal or replacement is often the only possible treatment option.9,10 Fortunately, the prevention and killing of adhered bacteria have been shown to decrease the incidence of implant-associated infections.4
To date, many clinically approved devices do not adequately prevent bacterial adhesion and biofilm formation.11 The development of anti-bacterial materials is thus important for addressing device-associated infections. This goal has been pursued through the development of both surface coatings that passively reduce protein/bacterial adhesion and biomaterials that actively release an antimicrobial agent.1 Passive antibacterial materials (e.g., polyethylene glycol, quaternary ammonium-modified interfaces)12–14 have a limited sphere of influence as they only affect bacteria in direct contact with the surface. Often, this sphere of bacterial efficacy is reduced upon protein adsorption.1 In contrast, materials that actively release antibacterial agents can impact bacteria both in contact with and in the vicinity of a surface. Antibacterial agents that have been effectively loaded into polymeric biomaterials for subsequent release include antibiotics,15,16 antibodies,17 silver ions,18 and nitric oxide (NO).19,20
Nitric oxide, a reactive, diatomic radical produced by immune cells by the body response to pathogens,21,22 has received considerable attention as an antibacterial agent due to its localized action and broad spectrum activity. Materials capable of storing and releasing NO have been the focus of intense research for the goal of reducing bacterial adhesion and implant-associated infections.23,24 Surface NO release has been shown to influence both bacterial adhesion and biofilm formation.25,26 Nablo and Schoenfisch reported that NO fluxes ≥30 pmol cm−2 s−1 inhibited Pseudomonas aeruginosa adhesion by 85%.20 Charville et al. subsequently examined the reduction of bacterial adhesion due to NO flux in the presence of adsorbed fibrinogen (Fg) to better mimic in vivo conditions.19 While the adsorbed fibrinogen enhanced bacterial adhesion, a NO flux-dependent reduction of bacterial adhesion was still observed for both Gram-positive and negative bacteria.
The potential to both reduce adhesion and kill adhered bacteria sets NO apart from other antibacterial agents. Hetrick and Schoenfisch previously reported the killing efficacy of NO for P. aeruginosa as a function of NO payload.27 Herein, we further these studies by investigating the NO fluxes necessary to inhibit bacterial adhesion and reduce the viability of adhered bacteria as a function of bacteria species and class. Such studies are critical for understanding the clinical potential of NO-releasing interfaces.
To reach the desired NO flux, the PVC-coated NO-releasing xerogels were incubated at 37 °C for 0.5–96 h. The substrates were then immersed in 4 mL of a 20 μg mL−1 solution of Fg in PBS (pH 7.4) for 90 min at 37 °C (gentle shaking). A concentration of 20 μg mL−1 Fg enabled the formation of a protein monolayer (and not protein agglomerates and/or multilayers) on the PVC.29 The PVC-coated xerogels were then removed from the Fg solution and immediately immersed in 4 mL of a bacterial suspension (108 CFU mL−1) at 37 °C for 60 min.
Initial experiments revealed that S. aureus, MRSA, and E. coli did not readily adhere to control PVC-coated substrates (<0.10 relative surface coverage). Low absolute adhesion for these strains would make accurate measurements of the relative adhesion to NO-releasing substrates difficult due to the error inherent in the optical microscopy evaluation. To enhance bacterial adhesion and mimic in vivo biofouling more closely, PVC-coated slides were pre-incubated in fibrinogen (Fg) solutions prior to bacteria exposure. Contact angles of Fg-adsorbed PVC-coated control and NO-releasing xerogels were both 55 ± 5° (decreased from 89°), confirming Fg adsorption and the expected surface alteration. Charville et al. previously reported that NO-release does not influence Fg adsorption onto PVC interfaces.19 Soaking the Fg-adsorbed surfaces in PBS for 24 h had no effect on the contact angle indicating that the Fg layer is not weakly bound.
The NO-release properties of the bare and PVC-coated S-nitrosothiol xerogels are provided in Table 1. As expected, the PVC topcoat did not significantly alter the NO-release properties of the xerogels since the NO release is thermally triggered and NO readily diffuses through PVC.28,34 The substrates released NO for at least 14 d at low NO fluxes (e.g., 0.16 pmol cm−2 s−1). The average NO flux employed for the bacteria studies ranged from 0.5–50 pmol cm−2 s−1, with 50 pmol cm−2 s−1 selected as the greatest flux due to unpreventable loss of NO during the pre-incubation of the xerogels in Fg prior to bacteria exposure. Prior to immersion in bacteria solutions, the greatest NO flux measured for the PVC-coated NO-releasing xerogels was 260 pmol cm−2 s−1 (Table 1). The NO flux changes by a large degree initially due to the pseudo first-order NO-release kinetics common for S-nitrosothiol-modified xerogels.28
NO-release properties | 40% MPTMS–MTMOS xerogel | PVC-coated 40% MPTMS–MTMOS xerogel |
---|---|---|
a Time to reach maximum NO flux. | ||
tmax![]() |
1.1 ± 0.2 | 1.2 ± 0.2 |
[NO]max (pmol cm−2 s−1) | 290 ± 70 | 260 ± 60 |
[NO]1 h (pmol cm−2 s−1) | 64 ± 10 | 58 ± 8 |
[NO]5 h (pmol cm−2 s−1) | 26 ± 5 | 22 ± 4 |
[NO]12 h (pmol cm−2 s−1) | 9 ± 1 | 8 ± 1 |
[NO]24 h (pmol cm−2 s−1) | 3.4 ± 0.5 | 3.2 ± 0.5 |
[NO]48 h (pmol cm−2 s−1) | 1.2 ± 0.1 | 1.1 ± 0.1 |
[NO]96 h (pmol cm−2 s−1) | 0.5 ± 0.1 | 0.5 ± 0.1 |
Total NO (μmol cm−2) | 2.6 ± 0.4 | 2.5 ± 0.3 |
Based on prior adhesion results,19,20,27 we predicted a negative correlation between bacterial adhesion and NO flux. Indeed, a NO flux-dependent decrease in bacterial adhesion was observed for all six bacterial strains investigated (Fig. 1) relative to controls (with 1.0 indicating identical adhesion to the control substrates). S. aureus, MRSA, E. faecalis, and E. coli showed a greater susceptibility to NO from 0–20 pmol cm−2 s−1 with little to no additional reduction in bacterial adhesion (surface coverage) at increased NO fluxes. This maximum plateau in reduced adhesion corroborates a previous report by Nablo and Schoenfisch where NO release resulted in a large initial reduction in bacterial adhesion but was followed by little additional effect beyond a certain NO flux threshold.20 In contrast, P. aeruginosa and S. epidermidis required greater NO fluxes to inhibit adhesion by 50% (20 and 35 pmol cm−2 s−1, respectively) and 80% (35 and 50 pmol cm−2 s−1, respectively). While increased NO fluxes were necessary to reduce adhesion for these particular strains, the NO release still decreased adhesion by >80%, analogous to the relative inhibition observed for other strains (Table 2).
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Fig. 1 The NO flux-dependent relative adhesion of (A) S. aureus, (B) MRSA, (C) S. epidermidis, (D) E. faecalis, (E) E. coli, and (F) P. aeruginosa to pre-adsorbed Fg PVC-coated xerogels after 1 h of exposure. A relative adhesion of 1.0 represents the adhesion of the strain to control (i.e., non-NO-releasing) substrates. Data are mean ± standard deviation. |
Bacteria strain | Gram class | Linear regression slope (relative adhesion per pmol cm−2 s−1)a | r2 | NO flux required to reduce adhesion by 50% (pmol cm−2 s−1) | NO flux required to reduce adhesion by 80% (pmol cm−2 s−1) | Absolute surface coverage to controls (%) |
---|---|---|---|---|---|---|
a Slopes calculated from NO fluxes between 0–20 pmol cm−2 s−1. | ||||||
S. aureus | + | −0.043 ± 0.005 | 0.922 | 10 | 20 | 17.1 ± 2.7 |
MRSA | + | −0.037 ± 0.005 | 0.914 | 10 | 20 | 16.8 ± 2.3 |
S. epidermidis | + | −0.017 ± 0.002 | 0.939 | 35 | 50 | 14.7 ± 1.9 |
E. faecalis | + | −0.043 ± 0.004 | 0.954 | 10 | 20 | 13.5 ± 2.7 |
E. coli | − | −0.040 ± 0.004 | 0.953 | 10 | 20 | 19.5 ± 3.5 |
P. aeruginosa | − | −0.036 ± 0.003 | 0.984 | 20 | 35 | 47.2 ± 4.2 |
Linear regressions of the relative bacteria adhesion (measured as surface coverage) as a function of NO flux were performed from 0–20 pmol cm−2 s−1 to compare the relative susceptibility of each bacterial strain to NO (Table 2). This range was chosen because all strains showed a linear (r2 = 0.91–0.98) reduction in adhesion, allowing for direct comparison between strains. This linearity only held true until ∼80% reduction in bacterial adhesion. All of the strains studied except S. epidermidis were characterized as having similar linear regression slopes, ranging from −0.036 to −0.043 relative bacterial adhesion per pmol cm−2 s−1 of NO. Slight variations in slope were independent of the class of bacteria (Gram-positive or Gram-negative) and likely an attribute of the broad-spectrum activity of NO arising from multiple mechanisms of action on bacteria (e.g., nitrosative and oxidative stress).35 In our study, S. epidermidis was the only bacterium with a relatively low susceptibility to NO with respect to bacterial adhesion (slope of −0.017). Charville et al. reported similar behavior when investigating the role of Fg and NO release on bacterial adhesion,19 S. epidermidis is indeed unique in that it has Fg-binding proteins such as serine-aspartate repeat G (SdrG) present on its surface.36 While S. aureus also has Fg-binding proteins (e.g., Clumping Factor A),37 the dissociation constant (KD) of SdrG is two orders of magnitude lower than Clumping Factor A, indicating a significantly increased binding strength.38 The large affinity of SdrG to Fg may decrease the susceptibility of S. epidermidis to the anti-adhesive effects of surface-derived NO. Despite the relatively low efficacy of NO in preventing S. epidermidis adhesion, NO release at fluxes >20 pmol cm−2 s−1 further diminished the bacterial surface coverage with an 81% reduction in adhesion compared to control surfaces for a flux of 50 pmol cm−2 s−1. Greater NO fluxes may further inhibit S. epidermidis adhesion; however, the maximum average NO flux that could be achieved over the 1 h study using the S-nitrosothiol-modified xerogels was limited to 50 pmol cm−2 s−1.
Initial NO flux (pmol cm−2 s−1) | NO payload (nmol cm−2) | ||
---|---|---|---|
6 h | 12 h | 24 h | |
0.5 | 9.5 ± 0.8 | 19 ± 2 | 38 ± 2 |
1.0 | 17 ± 2 | 33 ± 3 | 66 ± 4 |
2.5 | 40 ± 3 | 72 ± 5 | 105 ± 9 |
5.0 | 90 ± 9 | 140 ± 10 | 240 ± 20 |
10 | 150 ± 20 | 250 ± 20 | 370 ± 20 |
20 | 270 ± 20 | 410 ± 30 | 550 ± 50 |
35 | 390 ± 50 | 560 ± 50 | 740 ± 60 |
50 | 1300 ± 200 | 1500 ± 200 | 1700 ± 200 |
To ensure that only bacteria adhered to the PVC-coated xerogel and not to the sides or back of the glass substrates were enumerated, the outer PVC layer was physically removed from the glass slide and sonicated. These bacteria were then plated onto TSA plates and enumerated to quantify viable bacteria.27 Optical microscopy confirmed that this sonication step removed >93% of the adhered bacteria and thus the viability quantification was an accurate measurement. Relative adhered bacterial viability was calculated as a function of time by dividing the viable bacteria concentration at x = 6, 12, or 24 h by the viable bacteria at t = 0 for a given bacteria strain and NO flux (eqn (1)). A relative viability of 1 would thus indicate that the NO release
![]() | (1) |
To ascertain whether NO was solely preventing adhesion or also killing adhered bacteria during the 1 h adhesion period, relative adhesion and relative viability were compared for the S. aureus and E. coli strains (Fig. 2). The relative surface coverage as determined by microscopy and relative adhesion of viable bacteria were generally comparable at all NO fluxes after a 1 h exposure. For example, S. aureus relative viability at substrates with an average NO flux of 50 pmol cm−2 s−1 was ∼0.15 that of control substrates, in line with the ∼0.10 relative adhesion observed with microscopy (Fig. 2A). The positive agreement between surface coverage and viable bacteria counts immediately following the adhesion assay indicates that surface NO release initially only influences bacterial adhesion and not adhered bacterial cell death.
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Fig. 2 The NO flux-dependent relative adhesion (open) and viability (filled) immediately following the 1 h adhesion period for (A) S. aureus and (B) E. coli to pre-adsorbed Fg PVC-coated xerogels. A relative adhesion or relative viability of 1.0 represents the adhesion or viability of the strain to control (i.e., non-NO-releasing) substrates. Data are mean ± standard error of the mean. |
While NO works primarily to prevent bacterial adhesion initially (during ∼1 h), NO uniquely kills adherent bacteria at longer exposure times making it an unusual antibacterial agent. This time-dependent viability of bacteria adhered to the NO-releasing surfaces is provided in Fig. 3 and Tables 4–6 for 6, 12, and 24 h NO-release conditions. The reduction in adhered bacteria viability was dependent on NO payload for all strains, albeit time dependent. For example, nearly all strains (MRSA, S. epidermidis, E. faecalis, E. coli, and P. aeruginosa) exhibited reduced viability at NO-releasing substrates at 6 h. Unexpectedly, the viability of adhered S. aureus was minimal prior to the 24 h time point (Table 6). As indicated by the data, this strain of S. aureus may require a minimal NO threshold to induce killing. S. aureus is known to resist NO via metabolic activities and as such killing may require greater NO concentrations, although it is uncertain why MRSA was not affected in this manner.45
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Fig. 3 Relative viability of S. aureus, MRSA, S. epidermidis, E. faecalis, E. coli, and P. aeruginosa at 3 different initial NO fluxes (1.0, 10, and 50 pmol cm−2 s−1) at incubation times of 6, 12, and 24 h. |
Bacteria strain | Initial average NO flux (pmol cm−2 s−1) | |||||||
---|---|---|---|---|---|---|---|---|
0.5 | 1.0 | 2.5 | 5.0 | 10 | 20 | 35 | 50 | |
a Relative viability >1 indicates bacteria proliferation. | ||||||||
S. aureus | 1.05 ± 0.19a | 1.06 ± 0.19a | 0.85 ± 0.17 | 0.69 ± 0.28 | 0.61 ± 0.23 | 0.84 ± 0.31 | 0.86 ± 0.21 | 0.87 ± 0.23 |
MRSA | 0.93 ± 0.28 | 0.93 ± 0.30 | 0.46 ± 0.17 | 0.39 ± 0.12 | 0.42 ± 0.16 | 0.46 ± 0.15 | 0.45 ± 0.18 | 0.32 ± 0.12 |
S. epidermidis | 1.18 ± 0.30a | 0.84 ± 0.21 | 0.75 ± 0.22 | 1.01 ± 0.30a | 0.41 ± 0.18 | 0.28 ± 13 | 0.25 ± 0.10 | 0.24 ± 0.08 |
E. faecalis | 0.96 ± 0.30 | 0.94 ± 0.20 | 0.42 ± 0.05 | 0.24 ± 0.07 | 0.22 ± 0.06 | 0.27 ± 10 | 0.33 ± 0.10 | 0.29 ± 0.09 |
E. coli | 1.16 ± 0.10a | 1.16 ± 0.15a | 0.51 ± 0.17 | 0.21 ± 0.10 | 0.22 ± 0.08 | 0.27 ± 0.08 | 0.17 ± 0.05 | 0.16 ± 0.04 |
P. aeruginosa | 0.92 ± 0.06 | 1.10 ± 0.15a | 0.41 ± 0.18 | 0.20 ± 0.07 | 0.18 ± 0.06 | 0.20 ± 0.08 | 0.06 ± 0.02 | 0.05 ± 0.01 |
Bacteria strain | Initial average NO flux (pmol cm−2 s−1) | |||||||
---|---|---|---|---|---|---|---|---|
0.5 | 1.0 | 2.5 | 5.0 | 10 | 20 | 35 | 50 | |
S. aureus | 0.69 ± 0.06 | 0.61 ± 0.25 | 0.65 ± 0.09 | 0.39 ± 0.09 | 0.54 ± 0.13 | 0.56 ± 0.16 | 0.60 ± 0.15 | 0.49 ± 0.06 |
MRSA | 0.75 ± 0.18 | 0.82 ± 0.26 | 0.34 ± 0.13 | 0.34 ± 0.14 | 0.23 ± 0.08 | 0.54 ± 0.13 | 0.28 ± 0.10 | 0.21 ± 0.06 |
S. epidermidis | 0.72 ± 0.20 | 0.75 ± 0.22 | 0.40 ± 0.15 | 0.57 ± 0.17 | 0.34 ± 0.11 | 0.24 ± 0.09 | 0.20 ± 0.08 | 0.20 ± 0.08 |
E. faecalis | 0.91 ± 0.04 | 0.61 ± 0.10 | 0.34 ± 0.11 | 0.20 ± 0.08 | 0.21 ± 0.07 | 0.21 ± 0.06 | 0.16 ± 0.05 | 0.11 ± 0.03 |
E. coli | 0.95 ± 0.22 | 0.73 ± 0.22 | 0.38 ± 0.13 | 0.16 ± 0.04 | 0.14 ± 0.05 | 0.07 ± 0.02 | 0.10 ± 0.03 | 0.08 ± 0.02 |
P. aeruginosa | 0.57 ± 0.02 | 0.46 ± 0.10 | 0.33 ± 0.05 | 0.25 ± 0.12 | 0.16 ± 0.06 | 0.08 ± 0.02 | 0.05 ± 0.02 | 0.04 ± 0.01 |
Bacteria strain | Initial average NO flux (pmol cm−2 s−1) | |||||||
---|---|---|---|---|---|---|---|---|
0.5 | 1.0 | 2.5 | 5.0 | 10 | 20 | 35 | 50 | |
S. aureus | 0.67 ± 0.17 | 0.66 ± 0.19 | 0.69 ± 0.23 | 0.43 ± 0.16 | 0.25 ± 0.12 | 0.38 ± 0.12 | 0.22 ± 0.07 | 0.15 ± 0.06 |
MRSA | 0.66 ± 0.13 | 0.72 ± 0.18 | 0.27 ± 0.11 | 0.25 ± 0.09 | 0.21 ± 0.06 | 0.18 ± 0.06 | 0.19 ± 0.07 | 0.15 ± 0.04 |
S. epidermidis | 0.80 ± 0.12 | 0.80 ± 0.19 | 0.38 ± 0.12 | 0.45 ± 0.16 | 0.34 ± 0.13 | 0.20 ± 0.08 | 0.15 ± 0.05 | 0.14 ± 0.04 |
E. faecalis | 0.83 ± 0.07 | 0.50 ± 0.20 | 0.34 ± 0.02 | 0.21 ± 0.03 | 0.23 ± 0.02 | 0.22 ± 0.04 | 0.13 ± 0.05 | 0.09 ± 0.02 |
E. coli | 0.76 ± 0.11 | 0.36 ± 0.07 | 0.36 ± 0.08 | 0.14 ± 0.05 | 0.11 ± 0.04 | 0.08 ± 0.02 | 0.09 ± 0.03 | 0.05 ± 0.01 |
P. aeruginosa | 0.66 ± 0.03 | 0.34 ± 0.04 | 0.29 ± 0.11 | 0.13 ± 0.04 | 0.14 ± 0.04 | 0.04 ± 0.01 | 0.04 ± 0.01 | 0.02 ± 0.004 |
The lowest 24 h NO payload investigated (38 nmol cm−2) eradicated 17–34% of the adhered bacteria regardless of bacteria species. Conversely, the greatest 24 h NO payload (1700 nm cm−2) caused maximal reductions in adhered bacteria viability of 85, 85, 86, 92, 95, and 98% for S. aureus, MRSA, S. epidermidis, E. faecalis, E. coli, and P. aeruginosa, respectively (Table 6). Therefore, substrates with an initial NO flux of 50 pmol cm−2 s−1 delivering a payload of 1700 nmol cm−2 over 24 h inhibited adhesion of all strains by >80% with concomitant eradication of ≥85% of the adhered bacteria. After the 1 h adhesion period, the initial NO flux of 50 pmol cm−2 s−1 resulted in a bacterial viability of 5 × 105–1 × 106 CFU cm−2 for each of the six strains examined. As such, enough viable bacteria were present that could still colonize the material.8 Of note, significantly larger concentrations of bacteria were employed in this study compared to what might be encountered clinically.
The NO payloads necessary to decrease the adhered bacteria viability by 50 and 80% after 24 h are provided in Table 7. Cumulative NO release levels of 66–240 and 240–1700 nmol cm−2 were required to reduce adhered bacteria viability by 50 and 80%, respectively. P. aeruginosa and E. coli were the most vulnerable strains with respect to NO-based toxicity, a result that was previously reported for planktonic bacteria.46 Importantly, the amount of bacteria killed is not proportional to the amount of NO released.
NO payload that reduced adhered bacteria viability by 50% (nmol cm−2) | NO payload that reduced adhered bacteria viability by 80% (nmol cm−2) | |
---|---|---|
S. aureus | 240 ± 20 | 1700 ± 200 |
MRSA | 105 ± 9 | 550 ± 50 |
S. epidermidis | 105 ± 9 | 550 ± 50 |
E. faecalis | 66 ± 4 | 740 ± 60 |
E. coli | 66 ± 4 | 240 ± 20 |
P. aeruginosa | 66 ± 4 | 240 ± 20 |
The rate of the total NO delivery also affected bacteria viability. While the cumulative NO payloads of xerogels with initial NO fluxes of 2.5 and 0.5 pmol cm−2 s−1 were approximately equivalent at 6 and 24 h, respectively (38–40 nmol cm−2, Table 3), E. faecalis viability at 6 h to surfaces with the greater initial NO flux (0.42 ± 0.05) was significantly lower than the viability at 24 h to surfaces with an initial NO flux of 0.5 pmol cm−2 s−1 (0.83 ± 0.07). This data suggests the bactericidal properties of NO-releasing surfaces are dependent on the NO payload, rate of delivery, and duration of NO release.
While the NO payloads used in this study were bactericidal to multiple strains, the cytotoxicity to mammalian cells represents an important consideration for future therapeutic potential. Nablo and Schoenfisch previously reported that NO-releasing PVC-coated xerogels were only slightly cytotoxic to L929 fibroblasts after 24 h incubation after an initial NO flux of ∼50 pmol cm−2 s−1.47 These previous results reveal that NO fluxes capable of greatly reducing bacterial adhesion and viability typically avoid cytotoxic effects against mammalian cells.
This journal is © The Royal Society of Chemistry 2013 |