Sustained Nitric Oxide-Releasing Nanoparticles Interfere with Methicillin-Resistant Staphylococcus aureus Adhesion and Biofilm Formation in a Rat Central Venous Catheter Model

Abstract

Staphylococcus aureus is frequently isolated in the setting of infections of indwelling medical devices, which are mediated by the microbe's ability to form biofilms on a variety of surfaces. Biofilm-embedded bacteria are more resistant to antimicrobial agents than their planktonic counterparts and often cause chronic infections and sepsis, particularly in patients with prolonged hospitalizations. In this study, we demonstrate that sustained nitric oxide-releasing nanoparticles (NO-np) interfere with S. aureus adhesion and prevent biofilm formation on a rat central venous catheter (CVC) model of infection. Confocal and scanning electron microscopy showed that NO-np-treated staphylococcal biofilms displayed considerably reduced thicknesses and bacterial numbers compared to those of control biofilms in vitro and in vivo, respectively. Although both phenotypes, planktonic and biofilm-associated staphylococci, of multiple clinical strains were susceptible to NO-np, bacteria within biofilms were more resistant to killing than their planktonic counterparts. Furthermore, chitosan, a biopolymer found in the exoskeleton of crustaceans and structurally integrated into the nanoparticles, seems to add considerable antimicrobial activity to the technology. Our findings suggest promising development and translational potential of NO-np for use as a prophylactic or therapeutic against bacterial biofilms on CVCs and other medical devices.

Keywords: Staphylococcus aureus; antimicrobials; biofilms; nanoparticles; nitric oxide.

FIG 1

Nitric oxide-releasing nanoparticles (NO-np) inhibit methicillin-resistant S. aureus (MRSA) strain 6498 cells. (A) MRSA strain 6498 planktonic cells were grown on polystyrene microtiter plates for 24 h at 37°C in the absence (control; Ctrl) or presence of increasing NO-np (1.25, 2.5, 5, 10, and 20 mg/ml) concentrations. Each point represents the average of three spectrophotometric measurements (optical density at 600 nm [OD₆₀₀]), and error bars indicate standard deviations (SDs). Statistical significance (*, P < 0.0001) was calculated by analysis of variance (ANOVA). (B) The effect of NO (5 mg/ml) on MRSA growth kinetics was determined using spectrophotometry (OD₆₀₀) for 24 h. Each symbol represents the average of three measurements for control, np, or NO-np treatment, and error bars indicate SDs. Statistical significance (P < 0.05 in comparing the results of control, np, and NO-np treatments) was calculated by multiple t tests. *, higher OD compared to np group; #, higher OD compared to NO-np group. (A and B) The initial inoculum was 10⁶ staphylococci per well.

FIG 2

NO-np are effective against S. aureus planktonic and biofilm-associated cells. The levels of bacterial viability of six distinct S. aureus clinical isolates in biofilms and planktonic cells were determined by CFU counts. Both phenotypes were exposed to 1.25, 2.5, and 5 mg/ml of np or NO-np for 24 h, and their viability was compared to that of bacteria (5 × 10⁶ bacteria per ml) incubated in medium alone. For biofilm formation, the initial inoculum was 10⁶ staphylococci per well. The biofilms were allowed to form for 24 h. Each symbol represents the result for a single strain. Black lines are the averages of the results for the six isolates, and error bars denote SDs. Statistical significance (*, P < 0.05 in comparing the results for biofilms and planktonic cells) was calculated by multiple t tests and adjusted by using the Holm-Sidak method. This experiment was performed twice, with similar results obtained each time.

FIG 3

NO-np interferes with adhesion of MRSA strain 6498 to glass-bottom plates. (A) Adhesion to a solid substrate was investigated using poly-d-lysine-coated 35-mm glass-bottom plates and confocal microscopy. Bacteria (10⁶ per plate) were allowed to adhere for 90 min in the absence and presence of np or NO-np. After treatment, the plates were rinsed to remove nonadherent cells, attached bacteria were stained with SYTO9 (green fluorescence), images were taken, and the numbers of attached bacteria were counted. Then, the percentage of attached bacteria treated with np or NO-np was calculated relative to the count for the untreated control. Bars represent the averages of five replicates, and error bars denote SDs. Statistical significance (**, P < 0.01; ****, P < 0.0001) was calculated by ANOVA. (B) Representative images of adhesion by control and np- or NO-np-treated MRSA cells. Scale bar, 10 μm. This experiment was performed twice, with similar results obtained each time.

FIG 4

MRSA 6498 cells within mature biofilms are effectively killed by NO-np. Microbial biofilms were grown on polystyrene microtiter or glass-bottom plates for 24 h at 37°C and incubated in the absence and presence of np or NO-np. For biofilm formation, the initial inoculum was 10⁶ MRSA cells per plate. (A) The viability (percentage of control) of biofilm-associated cells was evaluated using the FDA assay. (B) The differences in biofilm thicknesses were examined after exposure to np or NO-np and compared with the biofilm thickness of the untreated control. (A and B) Bars represent the average results from three wells, and error bars denote SDs. Statistical significance (*, P < 0.05; **, P < 0.01) was calculated by ANOVA. (C) Confocal microscopy of MRSA 6498 strain biofilms after treatment with NO-np. Images of mature bacterial biofilms showed exopolymeric matrix (red; stained with concanavalin A-Texas Red conjugate) and bacterial cells (green; stained with SYTO9). Images were obtained after 24-h coincubation of the bacterial cells in the absence and presence of np or NO-np. (D) The thickness and morphology of each biofilm can be observed in the Z-stack reconstruction. The pictures were taken at a magnification of ×100. (C and D) Scale bar represents 20 μm for all images. (A to D) These experiments were performed twice, with similar results obtained each time.

FIG 5

NO-np prevent MRSA strain 6498 biofilm formation in central venous catheters (CVCs) implanted in rats. MRSA biofilms were grown in vitro using catheter material as a substrate for 24 h at 37°C and then treated with 5 mg/ml of np or NO-np and compared to untreated controls. For biofilm formation, the initial inoculum was 10⁶ MRSA cells per plate. The microbial mass and viability of biofilm-associated cells were evaluated using the CFU (A) and FDA (B) assays. (A and B) Bars represent the average results from three catheters, and error bars denote SDs. Statistical significance (*, P < 0.05; ***, P < 0.001) was calculated by ANOVA. These experiments were performed twice, with similar results obtained each time. (C) Mean bacterial burdens in in vivo catheters incubated with 10⁶ MRSA cells/ml for 48 h are shown. CVCs implanted in the animals were treated with 5 mg/ml of np or NO-np at 24 h postinfection. This experiment was performed once using three animals (average results from three 5-mm pieces of each catheter per rat) per group. In addition, statistical significance (*, P < 0.05) was calculated using ANOVA. Bars represent the average results from three catheters, and error bars denote SDs. (D) Scanning electron microscopy (SEM) examination of MRSA strain biofilm formation on catheters placed in the jugular vein of a Sprague-Dawley rat and treated with PBS or 5 mg/ml of np or NO-np. Scale bar represents 1 μm for all images.