Inhibitory effect of 405 nm laser light on bacterial biofilm in urethral stent

Abstract

The clinical use of urethral stents is usually complicated by various adverse effects, including dysuria, fever, and urinary tract infection (UTI). Biofilms (formed by bacteria, such as Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) adhering to the stent cause UTIs in stented patients (approximately 11%). The undesirable consequences of antibiotics use include bacterial resistance, weight gain, and type 1 diabetes, which occur when antibiotics are used for a long time. We aimed to assess the efficacy of a new optical treatment with a 405 nm laser to inhibit bacterial growth in a urethral stent in vitro. The urethral stent was grown in S. aureus broth media for three days to induce biofilm formation under dynamic conditions. Various irradiation times with the 405 nm laser light were tested (5, 10, and 15 min). The efficacy of the optical treatment on biofilms was evaluated quantitatively and qualitatively. The production of reactive oxygen species helped eliminate the biofilm over the urethral stent after 405 nm irradiation. The inhibition rate corresponded to a 2.2 log reduction of colony-forming units/mL of bacteria after 0.3 W/cm² of irradiation for 10 min. The treated stent showed a significant reduction in biofilm formation compared with the untreated stent, as demonstrated by SYTO 9 and propidium iodide staining. MTT assays using the CCD-986sk cell line revealed no toxicity after 10 min of irradiation. We conclude that optical treatment with 405 nm laser light inhibits bacterial growth in urethral stents with no or minimal toxicity.

Figure 1

Growth of bacterial biofilm: (a) urethral stents stained with crystal violet at various time points after culturing and (b) bacterial viability versus culture time.

Figure 2

Response of normal cells (CCD-986sk) to 405 nm laser irradiation: (a) cell viabilities of various irradiation times (CTRL = control; (^*p < 0.05 vs. CTRL; N = 5) and (b) temperature development during laser irradiation on surface of CCD-986sk suspension in DMEM media.

Figure 3

Inhibition of bacterial biofilm after 405 nm laser irradiation: (a) bacterial viability after exposure of 405 nm laser light at various irradiation times (CTRL = control; ^*p < 0.05 and ^**p < 0.001 vs. CTRL; N = 5) and (b) temperature development during laser irradiation on urethral stent surface.

Figure 4

S. aureus bacterial inhibition after exposure of 405 nm laser at various irradiation times: (a) bacterial cells on agar plates (CTRL = control) and (b) quantification of bacterial removal in log CFU/mL (^*p < 0.05 and ^**p < 0.01 vs. CTRL; N = 10).

Figure 5

Optical inhibition effect of 405 nm laser light against S. aureus in urethral stent: (a) ROS generation during treatment, (b) DPPH scavenging activity, and (c) concentration of protein leakage using Lowry method (^*p < 0.05 and ^**p < 0.001 vs. CTRL; ns = not significant; N = 5), and (d) SEM images of S. aureus after laser treatment (blue, orange, and red arrow represent thick biofilm, bacteria released from biofilm, and bacterial death with morphological damage (scale bar = 2 µm; 20 kX and scale bar of inlet = 20 µm; 2 kX).

Figure 6

Analysis of bacterial membrane integrity after laser treatment: (a) live bacteria stained with SYTO 9, (b) dead bacteria stained with PI, (c) quantification of pixel intensity for live bacteria (green intensity), and (d) quantification of pixel intensity in dead bacteria (red intensity) (^**p < 0.001 vs. CTRL; ns = not significant; N = 7; scale bar = 100 µm; 40X).

Figure 7

Schematic illustrations of bacteria preparation: (a) bacterial culturing in urethral stent under dynamic condition, (b) examples of urethral stent before (day 0) and after culturing, and (c) 405 nm laser treatment set up. (day 3; stained with crystal violet; TSB = tryptic soy broth media; SA = S. aureus suspension; WB = water bath; US = urethral stent; UM = used media;P1, P2 = pump, S1 = small silicone, S2 = large silicone, and DF = diffusing fiber).