Slow release of nitric oxide from charged catheters and its effect on biofilm formation by Escherichia coli

Abstract

Catheter-associated urinary tract infection is the most prevalent cause of nosocomial infections. Bacteria associated with biofilm formation play a key role in the morbidity and pathogenesis of these infections. Nitric oxide (NO) is a naturally produced free radical with proven bactericidal effect. In this study, Foley urinary catheters were impregnated with gaseous NO. The catheters demonstrated slow release of nitric oxide over a 14-day period. The charged catheters were rendered antiseptic, and as such, were able to prevent bacterial colonization and biofilm formation on their luminal and exterior surfaces. In addition, we observed that NO-impregnated catheters were able to inhibit the growth of Escherichia coli within the surrounding media, demonstrating the ability to eradicate a bacterial concentration of up to 10(4) CFU/ml.

Figures

FIG. 1.

Accumulation of NO production from impregnated catheters. Shown is the total accumulation of nitrites and nitrates, in water, produced from catheters impregnated with NO. Nitrites and nitrates were measured using Griess reagent. The nitrite concentration was calculated per 1 cm of catheter. The error bars indicate standard deviations.

FIG. 2.

NO production per day from impregnated catheters. Shown is production of nitrites and nitrates per 24 h, in direct correlation with NO production, during a 14-day period. (A) Production after storage of the catheter in sterile water. (B) Production after storage of the catheter in air for 7 days after impregnation and then immersion in sterile water. (C) Production after storage of the catheter in sterile urine. All storage media were changed each day. The nitrite and nitrate contents were measured using Griess reagent. The nitrite concentrations were calculated per 1 cm of catheter. The error bars indicate standard deviations.

FIG. 3.

Presence of E. coli on the surfaces of impregnated catheters. Comparison of E. coli colonization on NO-impregnated catheters and on control catheters after immersion of the catheters for 24 h in suspensions comprising 102 CFU/ml (I), 103 CFU/ml (II), or 104 CFU /ml (III) E. coli. Each of the images shows a three-compartment petri plate in which a selected immersed catheter was rolled over the surface of each compartment and then incubated at 37°C overnight.

FIG. 4.

Antimicrobial activity of impregnated catheters. Shown is a comparison of the growth of E. coli in media from catheters impregnated with NO versus media from control catheters after immersion of the catheters for 24 h in suspensions comprising 102 CFU/ml (I), 103 CFU/ml (II), or 104 CFU/ml (III) E. coli. Each of the images shows a three-compartment petri plate in which each compartment was surface plated with an aliquot of suspension from a selected catheter previously immersed in a selected E. coli suspension and then incubated at 37°C overnight. (A) Representative plates of samples. (B) Viable counts of the triplicate CFU. The hatched bars are data from the control, while the checkered bars are data from the impregnated catheters. The error bars indicate standard deviations.

FIG. 5.

Persistent antimicrobial effects of NO-impregnated catheters immersed in water. The data describe CFU formation with treated and untreated catheter sections that were previously immersed in water (changed each day) over 2 weeks. The catheter sections were immersed in 103 CFU/ml of E. coli for 1 min and transferred to PBS for 24 h, and then samples were plated. The hatched bars are data from the control, while the checkered bars are data from the impregnated catheters. The error bars indicate standard deviations.

FIG. 6.

Biofilm formation and biofilm-embedded bacteria on the inner lumen of the catheter after 24 h of urine flow. Shown is a comparison of colonized biofilm formation. (A) Biofilm formation showing the absorbance at 595 nm of the crystal violet that was attached to the catheter pieces after extraction with ethanol. The hatched bars are data from the control, while the checkered bars are data from the impregnated catheters. The error bars indicate standard deviations. (B) Biofilm-embedded bacteria. The image compares bacteria within the biofilms on control and impregnated catheters. Following 30 s of sonication, 1 μl (bottom) and 10 μl (top) of water surrounding a selected catheter piece were plated (in triplicate) and incubated for 24 h.

FIG. 7.

Presence of E. coli associated with the surfaces of impregnated catheters after 1 week of storage. Shown is a comparison of colonization of E. coli on control catheters and on catheters impregnated with NO after both sets had been stored for 7 days in 5 ml sterile air (I) or 5 ml sterile water (II). After storage, the catheter sections were immersed in 103/ml E. coli for 1 min, followed by 24 h of incubation in PBS, after which a selected section was rolled on the surface of a compartment in a selected petri plate and incubated at 37°C overnight.

FIG. 8.

Effect of catheter storage conditions on the antimicrobial activity of planktonic E. coli in surrounding catheter fluids. Comparison of colonization of E. coli on control catheters and on catheters impregnated with NO after both sets were stored for 7 days in in 50 ml sterile air (I) or 5 ml sterile water (II). After storage, the catheter sections were immersed in 103 CFU/ml E. coli for 1 min, followed by 24 h of incubation in PBS. Each of the images shows a three-compartment petri plate in which each compartment was surface plated with an aliquot of suspension from a selected catheter previously immersed in a selected E. coli suspension and then incubated at 37°C overnight. (A) Representative plates of samples. (B) Viable counts of the triplicate CFU. The hatched bars are data from the control, while the checkered bars are data from the impregnated catheters. The error bars indicate standard deviations.