Original Contribution Control of pathogen growth and biofilm formation using a urinary catheter that releases antimicrobial nitrogen oxides Hiroaki Kishikawa a , Anette Ebberyd a , Ute Römling b , Annelie Brauner b,c , Petra Lüthje b,c , Jon O. Lundberg a , Eddie Weitzberg a,n a Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden b Department of Microbiology, Tumor Biology, and Cell Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden c Division of Clinical Microbiology, Karolinska Institutet, S-171 77 Stockholm, Sweden article info Article history: Received 19 June 2013 Received in revised form 17 September 2013 Accepted 19 September 2013 Available online 29 September 2013 Keywords: Nitrite Nitric oxide Bacteria Urinary tract Infection Free radicals abstract Antibacterial nitrogen oxides including nitric oxide are formed from nitrite under acidic conditions. In a continuous-flow model of the urinary bladder we used the retention cuff of an all-silicone Foley catheter as a depot for export of nitrogen oxides. The cuff was filled with sodium nitrite (50 mM) and an acidic buffer solution (pH 3.6) and the growth of nine common uropathogens in the surrounding artificial urine was measured along with biofilm formation on the catheter surface. In experiments with control catheters (NaCl) bacteria grew readily and biofilm developed within hours in five of nine strains. In contrast, with test catheters bacterial counts were markedly reduced and biofilm formation by Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterobacter cloace was prevented, whereas Escherichia coli and Staphylococcus aureus were unaffected. We conclude that antibacterial nitrogen oxides generated in the retention cuff of a urinary catheter diffuse into urine and prevent the growth of urinary pathogens and biofilm formation. Although promising, future studies will reveal if this novel approach can be clinically useful for the prevention of catheter-associated urinary tract infections. & 2013 Elsevier Inc. All rights reserved. The use of indwelling catheters is considered a major risk factor for urinary tract infection (UTI), which is the most common hospital- acquired infection [1–4]. In this context, the vast majority of UTIs are catheter-associated (CAUTIs) and it has been estimated that with a Foley catheter, bacteriuria develops with a daily incidence of about 5 to 10% [3]. A central part of the pathogenesis of CAUTI is bacterial colonization and biofilm formation on the catheter surface [5, 6]. Biofilms protect bacteria from traditional antibiotics by representing a mechanical barrier but also by reduced bacterial metabolism within the biofilm. From here, bacteria can continuously seed into the urine. Several attempts have been made to minimize CAUTI, including the introduction of entirely closed drainage systems and prophylactic treatment with systemic antibiotics [7–9]. Another approach is to cover the catheter surface with a coating that prevents bacterial adhesion and biofilm formation [10–12]. Despite these efforts, CAUTIs are still very common in the clinical setting, and the costs of these potentially serious complications are substantial [13, 14]. Cells in the innate immune system produce nitric oxide (NO) and other reactive nitrogen intermediates (RNIs) to attack invading microorganisms [15]. We have been studying a novel approach, based on the antimicrobial effects of RNIs, to prevent the growth of bacteria in urine [16]. When inorganic nitrite (NO 2 ) is acidified, a variety of reactive RNIs are generated, including NO, a gas with antimicrobial properties [17,18]. In the presence of the reducing agent ascorbic acid, NO generation from nitrite is greatly enhanced [19]. Urinary pathogens such as Escherichia coli and Pseudomonas aeruginosa are killed if they are exposed to the combination of mildly acidified urine (pH 5 to 5.5) and nitrite, and the antibacterial effect is further enhanced by ascorbic acid [20,21]. NO is a tiny uncharged gaseous compound, and it diffuses readily over biological membranes. We previously tested the idea of using the retention cuff of an all-silicone Foley catheter as a source of diffusible RNIs generated from acidified nitrite [20]. In a stationary model of the urinary bladder we showed that filling the retention cuff with a nitrite and ascorbic acid solution (pH 5 to 5.5) generated measurable NO levels outside the membrane, which resulted in the killing of E. coli and P. aeruginosa reference strains in the surrounding urine [20,21]. Here we have further developed this concept in a more physiolo- gical continuous-flow model of the urinary bladder. Furthermore, we measured the release of NO and other nitrosating species as well as the antibacterial effect and biofilm formation after inoculation with nine different bacterial species isolated from patients with urinary tract infections. Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/freeradbiomed Free Radical Biology and Medicine 0891-5849/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2013.09.012 n Corresponding author. E-mail address: [email protected] (E. Weitzberg). Free Radical Biology and Medicine 65 (2013) 1257–1264
Transcript
Original Contribution
Control of pathogen growth and biofilm formation using a urinarycatheter that releases antimicrobial nitrogen oxides
Hiroaki Kishikawa a, Anette Ebberyd a, Ute Römling b, Annelie Brauner b,c, Petra Lüthje b,c,Jon O. Lundberg a, Eddie Weitzberg a,n
a Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Swedenb Department of Microbiology, Tumor Biology, and Cell Biology, Karolinska Institutet, S-171 77 Stockholm, Swedenc Division of Clinical Microbiology, Karolinska Institutet, S-171 77 Stockholm, Sweden
a r t i c l e i n f o
Article history:Received 19 June 2013Received in revised form17 September 2013Accepted 19 September 2013Available online 29 September 2013
Antibacterial nitrogen oxides including nitric oxide are formed from nitrite under acidic conditions. In acontinuous-flow model of the urinary bladder we used the retention cuff of an all-silicone Foley catheter as adepot for export of nitrogen oxides. The cuff was filled with sodium nitrite (50 mM) and an acidic buffersolution (pH 3.6) and the growth of nine common uropathogens in the surrounding artificial urine wasmeasured along with biofilm formation on the catheter surface. In experiments with control catheters (NaCl)bacteria grew readily and biofilm developed within hours in five of nine strains. In contrast, with test cathetersbacterial counts were markedly reduced and biofilm formation by Pseudomonas aeruginosa, Klebsiellapneumoniae, and Enterobacter cloace was prevented, whereas Escherichia coli and Staphylococcus aureus wereunaffected. We conclude that antibacterial nitrogen oxides generated in the retention cuff of a urinary catheterdiffuse into urine and prevent the growth of urinary pathogens and biofilm formation. Although promising,future studies will reveal if this novel approach can be clinically useful for the prevention of catheter-associatedurinary tract infections.
& 2013 Elsevier Inc. All rights reserved.
The use of indwelling catheters is considered a major risk factor forurinary tract infection (UTI), which is the most common hospital-acquired infection [1–4]. In this context, the vast majority of UTIs arecatheter-associated (CAUTIs) and it has been estimated that with aFoley catheter, bacteriuria develops with a daily incidence of about 5 to10% [3]. A central part of the pathogenesis of CAUTI is bacterialcolonization and biofilm formation on the catheter surface [5,6].Biofilms protect bacteria from traditional antibiotics by representinga mechanical barrier but also by reduced bacterial metabolism withinthe biofilm. From here, bacteria can continuously seed into the urine.Several attempts have been made to minimize CAUTI, including theintroduction of entirely closed drainage systems and prophylactictreatment with systemic antibiotics [7–9]. Another approach is tocover the catheter surface with a coating that prevents bacterialadhesion and biofilm formation [10–12]. Despite these efforts, CAUTIsare still very common in the clinical setting, and the costs of thesepotentially serious complications are substantial [13,14].
Cells in the innate immune system produce nitric oxide (NO)and other reactive nitrogen intermediates (RNIs) to attack invading
microorganisms [15]. We have been studying a novel approach,based on the antimicrobial effects of RNIs, to prevent the growthof bacteria in urine [16]. When inorganic nitrite (NO2
�) isacidified, a variety of reactive RNIs are generated, including NO,a gas with antimicrobial properties [17,18]. In the presence of thereducing agent ascorbic acid, NO generation from nitrite is greatlyenhanced [19]. Urinary pathogens such as Escherichia coli andPseudomonas aeruginosa are killed if they are exposed to thecombination of mildly acidified urine (pH 5 to 5.5) and nitrite,and the antibacterial effect is further enhanced by ascorbic acid[20,21]. NO is a tiny uncharged gaseous compound, and it diffusesreadily over biological membranes. We previously tested the ideaof using the retention cuff of an all-silicone Foley catheter as asource of diffusible RNIs generated from acidified nitrite [20]. In astationary model of the urinary bladder we showed that filling theretention cuff with a nitrite and ascorbic acid solution (pH 5 to 5.5)generated measurable NO levels outside the membrane, whichresulted in the killing of E. coli and P. aeruginosa reference strainsin the surrounding urine [20,21].
Here we have further developed this concept in a more physiolo-gical continuous-flow model of the urinary bladder. Furthermore, wemeasured the release of NO and other nitrosating species as well asthe antibacterial effect and biofilm formation after inoculation withnine different bacterial species isolated from patients with urinarytract infections.
The model consisted of a glass vessel (100 ml) maintained at 37 1Cby a water heating jacket (Fig. 1). The vessel was sterilized byautoclaving and an all-silicone Foley urinary catheter (Argyle, Sher-wood Medical, Ireland) was inserted aseptically via an outflow at thebase of the vessel. The catheter retention cuff was filled with aceticacid or with 0.9% NaCl solution (saline; 7 ml) and secured at the base,thereby sealing the outflow. Saline was chosen for control purposesbecause it is the most commonly used solution to secure the positionof a urinary catheter in the clinical situation. Artificial urine wasproduced as described before [22,23], with the following composi-tion (g/L): calcium chloride (CaCl2) 0.32, magnesium chloride hydrate(MgCl2 �6H2O) 0.43, sodium chloride (NaCl) 3.0, sodium sulfate(Na2SO4) 1.5, sodium citrate dihydrate (Na3C6H5O7 �2H2O) 0.43,sodium oxalate (Na2C2O4) 0.01, potassium phosphate (KH2PO4)1.86, potassium chloride (KCl) 1.06, ammonium chloride (NH4Cl)0.666, urea (CH4N2O) 16.66, gelatin 3.33 (Sigma, Sweden), andtryphton soya broth 20 (Oxoid, Basingstoke, UK). The artificial urinewas stored at �20 1C until use. The pH of the artificial urine was setto 6.3 or 5.5. Artificial urine was flowed through the system at a rateof 25 ml/h and drained via the tip of the catheter. This created aresidual volume of urine (30–40 ml) beneath the outflow at the tip ofthe catheter. Bacteria were added to the system via the urine supplyport and grew in the residual urine. We aimed at bacterial counts of105–106 CFU/ml at the start of the experiments. During the experi-ments samples from the residual urine were aspirated aseptically viaa catheter from a sampling port at the top of the glass vessel forestimation of bacterial growth and urinary pH. In each experimentthree sets of bladder models were used in parallel.
Bacterial cultures and growth media
The bacterial strains used in the study were P. aeruginosa, E. coli,Staphylococcus aureus, Staphylococcus saprophyticus, Proteus mir-abilis, Klebsiella pneumoniae, Enterobacter cloacae, Citrobacterfreundii, and Enterococcus faecalis, all isolated from patients witha urinary tract infection. The strains were obtained from theDepartment of Clinical Microbiology, Karolinska University Hospi-tal, Sweden. Bacteria were kept on agar plates at 4 1C. Before eachexperiment one to three colonies were taken from the plate andgrown aerobically in Mueller–Hinton broth for 16 h at 37 1C,resulting in 1.8–6 � 109 CFU/ml. This overnight culture was then
added to the bladder system to give a final density of approxi-mately 105 to 106 CFU/ml at the start of the experiment.
Determination of antibacterial activity and biofilm formation
To determine bacterial growth and survival in the artificial urine,urine aliquots from the continuous-flowmodel were collected, seriallydiluted, and transferred to agar plates. The agar plates were incubatedaerobically for 24 h, and then a viable count was performed.
Catheter biofilm formation was determined using the crystalviolet staining method as described before [24,25]. Briefly, at theend of the experiments (7.5 h postinoculation) the catheters wereremoved and the tips were cut in pieces, which were added to1 ml of crystal violet dye in water (0.4% wt/vol) for 10 min andthen washed three times in 1 ml distilled water, after which theywere added to 1 ml of dimethyl sulfoxide (DMSO; Sigma) andincubated for 4 h to resolve the crystal violet from the catheters’surface. Finally, the absorbance of each DMSO sample was mea-sured spectrophotometrically at 600 nm to estimate the amount ofadherent bacteria, referred to as biofilm formation. Biofilm fromcatheters incubated in artificial urine without bacteria was mea-sured and considered as background (OD600 ¼ 0.107). The effectsof nitrite on strains yielding absorbance levels below backgroundwere not evaluated.
Protocol
After the catheter retention cuff was filled (7 ml) with either aceticacid buffer, pH 3.6, or control solution (saline) the experiments werestarted by adding 20ml of inoculated urine (�105–106 CFU/ml) to thebladder model, after which the continuous flow of urine (25 ml/h) wasinitiated. At this time sodium nitrite (final concentration 50mM)was added to the catheters prefilled with acetic acid to start theformation of nitrogen oxides. After 3.5 h, another 100 ml of sodiumnitrite solution was added to the retention cuff to again obtain aconcentration of 50 mM. The dose and dosing interval were chosenbased on a number of pilot studies with various concentrations ofnitrite and acid. Urine was collected aseptically from the bladdermodel at the start of the experiment, before refilling of nitrite at 3.5 h,and at the end of the experiment at 7.5 h. Catheter tips were sampledat the end of the experiments for measurement of biofilm formation.
In separate experiments with P. aeruginosa, biofilm formationwas studied at three different urinary pH's, 5.5, 6.3, and 7.4, or theretention cuff was filled with the NO donor NOC 12 (Sigma,Stockholm, Sweden, 2 mM), and the effects on growth and biofilmformation in urine, pH 6.3, were tested.
Nitric oxide measurement
NO release from the urinary catheters was measured in thebladder model glass vessels without artificial urine in separateexperiments. The urinary catheters were secured at the base of thevessel and filled with test compounds (acetic acid, pH 3.6,þ nitrite 50 mM or acetic acid, pH 7.4, þ nitrite 50 mM). Airsamples (1 ml) were sequentially aspirated via the upper samplingport for 3.5 h. Each sample was immediately diluted with 49 ml ofNO-free air and then injected into a chemiluminescence NOanalyzer (CLD 77 AM; Eco Physics, Switzerland). The measuredNO concentration was multiplied by 50 to achieve the actualconcentration within the glass vessel. Ambient NO levels werebelow 5 ppb during all experiments.
Measurement of S-nitrosocysteine and nitrite formation
Acidified nitrite gives rise to NO and other reactive nitrogenintermediates, some of which are potent nitrosating and nitratingFig. 1. The continuous-flow model of the urinary bladder.
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agents [17]. Protonation of NO2� leads to generation of HNO2
(nitrous acid), which spontaneously yields N2O3 (dinitrogen tri-oxide), NO, and NO2 (nitrogen dioxide). N2O3 and NO2 are power-ful nitrosating and nitrating agents, respectively [26].
To investigate if such compounds were generated and releasedfrom the retention cuffs we used a spectrophotometrical methodbased on the nitrosation of the sulfhydryl group of cysteine,yielding S-nitrosocysteine (CysNO) [27].
Catheters were placed at the base of the glass vessel and theretention cuff was filled with test compounds (acetic acid, pH 3.6,þ nitrite 50 mM or acetic acid, pH 7.4, þ nitrite 50 mM). Fortymilliliters of saline with L-cysteine (50 mM; Sigma) was put intothe artificial bladder. Samples (100 ml) were sequentially drawnfrom the vessel for 3.5 h and were stored frozen for latermeasurement. To detect the formation of CysNO, absorbance at336 nm was measured using a plate reader (SpectraMax Plus384;Molecular Devices, USA).
In additional experiments (n ¼ 3) catheters were placed at thebase of the glass vessel and the retention cuff was filled with testcompounds (acetic acid, pH 3.6, þ nitrite 50 mM or acetic acid, pH 7.4,þ nitrite 50mM). Nitrite accumulation in artificial urine was mea-sured after 7.5 h using HPLC (ENO-20; Eicom) as described earlier [28].
Statistical analysis
GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA) wasused for statistical analysis. Mann–Whitney U tests were performedfor comparisons between the control and the acetic acid þ nitritegroups. Wilcoxon's matched pair test was used for changes comparedto baseline within each group. Student's t test was used for biofilm
formation by P. aeruginosa at various urinary pH's. A p value of o0.05was considered statistically significant. Bactericidal effect was definedas 43 log order reduction in bacterial numbers compared to baselineand bacteriostatic effect was defined as no increase in bacterial countsor a reduction in counts of o3 logs compared to baseline.
Results
Bacterial growth and survival
Under control conditions with saline in the retention cuff,bacterial growth in artificial urine was only slightly affected bythe urinary pH (Table 1 and Figs. 2 and 3). In contrast, with aceticacid and nitrite in the retention cuff growth of all bacterial specieswas inhibited at a urinary pH of 5.5, with bactericidal activityagainst P. aeruginosa, S. saprophyticus, and Enterob. cloacae.At urinary pH 6.3, viable counts for all species except E. coli werereduced, or growth was significantly inhibited, but bactericidalactivity was reached only against C. freundii. The NO donor NOC-12inhibited growth of P. aeruginosa compared to control in urine atpH 6.3 (107.6 and 1010.7 CFU/ml, respectively).
Biofilm formation
Biofilm formation was measured after 7.5 h exposure in urine atpH 6.3. There was a great variation in biofilm formation betweenthe species, and S. saprophyticus, Pr. mirabilis, C. freundii, and Enteroc.faecalis did not form detectable biofilm. Among the species thatgenerated biofilm within the time and under the conditions tested,
Table 1Bacterial growth in a continuous-flow model of the urinary bladder.
The data are shown as log 10 CFU/ml, medians, and 25th–75th percentile.n p o 0.05 compared with control group at 3.5 and 7.5 h.nn p o 0.01 compared with control group at 3.5 and 7.5 h.
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P. aeruginosa, K. pneumoniae, and Enterob. cloacae responded toacetic acid and nitrite with a significant reduction in biofilmformation, whereas the effect on biofilm formation by E. coli andS. aureus was not significant (Fig. 4). In the interspecies comparison,the biofilm-reducing effect did not correlate with the influence onbacterial growth under the same conditions. Moreover, in theexperiments in which biofilm formation by P. aeruginosa at variousurinary pH's was tested we found that biofilm inhibition by nitritewas not dependent on urinary pH (Fig. 5).
The NO donor NOC-12 prevented biofilm formation byP. aeruginosa compared to control in urine at pH 6.3 (OD600 ¼ 0.87 0.1 and 2.2 7 0.5, respectively).
Nitric oxide release
When the retention cuff was filled with acetic acid buffer, pH3.6, and nitrite 50 mM a rapid and sustained release of NO could
be detected (Fig. 6A). NO reached its maximum of 388 7 4 ppmafter 30 min and then gradually decreased but the levels were stillsignificantly elevated after 3.5 h (243 7 12 ppm). The release ofNO from acetic acid buffer, pH 7.4, and nitrite 50 mM was minimaland did not exceed 4 ppm. These data show that protonation ofnitrite leads to generation of NO gas that is able to diffuse throughthe silicone membrane of the retention cuff.
S-nitrosocysteine and nitrite formation
Filling the retention cuff with acetic acid buffer, pH 3.6, and nitrite50 mM led to a progressive accumulation of CysNO in the surroundingNaCl–cysteine solution (Fig. 6B). In contrast, when acetic acid buffer atpH 7.4 was used together with 50 mM nitrite no generation of CysNOcould be detected. Together, these data suggest that nitrogen speciesare formed inside the retention cuff with the capacity to induce
Urine pH 5.5
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Fig. 2. Effects of acidified nitrite on bacterial viable counts (log 10 CFU/ml) in urine (pH 5.5) after 3.5 and 7.5 h using a continuous-flow model of the urinary bladder.The retention cuffs of all-silicone Foley catheters were filled with acetic acid, pH 3.6, and nitrite 50 mM (nitrite) or saline (control). Data are shown as medians, n ¼ 4–8.For statistics see Table 1.
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nitrosation processes in the surrounding urine. These processes mayunderlie the observed antibacterial effects of acidified nitrite.
In additional experiments we investigated if nitrite itself couldpass across the silicon membrane. With 50 mM nitrite at pH 7.4 inthe retention cuff the concentration of nitrite in the surroundingswas 6 7 2 μM after 7.5 h. In contrast, at pH 3.6 in the cuff the
concentration was 1370 7 795 μM. Artificial urine itself contained
0.40 μM nitrite. These data suggest that nitrite itself does not to asignificant degree pass the silicone membrane.
Urine pH
When we measured urinary pH at the end of the experimentswe found a slight reduction in most strains irrespective oftreatment. In controls, urine pH decreased from 6.30 to 5.99 (p¼ 0.056) and in the nitrite group from 6.3 to 6.08 (p o 0.002)
However, the reduction in pH was similar in the two groups,suggesting that acidification of the surrounding urine in itself wasnot responsible for the growth inhibition.
Discussion
We have developed a novel concept for the delivery ofantibacterial nitrogen oxides into the urinary bladder of a cathe-terized patient. The retention cuff of an all-silicone Foley catheteris filled with acidified nitrite, and reactive gaseous nitrogen oxidesgenerated under these conditions diffuse from the cuff into thesurrounding urine. Whereas a tight latex material would preventdiffusion of these small molecules, the more permeable all-siliconmaterial used here allows for this to occur, thereby deliveringantibacterial RNIs to the catheter surface and the surroundingurine [20]. Here this approach was tested in a continuous-flow
Urine pH 6.3
P. aeruginosa
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Fig. 3. Effects of acidified nitrite on bacterial viable counts (log 10 CFU/ml) in urine (pH 6.3) after 3.5 and 7.5 h using a continuous-flow model of the urinary bladder.The retention cuffs of all-silicone Foley catheters were filled with acetic acid, pH 3.6, and nitrite 50 mM (nitrite) or saline (control). Data are shown as medians, n ¼ 4–8.For statistics see Table 1.
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model of the urinary bladder, looking at effects on growth andbiofilm formation of clinical isolates of nine bacterial speciesrepresenting the most common pathogens causing CAUTI. The
majority of the tested strains were sensitive to the treatment, witheither a bactericidal or a bacteriostatic effect observed in the urinesurrounding the catheter. In addition, biofilm formation on thecatheter surface was inhibited in three of the five strains thatdeveloped biofilm in our model.
The antibacterial effects of acidified nitrite are related to none-nzymatic formation of RNIs [29]. Nitrite itself is not toxic to bacteriabut when acidified it forms HNO2, which in turn will decomposespontaneously to a variety of RNIs including NO, N2O3, NO2, andS-nitrosothiols, all of which have antibacterial properties [30].Because bacteria are commonly exposed to the same RNIs whenthey are produced endogenously by activated immune cells [31],some bacterial species have developed mechanisms to protectagainst this attack. These defense systems include induction ofmolecules such as flavohemoglobins that bind or destroy the RNIsor enzymes, i.e., nitrite reductases and NO reductases that rapidlymetabolize RNIs [30,32,33]. The existence of such defense mechan-isms may explain why some strains were apparently less sensitivethan others in the current study. As an example, E. coli is known torapidly upregulate a variety of RNI-detoxifying enzymes in responseto nitrosative stress, thereby ultimately converting these species toless harmful compounds such as nitrate [17,34,35]. Whereas E. coli
Pseudomonas
Control Nitrite0.0
0.1
0.2
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0.4p=0.0051
Abs
orba
nce
E.coli
Control Nitrite0.0
0.5
1.0
1.5
p=0.16
S.aureus
Control Nitrite0.0
0.5
1.0
1.5
2.0p=0.16
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orba
nce
Klebsiella
Control Nitrite0.0
0.1
0.2
0.3
0.4
0.5p=0.023
Enterobacter
Control Nitrite0
1
2
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5p=0.016
Abs
orba
nce
Fig. 4. Effects of acidified nitrite on biofilm formation on urinary catheters exposed for 7.5 h to various bacteria in a continuous-flow model of the urinary bladder. The cuffsof the catheters were filled with acetic acid, pH 3.6, and nitrite 50 mM (nitrite) or saline (control). Biofilmwas measured as absorbance after crystal violet and extraction withDMSO. Only the five bacterial strains that produced significant biofilm are shown.
0.0
0.5
1.0
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B C N C N C N
5.5 6.3 7.5
***
***
Abs
orba
nce
Fig. 5. Biofilm formation on Foley catheters after exposure to P. aeruginosa for 7.5 hin urine with pH 5.5, 6.3, and 7.4. Retention cuffs were filled with saline (C) oracetic acid, pH 3.6, and nitrite 50 mM (N). Biofilm was measured as absorbanceafter crystal violet and extraction with DMSO. B, background level. Data are shownas the mean 7 SEM, n ¼ 2–8. ***p o 0.0001.
H. Kishikawa et al. / Free Radical Biology and Medicine 65 (2013) 1257–12641262
showed some resistance to acidified nitrite at a higher urinary pH, S.saprophyticus was among the most sensitive of the strains tested.The mechanism behind these differences in susceptibility to nitriteneeds to be explored in further studies but might be related todifferences in intrinsic nitrogen handling by the bacteria. E. colinormally expresses effective nitrate and nitrite reductases because ituses these anions for respiration in the absence of oxygen [34,36].Thus, this bacterium is already generating RNIs by itself and musttherefore constantly be prepared to degrade potentially toxic inter-mediates, including NO. S. saprophyticus on the other hand has avery low nitrate- and nitrite-reducing capacity [37,38] and wouldtherefore not be exposed to potentially toxic RNIs under normalconditions. These different metabolic strategies can explain theinterspecies differences observed in our experiments; strain-specific differences in the susceptibility to RNIs within one bacterialspecies cannot, however, be ruled out.
The antibacterial effect was potentiated if the surroundingurine was made slightly acidic. This may be explained by thelower pH causing additional direct stress to the bacteria. However,there was no difference in bacterial growth in the control experi-ments at pH 6.3 and 5.5, indicating that pH in itself was notaffecting bacterial growth in a major way. In addition, RNIs mayagain be formed in the acidic surrounding urine. For example,HNO2 formed inside the cuff could easily diffuse out because it isuncharged. When pH in urine is high, HNO2 will immediatelyconvert back to nontoxic nitrite but if the urine is slightly acidicthe HNO2 will survive longer, thereby generating additionalRNIs outside of the cuff close to the bacteria. The formation ofS-nitrosocysteine observed here is direct evidence for the export ofnitrosating agents from the cuff. Both HNO2 and N2O3, formedfrom acidified nitrite, may be responsible for this reaction [17].In addition, nitrosating compounds may be re-formed in the urine
after oxidation of NO. The fact that a pure NO donor had similarantibacterial activity in this model supports the notion that NOitself contributes to the observed effects. However, NO may alsoreact to form the other RNIs discussed above. Although there was apH-dependent effect of nitrite on bacterial growth, biofilm forma-tion by P. aeruginosa at three different urinary pH levels was notpH dependent (Fig. 5). This discrepancy could perhaps beexplained by higher concentrations of RNIs near the site ofgeneration in the retention cuff compared to a dilution in thesurrounding urine. Also, a more sensitive mechanism by which NOtargets biofilm formation independent of bacterial growth couldexplain these results.
The objective of this study was to provide proof-of-concept fora novel strategy in the prevention of CAUTI. However, a positiveurine culture of Z105 is among the criteria of an alreadyestablished infection. In our study we started the experimentswith 105–106 CFU/ml in the urine to be able to detect bactericidaleffects (43 log decrease). Despite these high initial bacterialcounts we were still able to provide bacteriostatic and bactericidaleffects in the majority of the investigated species. Further studiesare clearly needed to explore the usefulness of the current methodin prevention of CAUTIs in the clinic. A remaining question is tofind out if the antibacterial effects are sustained over time. Inaddition, although the inclusion of a continuous-flow model inthis study represents a step closer to the clinical situation, itremains to be proven if antibacterial effects are sufficient alsoin vivo. It is possible that the high nitrosating potential created bythis system may negatively affect the urinary bladder mucosa.Clearly, in vivo animal experiments have to be performed toelucidate this question. Nevertheless, the concept of filling theretention cuff with small antibacterial RNIs has several potentialadvantages. RNIs may be viewed as “natural” antibiotics becausethey are already part of the normal innate immune system asopposed to traditional antibiotics [15]. In addition, no systemicside effects are to be anticipated by this approach. An attractivepractical implication is that Foley all-silicone catheters already inplace in patients might be used and that the catheters may berefilled after insertion without disturbing the integrity of theclosed drainage system. Moreover, doses and intervals betweenreloading of the catheters might be varied depending on theclinical situation. Because the primary insertion of the catheter isrelated to introduction of bacteria and later development of CAUTI,a single administration in conjunction with this procedure mightbe of value.
In conclusion, antibacterial nitrogen oxides are generated in theretention cuff of a urinary catheter from the reaction of nitrite withacid. These nitrogen oxides diffuse into urine and prevent the growthof several urinary pathogens and biofilm formation on the cathetersurface. Future studies will reveal if this novel approach can be usedfor the prevention of catheter-associated urinary tract infections.
Acknowledgments
This work was supported by grants from the Swedish ResearchCouncil, Torsten and Ragnar Söderbergs Foundation, StockholmCity Council (ALF), and Funds from the Karolinska Institutet. J.O.L.and E.W. are listed on patents related to the therapeutic use ofinorganic nitrate and nitrite.
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Nitric oxide
S-nitrosocysteine
Part
s pe
r mill
ion
(ppm
)
Fig. 6. (A) Nitric oxide and (B) S-nitrosocysteine formation from the retention cuffof all-silicone Foley catheters filled with acetic acid (pH 3.6, filled circles, and pH7.4, open circles) and nitrite (50 mM). Nitric oxide was measured directly bychemiluminescence in the surrounding air and S-nitrosocysteine by absorbance at336 nm in solutions originally containing L-cysteine (50 mM).
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