From the Department of Surgical Science, Section of Urology and the Department of Physiology and Pharmacology Karolinska Institutet, Stockholm, Sweden

ANTIBACTERIAL EFFECTS

OF NITRITE IN URINE

Stefan Carlsson M.D.

Stockholm 2005

All previously published papers were reprinted with permission of the publishers. Printed by Repro Print AB, Box 21085 100 31 Stockholm, Sweden © Stefan Carlsson, 2005 ISBN 91-7140-237-3

To Anne, Emma, Adam and Fabian

”Hela bollen måste ligga stilla”

Ralf Edström

Antibacterial effects of nitrite in urine

Stefan Carlsson, MD From the Department of Surgical Science, Section of Urology

and the Department of Physiology and Pharmacology Karolinska Institutet, Stockholm, Sweden

Abstract

Urinary tract infections (UTI`s) are among the most common conditions causing individuals to seek medical care. In addition, catheter-associated infections account for most of the hospital-acquired UTI´s and is also a major source of resistant nosocomial pathogens. The majority of bacterial strains causing UTI have a nitrate-reducing capacity and since the 1920s, when the urinary nitrite test was first developed, this dipstick assay has been an important component of modern UTI diagnosis. In this thesis we have expanded the role of nitrite beyond diagnosis of infection and explored its role as an antimicrobial agent. From earlier studies it is known that acidification of nitrite results in the formation of nitric oxide (NO) and other reactive nitrogen oxides, which are toxic to a variety of microorganisms. The aim of the present thesis was to investigate NO formation and possible antibacterial effects of acidified nitrite-containing urine. We also sought to determine the antibacterial effect of a two-step procedure in which bacteria were first fed with nitrate to form nitrite, followed later by acidification of the urine. Finally we wanted to evaluate in vitro a novel concept for intravesical delivery of antibacterial NO via the retention balloon of a urinary catheter. NO formation was measured in headspace gas using a chemiluminiscence-technique, while antibacterial effects were evaluated by turbidity measurements and by viable counts. We show that large amounts of the antimicrobial gas NO are generated in mildly acidified nitrite-containing human urine and this NO formation is greatly enhanced by the addition of ascorbic acid. Furthermore, we show that mildly acidified nitrite-containing human urine has potent antimicrobial activity against three of the most common urinary pathogens and this inhibitory effect is further increased by ascorbic acid. We also found, that Escherichia coli self destructs when it is first allowed to generate nitrite and then transferred to mildly acidified urine. Translated into clinical terms, this could imply that ingestion of nitrate followed some hours later by acidification of urine could be a new approach for treatment of UTI. Finally, potent antibacterial effects were also observed in infected urine when the nitrite-derived nitrogen oxides were delivered via the retention balloon of a urinary catheter. In conclusion, we describe potent antibacterial effects of acidified nitrite-containing urine and also suggest a novel attractive alternative to prevent catheter-associated urinary tract infections. Key words: nitrite, nitrate, nitric oxide, urinary tract infection, catheter-associated urinary tract infection, antimicrobial, Escherichia coli. ISBN 91-7140-237-3

LIST OF PUBLICATIONS

This thesis is based on the following papers which are referred to in the text by their Roman numerals (I-IV):

I. JO Lundberg, S Carlsson, L Engstrand, E Morcos, NP Wiklund and E Weitzberg. Urinary nitrite: More than a marker of infection. Urology, 50:189-191, 1997.

II. S Carlsson, NP Wiklund, L Engstrand, E Weitzberg and JO Lundberg. Effects of pH, nitrite, and ascorbic acid on nonenzymatic nitric oxide generation and bacterial growth in urine. Nitric Oxide: Biology and Chemistry, 5:580-586, 2001.

III. S Carlsson, M Govoni, NP Wiklund, E Weitzberg and JO Lundberg. In vitro evaluation of a new treatment for urinary tract infections caused by nitrate-reducing bacteria. Antimicrobial Agents and Chemotherapy, 47: 3713-3718, 2003.

IV. S Carlsson, E Weitzberg, NP Wiklund and JO Lundberg.

Intravesical nitric oxide delivery for prevention of catheter-associated urinary tract infections. Antimicrobial Agents and Chemotherapy, (in press May 2005).

CONTENTS ABBREVIATIONS................................................................................................................................... 9

INTRODUCTION ................................................................................................................................... 10 URINARY TRACT INFECTIONS ................................................................................................................ 10

Aetiology ....................................................................................................................................................... 10 Bacterial virulence ....................................................................................................................................... 11 Host defense mechanisms ............................................................................................................................ 11 Diagnosis ...................................................................................................................................................... 12 Treatment and Antimicrobial resistance. .................................................................................................... 13 Prophylaxis and Acidification of urine ....................................................................................................... 13 Catheter-associated urinary tract infection and Biofilms ........................................................................... 14

NITRIC OXIDE....................................................................................................................................... 15 The history of nitric oxide. ........................................................................................................................... 15 Enzymatic formation of NO ......................................................................................................................... 16 Nonenzymatic formation of NO................................................................................................................... 16 Nitrate in medicine ....................................................................................................................................... 17 Metabolism of nitrate/nitrite ........................................................................................................................ 18 Chemistry of NO, nitrate and nitrite ............................................................................................................ 18 Beneficial effects of NO ............................................................................................................................... 20 Beneficial effects of acidified nitrite............................................................................................................ 21 NO in inflammation and host-defense ........................................................................................................ 22 NO and inflammation in the lower urinary tract ........................................................................................ 23 Bacterial defense against RNIs.................................................................................................................... 24

AIMS...................................................................................................................................................... 25

MATERIAL AND METHODS ................................................................................................................ 26 STUDY SUBJECTS................................................................................................................................. 26 CHEMICAL ANALYSIS OF NO, NITRITE AND NITRATE ............................................................................... 26

Analysis of NO in gaseous phase by chemiluminescence ........................................................................... 26 Quantification of nitrite and nitrate by chemiluminescence ...................................................................... 27 Quantification of nitrite by capillary electrophoresis ................................................................................. 28

BACTERIAL CULTURES AND MEDIA ........................................................................................................ 28 ANALYSIS OF BACTERIAL GROWTH........................................................................................................ 29

Turbidity measurements............................................................................................................................... 29 Viable counts measurements ....................................................................................................................... 30

STATISTICS .......................................................................................................................................... 31 RESULTS AND COMMENTS ............................................................................................................... 32

NO FORMATION FROM NITRITE IN URINE ................................................................................................ 32 EFFECTS OF NITRITE ON BACTERIAL GROWTH ........................................................................................ 33

Effects of sodium nitrite ............................................................................................................................... 33 Bacterial growth after pre-incubation with nitrate ..................................................................................... 35 Delivery of antibacterial nitrogen oxides via a catheter retention balloon ................................................ 37

GENERAL DISCUSSION...................................................................................................................... 38 ACIDIFIED NITRITE AND ANTIBACTERIAL EFFECTS IN URINE ..................................................................... 38

What is the mechanism of action?............................................................................................................... 38 Bacterial defense systems............................................................................................................................. 40

POSSIBLE CLINICAL USEFULNESS ......................................................................................................... 41 Treatment of UTI ......................................................................................................................................... 41 Prevention of CAUTI ................................................................................................................................... 43

CONCLUSIONS .................................................................................................................................... 45

ACKNOWLEDGEMENTS ..................................................................................................................... 46

REFERENCES ...................................................................................................................................... 48

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ABBREVIATIONS ABU asymptomatic bacteriuria BCG bacillus calmette-Guérin CAUTI catheter-associated urinary tract infection CE capillary electrophoresis CFU colony-forming units cGMP cyclic guanosine monophosphate cNOS constitutive nitric oxide synthase E. coli Escherichia coli EDRF endothelium-derived relaxing factor eNOS endothelial nitric oxide synthase HNO2 nitrous acid H2O2 hydrogen peroxide HPLC high-performance liquid chromatography IL-8 interleukin 8 iNOS inducible nitric oxide synthase L-NAME NG-nitro- L-arginine-methylester L-NMMA NG-monomethyl-L-arginine LPS lipopolysacharides MBC minimum bactericidal concentration MIC minimum inhibitory concentration mRNA messenger ribonucleic acid NADPH nicotineamide adenine dinucleotide phosphate NO2

- nitrite

NO3- nitrate

NO2 nitrogen dioxide N2O3 di-nitrogen trioxide N2O4 di-nitrogen tetroxide nNOS neuronal nitric oxide synthase NO nitric oxide NOS nitric oxide synthase O2

- superoxide OH hydroxyl radical ONOO- peroxynitrite PDE-5 phosphodiesterase type-5 PMNL polymorphonuclear leukocytes PHOX phagocyte oxidase ppm parts per million ppb parts per billion pO2 partial pressure of oxygen RNI reactive nitrogen intermediates ROS reactive oxygen intermediates RSNO S-nitrosothiols sGC soluble guanylyl cyclase TMP trimethoprim SMX sulfamethoxazole SOD superoxide dismutase UTI urinary tract infection

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INTRODUCTION

Urinary tract infections Urinary tract infection (UTI) is defined as an inflammatory response of the urothelium to

bacterial invasion [1]. Acute uncomplicated UTI with an estimated incidence of 150 million

yearly on a global basis is among the most common conditions causing individuals to seek

medical care [2]. At the age of about thirty, 50% of all women report having had a UTI. In

contrast, only 20% of men in their seventies have experienced a UTI [3, 4] Approximately

25% of women who have had an episode of acute cystitis develop recurrent UTI [5]. UTI

affects up to 10% of the childhood population and is an important cause of morbidity [6]. It is

a heterogeneous disease which can be sub-divided into a variety of clinical conditions ranging

from the asymptomatic presence of bacteria in the urine to severe, sometimes life-threatening,

infections of the kidney with septicaemia [7].

Aetiology

Microorganisms reach the urinary tract by the ways of the ascending route and via the

haematogenous or lymphatic routes. The ascending route via urethra from periurethral

colonization is the most common pathway [8]. This explains the greater frequency of UTI in

women since the female urethra is short and situated in close proximity to the vaginal

vestibule and rectum. It also explains the increased risk of infection following bladder

catheterization or instrumentation. Other risk factors, apart from gender and the use of

catheters/surgical instruments, are vaginal intercourse, spermicide use and underlying

conditions affecting the urinary tract such as diabetes and pregnancy. Obstruction of urinary

flow (e.g. because of prostate enlargement) and diminished ability to empty the bladder (e.g.

because of spinal cord injury, decreased bladder contractility or neuropathy) also increase the

risk of UTI [4]. Haematogenous infections of the urinary tract can be caused by

Staphylococcus aureus, Candida species or Mycobacterium tuberculosis. These infections are

uncommon in normal adults, but can occasionally occur in immunocompromised patients and

in neonates [9, 10]. UTI via the lymphatic route is probably not of importance, but may occur

in unusual circumstances such as a severe bowel infection or retroperitoneal abscess [10, 11].

Escherichia coli (E. coli) is by far the most common cause of UTI, accounting for 80-85 % of

community-acquired infections and 50% of hospital-acquired infections. Other gram-negative

enterobacteriaceae including Proteus, Klebsiella and gram-positive Enterococcus faecalis

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and Staphylococcus saprophyticus are responsible for the remainder of most community-

acquired infections [12, 13]. In hospital-acquired UTI E. coli is still the leading cause of UTI,

but other bacteria such as Pseudomonas aeruginosa, Klebsiella species and Proteus mirabilis

are seen more frequent as causative agents [14].

Bacterial virulence

Uropathogenic bacteria are selected from the faecal flora by presence of virulence factors that

enable them to adhere to and colonize the perineum and urethra and migrate to the urinary

tract [15, 16]. These virulence factors include adhesins, siderophores, toxins, polysaccharide

coating, proteases and invasins. However, no single virulence factor is common to all clinical

UTI isolates and none of these factors is absolutely necessary for development of UTI

pathogenesis [17]. The more compromised the natural defense mechanism (e.g. obstruction,

bladder catheterization, diabetes with poor blood glucose control), the fewer virulence factors

are needed for any bacterial strain to induce infection. This is supported by the observation

that bacteria isolated from patients with a complicated UTI frequently fail to express virulence

factors [17-19]. There has also been suggested that if a bacterial strain had a doubling time of

about 50 minutes or less in urine, it could maintain itself in the normal bladder without

adhering [20].

Host defense mechanisms

Flow of urine through the urinary tract and voiding are effective natural bladder defenses

against infection [8, 21]. In addition, low pH, urea and high urine osmolality can be inhibitory

to bacterial growth. Both concentration of urea and urine osmolality are decreased by urine

dilution, which has led some authors to suggest that diuresis actually could favour bacterial

proliferation [22-24]. The asymptomatic bacteriuria (ABU) strains do not activate a host

response because these strains stop expressing adherence factors once they are established in

the urine thereby avoiding the antibacterial defense [25]. In contrast, the uropathogenic

strains, continue to adhere and activate the uroepithelial cells to release cytokines that recruit

polymorphonuclear leukocytes (PMNL) to the site of infection. During their passage across

the mucosa , the PMNL kill the bacteria [26]. The cytokine interleukin-8 (IL-8) seems to be

the main driving force for the PMNL to cross the urinary tract epithelium and the emergence

of leukocytes in the urine, known as pyuria , is a classical sign of UTI [25]. The attachment of

a uropathogenic strain may result in exfoliation of host bladder epithelial cells as part of an

innate host defense system [27]. Another host defense strategy is to produce soluble receptor

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molecules that compete with bacterial adhesins and block bacterial attachments [28]. The

vaginal flora, dominated by Lactobacillus species, is an additional important host defense

system against UTI. Particularly the Lactobacillus species that produce hydrogen peroxide

(H2O2) seems to have a protective effect [29]. Lactobacilli have also been shown to generate

an acidic vaginal pH, which appear to be of importance for lowering the rate of UTI [30].

Unfortunately, use of antibiotics and intravaginal antimicrobials including antimycotics and

spermicides reduces the vaginal flora and increases susceptibility to UTI [31].

Diagnosis

Urine cultures are still the golden standard to diagnose UTI as it has been for decades,

although the number of bacteria considered relevant for diagnosis has been changed since the

classical “Kass criteria” of 105 bacteria/ml were described in 1956 [32]. Stamm and co-

workers showed in 1982 that a count of ≥ 105/ml identified only about 50% of dysuric

women with bacteriuria, whereas a count of ≥ 102/ml had a predictive value of 0.88 for

diagnosing a probable urinary tract infection [33]. The diagnostic criteria for UTI in acute

uncomplicated cystitis in women is ≥ 103 colony-forming units (CFU) of uropathogen/ml of

mid-stream sample of urine according to EAU guidelines [2, 34]. Since the 1920s, when the

urinary nitrite test was first developed, this dipstick assay has been an important component

of modern UTI diagnosis [35, 36]. Most dipstick assays will indicate positive nitrite (change

of colour) when urinary nitrite levels exceed 7-25 µM, as a result of bacterial nitrate-reducing

capacity [37, 38]. Today the common urine dipstick test in use detects nitrite and leukocyte

esterase. These tests are rapid and cheap alternatives, but show less accuracy compared with a

quantitive culture [39]. Another clear disadvantage with the dipstick assays is that they do not

give information about the exact species that caused the UTI or the antimicrobial

susceptibility profiles. The majority of bacterial strains causing UTI have a nitrate-reducing

capacity, like for example E coli, Pseudomonas aeruginosa and Proteus mirabilis.

Staphylococcus saprophyticus can also express nitrate-reductase, but normally very low levels

[40]. However, streptococci, including strains of Enterococcus faecalis, do not have nitrate

reductase enzymes and lack the capacity to reduce urinary nitrate to nitrite [41, 42]. Recently,

a meta-analysis demonstrated that sensitivities of the combination of both nitrites and

leukocyte-esterase vary between 68 and 88% in different patient groups. The sensitivity was

also analysed in different patient settings and found to be highest in studies carried out in

family medicine (90%). The sensitivity of the urine dipstick test for nitrite alone was 45-60%

with higher level of specificity (85-98%) [43]. Reasons for false negative results using

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dipstick test for nitrite i.e., negative nitrite assay in urine with more than 105 nitrate-reducing

organisms/ml could be urinary pH below 6.0, urobilinogen, lack of dietary nitrate or intake of

ascorbic acid [37]. Also high frequency of micturition might be a reason for false negative

results, since an incubation at 37◦ C of four to six hours is preferable for generation of

detectable nitrite in infected urine samples [41].

Treatment and Antimicrobial resistance.

In treatment of uncomplicated cystitis, a short course of antibiotics are highly effective and

also desirable because of the improved compliance that they promote, their low cost and the

low frequency of adverse reactions. According to American guidelines, trimethoprim-

sulfamethoxazole (TMP-SMX) for three days is considered the current standard therapy [44].

TMP alone and other fluoro-quinolones (ofloxacin, norfloxacin, ciprofloxacin, and fleroxacin)

are equivalent to TMP-SMX [44]. Pivmecillinam and nitrofurantoin are alternative oral drugs,

especially in situations in which fluoroquinolones are not indicated [45, 46]. In Sweden,

clinical practice guidelines have been developed by the STRAMA-group, which recommends

pivmecillinam or nitrofurantoin or TMP for seven days as a standard treatment of

uncomplicated cystitis in women [47]. However, there are other Swedish studies supporting

short-term treatment for 3-5 days of uncomplicated lower urinary tract infections in women

[48, 49]. A growing problem of worldwide concern is the increasing resistance of pathogens

to conventional antibiotics [50]. In UTI much of the increase in bacterial resistance is in acute

uncomplicated cystitis with increasing TMP-SMX and β-lactam resistance. Of more concern,

however, are the emerging issues of fluoroquinolone resistance and multidrugresistance

among community-acquired urinary isolate. Important strategies to help slow the progression

of resistance is a judicious use of antibiotics and to develop novel methods for the prevention

of UTI [51].

Prophylaxis and Acidification of urine

Urinary acidification has long been used as an aid in the treatment and prevention of urinary

tract infections. Various agents have been used in attempts to lower urinary pH, these include

mandelic acid, gluconic acid, ammonium chloride, methionine and ascorbic acid (vitamin C)

[52-55]. Urinary acidification with vitamin C has traditionally been used as a household

remedy, although evidence to support its efficacy has largely been anecdotal and any

antibacterial action still remains unclear [56]. Effective acidification of urine has been poor

and antibacterial effects variable [57-59]. Methenamine hippurate, a weak base that slowly

14

hydrolyzes in acidic urine to ammonia and the nonspecific antibacterial formaldehyde, are

often used for the prevention of urinary tract infection. However, there is not enough evidence

to conclusively support the use of methenamine hippurate for UTI prophylaxis [60]. Today

most prophylaxis of UTI consists of antimicrobial therapy. The increasing prevalence of E.

coli isolates that are resistant to antimicrobial agents, however, has stimulated interest in

novel, non-antibiotic methods for preventing UTI. Natural compounds like cranberry products

have been more popular and used as a household remedy for treating and preventing UTI

[61]. It has been postulated that acidification of urine is an important means by which

cranberry products might protect against UTI. However, effective acidification of urine

through cranberry ingestion has not been proved [62]. The mechanism of action is likely to be

a tannin (proanthocyanidin) that block adherence of P-fimbriated E. coli [63]. Although there

are some clinical trials that have shown positive effects with cranberry ingestions in

preventing UTI a Cochrane review in 2001 concludes that there is no compelling evidence to

recommend cranberries for prevention of UTI [64].

Catheter-associated urinary tract infection and Biofilms

Catheter-associated urinary tract infection (CAUTI) accounts for most of the hospital-

acquired UTI [65]. In medical intensive care units in the US as much as 95% of urinary tract

infections were associated with urinary catheters [66]. CAUTI is also a major source of

resistant nosocomial pathogens [67]. The incidence of bacteriuria in catheterized patients

varies between 3% and 10% per day [68]. A biofilm is a collection of microbial organisms on

a surface that is surrounded by an extra cellular matrix composed primarily of poly-saccharide

materials. Urinary catheters can develop biofilm in two ways. The extra luminal route, by

direct inoculation at time of catheter insertion or by migration in the mucous sheath

surrounding the catheter, is the most common way (about 70%) [69]. Adherent bacteria

aggregate, multiply and form a biofilm within a few hours [70]. Microorganism may also

enter the catheter from the internal lumen of the catheter, but this intra luminal route

presupposes a contamination of the closed drainage system [71]. The biofilm provides a

protective environment for the microorganisms and make them less susceptible to

antimicrobial agents. The reason for this poor effect of antimicrobial agents is that the

bacteria in the biofilm grow much slower than bacteria growing freely within the urine and

also that the extra cellular matrix prevent penetration of antimicrobial agents into the biofilm

[72]. Another problem with biofilm production is that freely growing bacteria found in

cultures obtained from catheterized patients may not reflect bacterial population growing

15

within the biofilm [73]. Guidelines recommend aseptic technique during catheter insertion

and stress the importance of using a closed urinary drainage system to minimise the

development of CAUTI [74]. Other approaches in preventing these infections include

systemic antibiotics and anti-infective catheters [70] . Silver alloy catheters have been used

and the result of a meta-analysis indicates that they likely prevent bacteriuria and despite a

higher cost also seem economically efficient when used in patients receiving indwelling

catheterization for 2 to 10 days [75, 76]. Impregnation with salicylic acid and even

electrification have been showed to reduce bacterial adhesion to urethral catheters [77, 78].

Additional methods are suprapubic catheters and clean intermittent catheterization.

Suprapubic catheters are mainly used in patients requiring long-term catheterization and

several trials have indicated that patients have lower risk of bacteriuria and higher rate of

satisfaction compared with those using indwelling catheters [79]. Clean intermittent

catheterization is associated with a decreased risk for UTI compared with long-term

indwelling catheters [80]. Despite these preventive efforts CAUTIs are very common and

result in substantially increased health care costs and leads to substantial morbidity and

mortality with data suggesting an almost threefold increase in mortality even when comorbid

conditions and other factors are accounted for [81, 82].

Nitric Oxide The history of nitric oxide. In 1980, Furchgott and co-workers showed that acetylcholine-induced relaxation of blood

vessels was dependent on the endothelium and as a consequence the endothelium-derived

relaxing factor (EDRF) was discovered [83]. Murad and co-workers had three years earlier

shown that nitroglycerine and nitroprusside cause relaxation of smooth muscle cells via

activation of the enzyme soluble guanylyl cyclas (sGC) which increase cyclic guanosine

monophosphate (cGMP) [84]. Murad and co-workers then found that Nitric Oxide (NO) was

the mediator of this cGC activation and, in 1983, that EDRF induced relaxation in smooth

muscle cells via increased cGMP [85, 86]. Eventually in 1987, Ignarro and Moncada with co-

workers independently showed that NO is identical to EDRF [87, 88]. With these Nobel Prize

rewarded discoveries (Furchgott, Ignarro and Murad in 1998), NO went from being regarded

merely an atmospheric pollutant to a highly significant biological molecule. The discovery of

NO led to an explosion of research regarding the biological significance of this small diatomic

molecule with, until today, more than 65 000 publications on the topic.

16

Enzymatic formation of NO

Mammalian cells synthesize NO enzymatically from the amino acid L-arginine and molecular

oxygen by nitric oxide synthase (NOS) [89] (fig 1). This enzymatic reaction is nicotineamide

adenine dinucleotide phosphate (NADPH)-dependent and results in formation of equimolar

amounts of L-citrulline [90]. NOS also needs the cofactors heme, flavin adenine dinucleotide,

flavin mononucleotide and tetrahydrobiopterin [91]. At present, three main isoforms of the

enzyme have been identified, out of which two are expressed constitutively and one isoform

is inducible. The isoforms of the enzymes were named after the tissue in which they were

initially found [92]. The two constitutively expressed (cNOS) are neuronal NOS (nNOS) and

endothelial NOS (eNOS) and activation of these enzymes requires calmodulin, which in turn

is controlled by the intracellular concentration of Ca2+ [93, 94]. Within seconds from

stimulation with agonists such acetylcholine or bradykinin, cNOS produce femtomolar to

picomolar concentration of NO. The third isoenzyme, iNOS, is on the other hand independent

of free Ca2+and its expression can be induced by bacterial products such as lipopolysaccharide

and proinfammatory cytokines (e.g. tumour necrosis factor-α, interleukin-1β and interferon-γ)

a process involving transcription factors (e.g. nuclear factor-κB) and thereby increasing iNOS

mRNA [95, 96]. The time that is required for iNOS mRNA and protein to be synthesized

results in a delay of several hours between cell activation and NO synthesis. Once iNOS is

expressed, which can happen in a wide variety of human cells, it can produce nanomolar

concentration of NO and this production is sustained over a prolonged period of time.

Nonenzymatic formation of NO

Chemical synthesis of NO in vivo was first described 1994 when Benjamin and co-workers

and Lundberg and co-workers independently showed NO production in the stomach, which

relies on the secretion of nitrate in saliva and bacterial conversion to nitrite on the tongue with

reduction to NO by stomach acid [97, 98]. The dorsal surface of the tongue harbours a

specialized flora of symbiotic nitrate-reducing facultative anaerobic bacteria [99].

Nonenzymatic formation occurs in the stomach at low pH, when the nitrite ion (NO2-) is

converted to nitrous acid (HNO2), which subsequently decomposes to various nitrogen oxides

including NO in the parts per million (ppm) range [97, 98] (fig 1).

17

Fig 1. Enzymatic and nonenzymatic NO formation.

Apart from pH, this nonenzymatic formation depends on pO2, proximity to heme containing

proteins, redox state and thiol concentration [100]. Reducing agents, like ascorbic acid, will

further enhance this reaction. Ascorbic acid and ascorbate ion will generate NO over a wide

pH range in aqueous solution by rapid reduction of nitrous acid (HNO2) and formation of

dehydroascorbic acid [101] (fig 1). In addition to the stomach, in vivo non-enzymatic

formation of NO has also been shown in the oral cavity, the heart, and on the skin [99, 102-

104]. The enzyme nitrate reductase is not found in humans, but a number of commensal

bacteria inhabiting mucosal surfaces have multiple nitrate reductases and can catalyse the

reduction of nitrate to nitrite especially during oxygen limitation. There are now increasing

data suggesting that these nitrate-reducing commensals have a true symbiotic role in

mammals [105, 106]. Other enzymes in humans, that can reduce nitrite to NO are

mitochondrial enzymes, xantineoxidase and cytochrome P450 [107-110]. In addition, deoxy-

hemoglobin has recently been shown to reduce nitrite to NO and thereby causing

vasodilatation in the human circulation [111].

Nitrate in medicine

During the 19th century inorganic nitrate was a very common component in many galenic

preparations and was widely used in treatment of different disorders (eg. oedemas, pain and

inflammation) [112]. In the early decade of the 20th century it was shown that inorganic

nitrate could cause methaemoglobinaemia in infants and its therapeutic use clearly fell into

Enzymatic NO formation L-Arginine + NADPH + O2 L-Citrulline + NO + NADP+

NOS

Nonenzymatic NO formation NO2

- + H+ HNO2 Nitrite Nitrous acid

2HNO2 N2O3 + H2O Nitrous acid Dinitrogen trioxide

N2O3 NO + NO2 Dinitrogen trioxide Nitrogen dioxide

Ascorbic acid + 2HNO2 Dehydroascorbic acid + 2NO + 2H2O Nitrous acid

18

disgrace [113]. Organic nitrates, like glyceryl trinitrate and isorbididinitrate on the other hand,

have been used therapeutically for over 100 years and are still in use in the treatment and

prophylaxis of angina pectoris [114]. Since around 1950 when inorganic nitrate was suspected

to be associated with development of gastric cancer its bad reputation grew even worse [115].

The metabolism of nitrate and nitrite can result in N-nitrosoamines which are carcinogenic in

cell cultures and animal models [116]. However, despite numerous studies, there is still no

clear evidence in humans for a link between inorganic nitrate intake and gastric cancer and

most studies show no relationship between a high intake of nitrate and gastric cancer in

humans [117-120]. More recent studies even indicate the opposite, that in humans the

bacterial metabolism of nitrate to nitrite and the subsequent formation of biologically active

nitrogen oxides could in fact be beneficial [105, 121].

Metabolism of nitrate/nitrite

Green vegetables such as lettuce and spinach, root vegetables such as beetroot and drinking

water are the main sources of exogenous nitrate intake [122]. Exogenous intake of nitrite is

much smaller, but nitrite can be found in some food as a ingredient in red meat curing giving

cured meat and hot dogs their red colour (nitrosylated myoglobin) and protecting it from

oxidation and spoiling [123]. Nitrite is particularly effective against the pathogen Clostridium

Botulinum [124]. About 90% of ingested nitrite comes from nitrate in saliva and less than

10% comes from food [125]. The main source of endogenous nitrate in mammals is the L-

arginine-NO-pathway, which in fasting humans constitutes the major source of nitrate/nitrite

in plasma [126]. After ingestion, nitrate is absorbed from the stomach and proximal small

intestine into plasma, where it mixes with endogenously synthesized nitrate [127]. It is then

concentrated, by a factor of ten, from the plasma into the saliva [128]. By this entero-salivary

circulation of nitrate, about 25% of plasma nitrate is actively taken up by the salivary glands

and secreted with saliva [129]. About 1/5 of this recycled nitrate (approximately 5% of the

total ingested nitrate) is converted to nitrite by oral cavity organisms [130]. Studies with

isotope labelled administrated nitrate have shown that only 60% is recovered in the urine.

Consequently, the exact fate of all the nitrate in the body is still unresolved [131].

Chemistry of NO, nitrate and nitrite

The gas NO, with a molecule weight of only 30 Dalton, is certainly one of the smallest

biological mediators. Due to it small size, uncharged character and lipid solubility it diffuses

easily and rapidly over biological membranes. NO is also a free radical in that it has an

19

unpaired electron and is therefore extraordinary labile, with a half-life of only about 1-5

seconds in most biological systems [132]. NO exhibits a low level of solubility in water

(approximately 9 times more lipid soluble than water soluble) and when inserted into a

physiological buffer, the major part of the gas immediately appears in the headspace [133].

Despite being a radical the reactivity of NO under physiologically circumstances is quite

limited to either a reaction with other radicals, with oxygen or with transition metals [134].

The in vivo reaction of NO and superoxide (O2-), that forms peroxynitrite (ONOO-), is an

example of two relatively unreactive free radicals that will produce a much more reactive

species (fig 2). Peroxynitrite is a potentially cytotoxic substance that can be protonated to

peroxynitrous acid (OONOH), which in turn dissociates to oxidizing hydroxyl radical (OH)

and nitrogen dioxide radical (NO2) within seconds at physiological pH [135] (fig 2).

However, eukaryotic cells contain large amounts of superoxide dismutas (SOD), an enzyme

that keep concentration of O2- remarkably low [136]. High concentrations of gaseous NO can

react with oxygen to form NO2, a poisonous gas called “brown gas” (fig 2). However, the

reaction is of second order with respect to NO which means that it will be slower when NO

concentrations are low for example in vivo (normally 100-300 nM and no more than 1-3 µM

during inflammation). Much more common in biological fluids, with oxygen present, is the

rapid oxidation of NO to nitrite (NO2-) or nitrate (NO3

-) [137]. One of the most important

examples of a reaction with metals is when NO interacts with the heme group in the enzyme

sGC and thereby increases cGMP, which in turn mediates many of the biological actions of

NO [138]. Another example of reaction with metals occurs in the blood, where NO reacts

with Hb(Fe2+)O2 to form NO3- and methaemoglobin. This is suggested to be an important

pathway for NO elimination [139] (fig 2). NO can also react with thiol groups allowing the

formation of S-nitrosthiols (RSNOs) that may represent a physiologically important source of

NO [140]. Acidification of nitrite will generate nitrous acid (HNO2), which will

spontaneously yield di-nitrogen trioxide (N2O3), NO and nitrogen dioxide (NO2) [100]. The in

vivo chemistry of these reactive nitrogen intermediates (RNIs), many of which have

biological effects, is very complex and still not fully characterized [141, 142]. Until recently,

the above mentioned oxidation of NO to the inert and stable end products nitrite and nitrate

was generally accepted as a mechanism for inactivation of NO. However, several recent

studies have shown that different pathways exist to recycle nitrite back into bioactive NO in

blood and tissue [97, 98, 102, 108, 110, 111, 143].

20

Fig 2. NO reaction with oxygen, superoxide and oxyhaemoglobin. Nitrate reduction by

bacterial nitrate reductase (NR).

Beneficial effects of NO

NO is a unique messenger molecule. In the autonomic nervous system NO functions as a

major non-adrenergic non-cholinergic neurotransmitter. Moreover, NO has a particularly

important role in relaxation of smooth muscle in the gastrointestinal and urogenital tracts and

NO is responsible for the smooth muscle relaxation of corpus cavernosus leading to penile

erection. These important smooth muscle relaxations are mediated by the reaction of NO with

sGC leading to increased cGMP [83, 144, 145]. Sildenafil (Viagra®), a drug that inhibits

phosphodiesterase type-5 (PDE-5) and thereby inhibit the degradation of cGMP leading to a

prolonged effect of cGMP and better erection, has revolutionised the treatment of erectile

dysfunction [146]. In the urinary tract of animal models, NOS activity has been found in the

urothelium, smooth muscle, striated muscle, nerves and blood vessels [147]. In addition, NO

is an important mediator in dilation of bladder neck, urethra and urethral sphincter during the

micturation reflex [148]. NO is also a potent vasodilator and inhibitor of platelet aggregation

and adhesion [149-151]. Inhibitors of NO synthases have been very useful to explore the

biological activities of NO. Two L-arginine analogues, NG-monomethyl-L-arginine (L-

Nitric oxide oxidation 2NO + O2 2NO2

Nitric oxide oxygen Nitrogen dioxide NO + O2

- OONO- Nitric oxide Superoxide Peroxynitrite OONO- NO3

-

Peroxynitrite Nitrate OONO- + H+ HONOO OH + NO2 Peroxynitrite Peroxynitrous acid Hydroxyl radical Nitrogen dioxide NO + Hb(Fe2+)O2 NO3

- + MetHb(Fe3+) Nitric oxide Oxyhaemoglobin Nitrate Methaemoglobin

Nitrate reduction

NO3

- + e- + 2H+ NO2- + H2O

Nitrate Nitrite NR

21

NMMA) and NG-nitro- L-arginine-methylester (L-NAME), that lack selectivity for the NOS

isoforms, have been shown to increase blood pressure in normotensive humans, confirming

that NO is important in the regulation of vascular tone [152, 153]. There are also several NO-

donors currently being used in various types of experimental studies [154]. Some of these

different NO-donors appear to be of interest for their potential therapeutic applications, for

example the diazeniumdiolates (formerly NONOates), that have been used in studies

regarding inhibiting restenosis after angioplasty, preparing thromboresistant medical devices,

reversing vasospasm, and relieving pulmonary hypertension [155]. Another very exciting

group of NO-donors, are the NO-non-steroidal anti-inflammatory (NO-NSAIDs) drugs, which

are characterized by a reduction in gastrointestinal side effects [156, 157]. Since NO is also

thought to play a role in detrusor instability, a NO-releasing and prostaglandin synthesis-

inhibitory-drug has been used in patients with neurogenic bladder instability with promising

results [157].

Beneficial effects of acidified nitrite.

Dietary nitrate has an important role in protection against ingested pathogens. After a high

nitrate containing meal, like a portion of lettuce, the levels of nitrite and nitrate in saliva

increase. When this saliva enters the acidic environment in the stomach, it results in an

increased NO concentration from basal levels of 20 parts per million (ppm) to > 400 ppm.

The acid environment alone is not sufficient for killing of pathogenic bacteria in the stomach.

However, after mixture of salivary nitrite in gastric juice, most pathogenic bacteria are killed

within a hour in vitro [105, 127]. Dietary nitrate and the above described acidified-nitrite-

derived NO are also of great importance as a gastroprotective agent. Bjorne and co-workers

have shown in a rat in vivo model that mucosal blood flow and mucus secretion increased

after luminal application of nitrite-rich saliva, whereas saliva from a fasting individual had no

effect [158]. Acidified nitrite has antimicrobial effect on periodontal bacteria, which might

explain some of the protective effects of normal saliva against dental caries [159].

Commensal skin bacteria have been shown to produce NO by the reduction of sweat nitrate to

nitrite and its subsequent conversion to NO by the acidic environment [103]. In vitro studies

have shown that acidified nitrite is microbiocidal to common cutaneous pathogens [160]. As

described earlier, acidification of nitrite results in production of a complex mixture of

nitrogen oxides including nitrous acid (HNO2), di-nitrogen trioxide (N2O3), nitrogen dioxide

(NO2) and NO. All these nitrogen oxides may act as nitrosating agents and can rapidly react

with reduced thiols to form nitrosothiols. Nitrosothiols as well as many other of these nitrogen

22

oxides can kill microbes and therefore the exact mechanism responsible for the above

described in vivo antibacterial effects of acidified nitrite is not fully understood and needs to

be further investigated.

NO in inflammation and host-defense

In 1983, it was shown that that E. coli lipopolysaccharides (LPS) stimulates urinary nitrate

excretion and it was proposed that this mammalian nitrate biosynthesis was the result of

oxidation of reduced nitrogen compounds [161]. In 1985, Marletta and Stuehr reported that

LPS-stimulated peritoneal macrophages from mice, synthesize nitrite and nitrate from an

unknown precursor molecule [162]. Three years later the same group showed that NO, was

produced and that L-arginine, was the precursor [162, 163]. Independently in 1987, Hibbs and

co-workers, showed that the anti-tumour effect of macrophages were abolished when arginine

was removed from the medium [164]. In addition to macrophages, cytokine-induced high-

output NO synthesis, has been found in most somatic cells involved in cell-mediated immune

reactions [165]. Today, NO is clearly regarded as a central component of innate immunity and

an effective antimicrobial agent [166]. On the other hand, the large amounts of NO resulting

from increased iNOS expression, may also contribute to the morbidity of infection by acting

as vasodilator, myocardial depressant and cytotoxic mediator [167]. Not all of the host–

derived NO is synthesized by NOS. As described earlier, dietary nitrate can be reduced by

oral bacteria to nitrite and then in the acidic environment in the stomach further reduced to

NO and other RNIs may also be formed. The first line of host defense is the innate immune

response, where phagocytic cells are very important components. In addition to the iNOS

pathway that generate NO radicals, phagocytic cells use another important antimicrobial

system, the NADPH phagocyte oxidase (PHOX) pathway responsible for superoxide (O2-)

generation. Superoxide (O2-) is like hydrogen peroxide (H2O2) and hydroxyl radical (OH)

intermediate reduction products of oxygen en route to water and they are commonly called

“reactive oxygen species” (ROS) [168]. The microbial targets of ROS and RNI are mainly

thiols, metal centres and DNA. RNI mainly inhibit respiration and interfere with DNA

replication, whereas ROS work mainly through direct DNA damage. The antimicrobial

actions of RNI are more complex than those of ROS and multiple cellular targets are almost

certainly involved, although the exact mechanism for the antimicrobial effects are still not

known [167, 169, 170]. ROS/ RNI and phagocytic cells represent nonspecificity in the

immune system and are effective killers of microbes and essential for clearing invading

microbes from an infected area. Phagocytes are quick to sacrifice themselves and surrounding

23

host tissue to prevent microbial metastasis, thus resulting in further tissues damage [169] .

Increased NO production in vivo has been described in many chronic inflammatory processes,

such as for example cystitis [171], asthma [172], colitis [173] and rheumatoid arthritis [174].

NO has been suggested to be very useful as a non-invasive marker of several inflammatory

diseases, although the role of NO in chronic inflammatory processes remains to be elucidated

[175]. However, there are now studies questioning whether the sustained iNOS expression

seen in many chronic inflammatory diseases, for example psoriasis, is actively producing

significant amounts of NO in vivo and recent evidence, in fact, suggest that lack of NO

formation in this disease is of importance in the pathophysiological process [176].

NO and inflammation in the lower urinary tract.

In studies of the mouse urinary tract, as discussed earlier, it has been shown that the

uropathogenic strains adhere to the uroepithelial cells and activate them to release cytokines.

The cytokines recruit polymorphonuclear leukocytes (PMNL) that clear the urinary tract from

the infection [177]. In humans, increased iNOS activity has been detected during UTI from

neutrophils in the urine and the iNOS activity persist for a period of time after initiation of

antibiotic treatment [178]. Luminal NO in the urinary bladder is increased in patients with

cystitis of different aetiology [179]. Cytokines, as well as bacillus calmette-Guérin (BCG),

have been shown to increase iNOS activity in vitro in human uroepithelial cells [180, 181].

However, studies with UTI in iNOS-deficient mice have shown that there were no differences

in bacterial clearance or persistence between mice with intact iNOS capacity compared with

the deficient genotype [182]. It has been shown that human bladder smooth muscle cells

respond to LPS and inflammatory cytokines with increased iNOS activity and collagen type

III expression. The authors suggest that iNOS may be a critical mediator of the bladder wall

fibrotic response to chronic UTI [183]. In ifosfamide induced hemorrhagic cystitis in mice,

NO produced by iNOS, is suggested to be responsible for urothelial damage and inflammation

[184]. In conclusion, the exact role of NO in human UTI is not clear, but taking the well-

known antimicrobial properties into account it is very likely that NO in some way enhance

local host defense in the bladder.

24

Bacterial defense against RNIs.

Since protection against ROI have been studied for over 100 years, detoxification-enzymes

like microbial catalase, SOD and peroxides that convert ROS to less toxic species have been

well documented [185]. In contrast, until about seventeen years ago, RNI production in

mammals was not thought to exist, because RNI was regarded to be too toxic [169].

Knowledge about resistance to RNI is therefore a relatively new concept. However, some

pathways for microbial defense against RNI have now been identified and include scavenge

of RNIs, suppressed RNI production, repair of nitrosative damage and enzymatically

detoxification of RNIs. The most studied bacterial enzymatic detoxification defense to NO

and other RNIs are in E coli, where nitrite reductases and NO reductases, as well as a

flavohaemoglobin, have been characterized [105, 186]. Studies in E coli have also

demonstrated specific antioxidant regulons. One example is a family of approximately 12

genes (even including the SOD gene) called the soxRS regulon, which may protect bacteria

against NO [187].

25

AIMS

The general aim of this thesis was to investigate antibacterial effects of nitrite in urine.

The specific aims were:

• To investigate the changes in NO formation in relation to different concentrations of

nitrite and different pH-levels with or without ascorbic acid in normal and infected

urine.

• To study antibacterial effects of acidified nitrite and ascorbic acid against three

common urinary pathogens at different urinary pH levels.

• To evaluate the minimal bacteriostatic and the minimal bactericidal concentrations of

acidified nitrite and ascorbic acid and to compare the effects with conventional

antibiotics.

• To evaluate the antibacterial effect of a new two-step procedure in which bacteria are

first fed with nitrate, followed by acidification of the urine.

• To evaluate the in vitro antibacterial effect of a novel concept for delivery of NO via a

catheter retention balloon into the surrounding urine.

26

MATERIAL AND METHODS

Study subjects In paper I urine was collected from 8 patients (41-70 years old) with bacteriuria as confirmed

by urinary cultures. Urine was also collected from 5 healthy subjects before and after

ingestion of vitamin C ( 2g/day) for 2 days.

In paper III 8 healthy subjects mean age 37 years (range 25-47) fasted overnight. Basal

urinary samples were collected and then the subjects ingested sodium nitrate (10 mg/kg)

dissolved in 150 mL water. Urinary samples were collected at 1, 2 and 3 hours.

Chemical analysis of NO, nitrite and nitrate Analysis of NO in gaseous phase by chemiluminescence (paper I-II and IV)

In this thesis we have used two chemiluminescence NO analysers when measuring gaseous

NO. In paper I and II CLD 700, Eco Physics, Dürnten, Switzerland was used and in paper

IV, Aerocrine AB, Stockholm, Sweden was used. Measurement of gaseous NO with

chemiluminescence, is based on the reaction of NO with an excess of ozone (O3) to produce

nitrogen dioxide (NO2), which is partly excited (NO2*). When NO2

* returns to its ground state

the excess energy is released as a photon (hv) and light (luminescence) in the 640-3000 nm

wavelength range is emitted, which is measured by a sensitive photomultiplier tube. The

intensity of this luminescence is proportionally converted into an electrical signal and

displayed as NO levels with a response time of less than 0.7 s. The chemiluminiscence assay

is extremely sensitive for NO and has a detection limit of 1 part per billion (ppb) and a linear

response for concentrations between 1-100 000 ppb. The assay also shows a high specificity

for NO without interference from other nitrogen oxides [188]. When ≥50 ml of NO-

containing gas (0.5-20 ppm) was injected into the NO analyser the recovery of the NO signal

was 100%. In this thesis we made use of the well documented properties of NO, as a gas with

poor solubility in water and extremely high diffusibility. Thus, when NO is injected into a

physiological buffer, >80% immediately appears in the gas space (head space) [133]. NO has

also been shown to diffuse through the inflatable retention balloon of a urinary catheter [133,

179, 189]. In paper I and II, urinary samples (10 ml) were incubated at 37◦ C in closed

syringes with a headspace of 50 ml. After 30 min, the headspace gas was removed and

immediately injected into the chemiluminescence analyser (CLD 700, Eco Physics, Dürnten,

Switzerland). Ambient NO levels were below 5 ppb. In paper I the urinary samples consisted

27

of 10 ml infected urine or 10 ml control urine with or without addition of 100 µM sodium

nitrite and NO release was measured at different pH levels. NO was also measured before and

after ingestion of vitamin C (2g/day) for 2 days in 10 ml urinary samples with addition of 100

µM sodium nitrite at different urinary pH levels (pH 4-7). In paper II NO was measured in

10 ml urinary samples consisting of sodium nitrite (50-500 µM) with or without addition of

vitamin C (10 mM) at different urinary pH levels (pH 4.5-6.0). Ambient NO levels were

below 5 ppb in all experiments.

In paper IV An all-silicone catheter (Argyle®, Sherwood Medical, Tullamore, Ireland) was

placed in a bladder model (50 ml flask) and the retention balloon was filled with 10 ml of

saline containing ascorbic acid (10 mM) and sodium nitrite (5 mM) at pH 2.5. The flask was

then closed and synthetic NO-free air was flushed via an inlet at a rate of 4 L/min. Headspace

NO concentration was continuously measured from an outlet by a rapid-response chemi-

luminescence system (Aerocrine AB, Stockholm, Sweden) at room temperature (20° C ).

Ambient NO levels were below 5 ppb in all experiments.

Quantification of nitrite and nitrate by chemiluminescence

In paper III and paper IV nitrite and/or nitrate sample concentrations were determined by

chemiluminescence after reductive cleavage and subsequent determination of the NO released

into the gas phase. The samples were directly introduced via a gas-tight syringe into a

reduction solution of a micro reaction purge vessel coupled with a condenser and heating

jackets unit (Sievers, Boulder, CO, USA). The condenser jacket temperature was controlled

by a continuous flow of cold water while the temperature of the heating jacket was controlled

by a flow of warm water regulated by a constant-temperature circulating bath (MGW Lauda

M3). Nitrogen gas, at flow of 192 mL/min, was used as the carrier gas of NO. The flow could

be adjusted with a needle valve integrated with the purge vessel and the outlet of the gas

stream was passed through a scrubbing bottle containing sodium hydroxide (1 M, 0°C) in

order to trap traces of acid before transfer into the NO analyzer. A chemiluminescence NO

analyser (Aerocrine AB, Stockholm, Sweden) was used to display the NO signals and collect

the data, which were further manipulated with Origin for Windows, Version 7.0 (Microcal,

Northampton, MA) and reported as area under the curve. Nitrite was determined according to

Feelish et al [190]. The reducing mixture, consisting of 45 mmol/L potassium iodide (KI) and

10 mmol/L iodine (I2) in glacial acetic acid, was kept at a constant temperature of 56 °C and

continuously bubbled with nitrogen gas. The amount of nitrite in a given sample was

quantified by simple subtraction of the peak areas of sample aliquots pretreated with

28

sulfanilamide from that of untreated aliquots (10% (v/v) of a 5% solution of sulfanilamide in

1N HCl is added to the biological sample (final concentration 29 mmol/L) and incubated for

15 min at room temperature. Under these conditions, nitrite reacts with sulfanilamide to form

a stable diazonium ion that is not converted to NO. Nitrate was reduced to NO with a solution

of Vanadium(III)chloride in hydrochloric acid 1N (saturated solution) at 95°C. Since

Vanadium(III)/HCl will also convert nitrite to NO the amount of nitrate was quantified by

subtraction of the nitrite concentration calculated before.

Quantification of nitrite by capillary electrophoresis

Capillary electrophoresis (CE) has been used in the analysis of several ions and

micromolecules, because of its high efficiency of separation and short analysis time compared

to other available techniques such as high-performance liquid chromatography (HPLC). In

paper I CE was used to analyse urinary nitrite concentrations in 8 patients with bacteruria.

After collecting midstream urine the samples were stored at -80◦ C until analysis. The CE

system used was Hewlett Packard capillary electrophoresis system (HP 3D, Waldbrunn,

Germany) with UV detection at 214 nm. The background electrolyte consisted of 25 mM

sodium sulphate and 5% NICE-Pack OFM Anion-BT, dissolved in freshly brought Milli-Q

water. Samples were injected hydrodynamically by 30 mbar for 60 seconds onto extended

light path capillaries (104 cm effective lenght and 75 µm ID). The analysis was done at an

applied negative voltage of 300 kV/m and the temperature of the capillaries was set to 40◦ C.

Data were analyzed with a HP 3D CE Chem Station data system. Standard curves for nitrite

was plotted. Before being used for the first time, capillars were conditioned with sodium

hydroxide (1 and 0.1 M) and the background electrolyte for 5 min each while the temperature

of the capillary was set to 60◦ C.

Bacterial cultures and media

The strains used in this thesis were E. coli U1106024 (paper IV) isolated from a patient with

urinary tract infection and reference strains E. coli ATCC 25922 (paper I-IV) , P. aeruginosa

ATCC 27853 (paper II), S. saprophyticus SS2 (paper II), all obtained from the department

of Clinical Microbiology, Uppsala, Sweden. In paper III E. coli RK 4353 and a RK 4353

mutant, lacking all three known nitrate reductase enzymes were used [191]. The RK 4353

strains were a generous gift from Dr. JA. Cole, Birmingham, UK. Before each experiment

29

bacteria were grown aerobically in Mueller-Hinton broth for 6 hours at 37° C, resulting in 2-5

x 108 colony-forming units (CFU)/mL.

All growth experiments in this thesis were carried out in pooled urine. Midstream urine was

collected from healthy subjects (paper I: 5 subjects, paper II: 8 subjects, paper III: 10

subjects, paper IV: 4 subjects) pooled, divided into batches (50 ml) and immediately frozen

(-20 C) until use.

Analysis of bacterial growth

Turbidity measurements

The basic concept for turbidity measurements is that bacteria scatter light in proportion to

their number. The turbidity or optical density of a suspension of cells is directly related to the

cell mass or cell number, after construction and calibration of a standard curve. The method is

efficient, reproducible and has been widely used as a rapid screening method for large number

of antimicrobial agents [192, 193]. In paper I-II the bacterial strains were grown in Mueller-

Hinton broth for 6 hours at 37° C, resulting in 2-5 x 108 colony-forming units (CFU)/mL. The

strains were diluted to a bacterial density of 106 CFU/mL in the pooled urine. After 2 hours of

incubation at 37° C in a closed tube (in pooled urine with various amounts of nitrite, with or

without addition of vitamin C and at various urinary pH-levels), 40 µL of the bacterial

suspension was transferred to micro well plates containing 360 µL sterile filtered control

urine (pH 6). After this dilution (1/10) and transfer to the recovery medium (filtered control

urine, pH 6), bacterial growth was measured continuously for 10 hours (paper I) and 20

hours (paper II) by vertical photometry (optical density) at a wavelength of 540 nm in a

computerized incubator for bacteria (Bioscreen C, Labsytems, Helsinki, Finland). In paper

III the bacterial culture was diluted to a bacterial density of 2-5 x 106 CFU/mL in flat-bottom

micro well plates (96 wells of 300 μL) containing the pooled urine and various amounts of

nitrite, with or without addition of vitamin C and at various urinary pH-levels. Note that in

paper III, there is no use of a recovery medium. Bacterial growth was measured continuously

for 20 hours at 37° C by vertical photometry (optical density) at a wavelength of 540 nm in a

computerized incubator (Molecular Devices Spectra Max 340 Sunnyvale, CA). Minimum

inhibitory concentration (MIC) was defined as the lowest concentration at which no visible

growth had taken place after 20 hours. MICs of nitrite and ascorbic acid were determined. We

also determined MIC values for nitrite in combination with a fixed concentration of ascorbic

30

acid (10 mM). MIC was also determined for nitrofurantoin and trimethoprim using the same

urine.

Viable counts measurements

Viable count involve spreading (plating out) a sample of a culture on a nutritient agar surface.

The sample is diluted in a non-toxic diluent (e.g. phosphate-buffered saline) before plating.

Each viable unit grows and forms a colony. Each colony that can be counted is called a CFU

and the number of CFU´s is related to the viable number of bacteria in the sample. An

excellent correlation has been shown by Domínguez and co-workers between antibacterial

effects by turbidity (Bioscreen C) and viable counts methods for ciprofloxacin and standard

strains of E. coli and P aeruginosa [194].

In paper III we used the viable count method for determination of bactericidal activity of

various amounts of nitrite, with or without addition of vitamin C and at various urinary pH-

levels in the pooled urine. In paper IV we used the viable count method for determination of

bactericidal activity of a novel in vitro concept of delivery of NO via a catheter retention

balloon into the surrounding urine. An all-silicone catheter (Argyle, Sherwood Medical,

Tullamore, Ireland) was placed in the flask and the retention balloon was filled with 10 ml of

saline containing ascorbic acid (10 mM) and sodium nitrite (5 mM). The acidity of the

solution was adjusted to pH 2.5 using hydrochloric acid (3 M). Ascorbic acid and nitrite were

prepared and mixed immediately before administration. Ascorbic acid solution alone (pH 2.5)

was used in control catheters. After filling the retention balloons the catheter was gently

pulled outwards and fixed at the neck whereby the flask-opening was sealed off. Then the

flasks were turned up-side-down and incubated at 37 degrees for 24 h.

After 20 hours (paper III) or 24 hours (paper IV) of bacterial growth/incubation (at 37° C,

10 μL urine was diluted in series with PBS (pH 7.3) and transferred to agar plates. In separate

experiments (paper IV) we also tested shorter exposure times of 5, 30, 60 and 120 min. The

agar plates were incubated for 24 hours and then a viable count was performed (counting

CFU/ml). MBC (Minimum Bactericidal Concentrations) was defined as the concentration

where at least 99.9% (a reduction of > 3 log CFU/ml) of the original inoculate was killed.

In paper IV we also compared the effects of nitrite with another NO-releasing compound

DETA NONOate, which is considered to be a rather pure NO donor. In these experiments the

sodium nitrite was replaced with DETA NONOate (0.05, 0.5 or 5 mM) and all other test

parameters were identical. DETA NONOate was prepared immediately before the start of the

experiment. We then used the viable count method for determination of bactericidal activity

31

as described above. To exclude any antibacterial effect of the parent compound (a polyamine),

the same experiment was also performed with DETA NONOate solution that had been

prepared and stirred for 72 h in open air to release all NO (complete NO release was

confirmed with chemiluminescence showing that the NO signal at 24 h was <0.1 % of the

initial peak level).

Statistics Experimental data are expressed as mean ± S.E.M (paper I-II) and as mean ± S.D. (paper III

–IV). Statistical significance was tested according to Mann-Whitney U test (paper I). For

calculation of statistical difference between repeated measurements analysis of variance

(ANOVA) was used (paper II).

32

0

1

2

3

4

5

6

6,05,5

5,04,5

BasalAA

50+AA100+AA

500+ AA

log

NO

(ppb

)

RESULTS AND COMMENTS

NO formation from nitrite in urine (paper I-II, IV)

Infected urine may contain considerable amounts of nitrite as a result of bacterial nitrate

reductase activity. We sought to determine whether NO was generated from acidified nitrite-

containing urine. We found that at a basal urinary pH (5.5-6.5), NO formation from both

control urine and infected urine was low. In contrast, large amounts of NO were generated

from infected urine (containing 8 to 400µM nitrite) when the urine was acidified to pH 4.5-

5.0 and from acidified non-infected urine if nitrite (100 µM) was added. Urinary NO

formation was strongly pH dependent in the presence of nitrite and was markedly enhanced

by addition of ascorbic acid. We also found that increasing concentrations of nitrite (0-500

μM) caused a dose-dependent enhancement of NO formation that again was greatly enhanced

by the addition of ascorbic acid (Fig 3 and 4).

Fig 3. Urinary NO formation (ppb) in urine from different concentrations of sodium nitrite (0-

500 µM) with the addition of ascorbic acid (AA ,10mM) or from AA alone, at varying urinary

pH values (4.5-6.0).

33

pH 6.0 pH 5.5 pH 5.0 pH 4.5

log

NO

ppb

1

2

3

4

5

6nitritenitrite + AA

Fig 4. NO formation in urine from nitrite (500 µM) at different urinary pH values (4.5-6.0) with

and without the addition of ascorbic acid (AA ,10mM).

We also studied whether a nitrite/ascorbic acid-containing solution when placed in a catheter

retention balloon would generate NO that could be measured outside the balloons. Indeed,

NO did traverse the silicone membrane and measured levels peaked initially at about10 ppm

in the headspace gas. If the balloon was filled with nitrite alone (pH 7) or ascorbic acid alone

(pH 2.5) no NO signal was noted (NO < 0.001 ppm). These results shows that a tiny

uncharged gas such as NO can easily diffuse through the silicon membrane of the retention

balloon of a silicone urinary catheter (Argyle®), which is in accordance with previous findings

when a similar catheter was used as a sampling device [133, 179, 189].

Effects of nitrite on bacterial growth Effects of sodium nitrite (paper I-III)

Urine is an excellent culture medium for bacteria that cause urinary tract infections [32].

Indeed, at a basal urinary pH and even in slightly acidic urine the bacterial growth was very

good. In contrast, the growth of E. coli, P. aeruginosa and S. saprophyticus was dose-

dependently inhibited by nitrite in acidified urine. The inhibitory effect of nitrite was greater

at lower pH. Addition of ascorbic acid (10 mM) further enhanced the inhibition of bacterial

growth by nitrite. Increasing concentrations of nitrite (0-500 μM) with a fixed concentration

of ascorbic acid (10mM) caused a dose-dependent inhibition of E.coli growth which

correlated to the NO formation observed (Fig 5). Note that by this method the bacteria are

transferred to a recovery medium after 2 hours of nitrite exposure and thus we are studying

post-antibiotic effects.

34

Fig 5. Growth of E.coli, after 2 hours of incubation at varying pH values (4.5-6.0) and nitrite

concentrations (0-500 µM) with the addition of ascorbic acid (AA ,10mM) and AA alone.

Bacterial growth is expressed as the time needed for the bacteria to growth to A50 (A50 was

defined as 50% of the maximal absorbency of the controls).

In the following experiments we studied the effect of continuous exposure to acidified nitrite.

Again we found that the growth of E.coli ATCC 25922 was inhibited by sodium nitrite in

acidified urine and that addition of ascorbic acid (10 mM) further enhanced the inhibition of

bacterial growth by sodium nitrite (Fig 6). With the prolonged nitrite exposure the

antibacterial effect was more pronounced which also allowed for calculations of MIC and

MBC. The MICs of sodium nitrite alone was 160 μM at pH 5 and 1280 μM at pH 5.5. With

the addition of ascorbic acid (10 mM) MICs for nitrite were reduced to as low as 20 μM at pH

5 and 160 μM at pH 5.5. Ascorbic acid itself showed poor antibacterial effects with MICs >

40 mM. Bactericidal effects (MBCs) of sodium nitrite + ascorbic acid for E. coli ATCC

25922, was 40 μM at pH 5.0 and 320 μM at pH 5.5.

Taken together, in paper I and II the antibacterial effects of acidified nitrite were a result of

post antibiotic treatment, since the bacteria were only exposed for two hours before being

transferred to recovery medium. In contrast, in paper III, when the bacteria were exposed to

acidified nitrite and ascorbic acid during the entire experiments (20h), the effects were much

more pronounced and in fact for urinary pH ≤ 5.5 the effects were bactericidal.

2

4

6

8

10

12

14

16

18

20

6,05,5

5,04,5

BasalAA

50+AA100+AA

500+AA

Tim

e to

reac

h A

50 (h

ours

)

35

Fig 6. Growth of E.coli in urine at pH 5.5 with different amounts of sodium nitrite (20-160 µM)

and a fixed concentration of ascorbic acid (AA, 10 mM).

Bacterial growth after pre-incubation with nitrate (paper III)

Having confirmed that exogenous nitrite had antibacterial effects in urine, we wanted to go on

and study if nitrite generated by the bacteria themselves could be turned against them in the

form of toxic RNIs. We first studied how much nitrate that would accumulate in urine after

ingesting 10 mg/kg of body weight sodium nitrate (corresponds to the amounts found in about

300g of spinach). Basal nitrate levels in urine were about 0.7 mM in fasting individuals and

these levels increased about 10-fold already one hour after ingestion of sodium nitrate. Now

knowing the amount of nitrate that could easily be achieved in urine, we continued with

studying how much nitrite the nitrate reducing bacteria, E. coli ATCC 25922, could form

from this nitrate. For comparisons we used a E. coli mutant that lacked all nitrate reducing

enzymes. When E. coli was incubated for 20 hours in basal urine (nitrate 0.7 mM) about 2 µM

nitrite was generated. When the basal urine had been supplemented with 1 mM sodium nitrate

the nitrite formation increased to 370 µM. Maximum formation of nitrite was seen after

supplementation with 3 and 10 mM sodium nitrate (2470 and 2400 µM nitrite respectively).

When the mutant lacking all three nitrate reductases was incubated for 20 hours with

maximum substitution of sodium nitrate (10 mM) the formation of nitrite was negligible. Note

that resulting nitrite concentrations after transferring the cultures to the acidic urine in the

second part of the experiment were 1/10 of the values above, in other words 37, 247 and

240µM nitrite respectively.

Time (hours)

0 5 10 15 20

Opt

ical

den

sity

0,10

0,15

0,20

0,25

0,30

0,35

0,40

Basal20 µM nitrite + AA40 µM nitrite + AA80 µM nitrite + AA160 µM nitrite +AA

36

Fig 7 Growth of two strains of E. coli. A control strain and a mutant lacking nitrate reductase.

The bacteria were first grown at pH 7 with sodium nitrate (10mM). After 20 hours the cultures

were transferred to acidified urine (pH 5.0) and bacterial survival was monitored by viable

counts.

Having shown that E. coli ATCC 25922 was able to produce significant amounts of nitrite

from urinary nitrate, we then continued with studying whether acidification in a second step

would result in antibacterial effects. We first incubated E. coli in urine at pH 7.0 with 0-10

mM sodium nitrate added, which resulted in accumulation of nitrite in the medium.

Subsequent transferring of the nitrite-rich culture to acidic urine resulted in a marked decrease

in viable counts. This effect was dose-dependent with more effective killing rates at higher

pre-incubation levels of sodium nitrate. At the highest level of nitrate (10 mM), a final

reduction of more than 3 logs CFU/ml (bactericidal effect) was observed after transfer to

acidic medium. If bacteria were pre-incubated in basal urine without addition of sodium

nitrate, no inhibition was noted when the culture was transferred to the acidic urine. This

indicates that a normal diet in a patient treated with urinary acidification is probably

insufficient to excrete enough nitrate in the urine to achieve antibacterial effects.

In experiments with the mutant lacking nitrate reductases and a control strain both strains

grew similarly well at pH 7.0 with 10 mM sodium nitrate added. When transferring the

cultures to acidic urine containing ascorbic acid the control strain was effectively killed while

the mutant retained full viability (fig 7). Taken together, in paper III we have shown that E.

Control strain

Nitrate reductase mutant

20 h

pH 7.3

20 h

pH 5

Overnight Culture

Urine + nitrate Nitrite↑↑

20 h

pH 7.3

20 h

pH 5

Overnight culture

Urine + nitrate Nitrite≈0

Time hours0 5 10 15 20

log

CFU

/ml

2

3

4

5

6

7

8

9

10

Time hours0 5 10 15 20

log

CFU

/ml

2

3

4

5

6

7

8

9

10

37

coli is effectively killed when it is first allowed to generate nitrite in nitrate rich urine at

neutral pH and then transferred to mildly acidified urine. In contrast, a mutant lacking a

nitrate reducing capacity, retained full viability by this procedure.

Delivery of antibacterial nitrogen oxides via a catheter retention balloon (paperIV)

A model of a catheterized urinary bladder was used to study a novel approach for intravesical

delivery of an antibacterial agent. Bacteria were grown in urine outside of the retention

balloon thereby simulating a catheterized bladder with infection. When the catheter balloon

was filled with ascorbic acid without nitrite (pH 2.5), the bacterial counts of the reference

strain had increased from 2.5x106 to 2.7x108 CFU/ml and the clinical isolate strain had

increased from 9.0x105 to 2.0 x108 CFU/ml after 24 h. In contrast, when using test catheters

with ascorbic acid and nitrite (pH 2.5) both tested strains were effectively killed (fig 8).

To further try to pinpoint the compound responsible for antibacterial effects of acidified

nitrite we also studied the effects of a pure NO donor. A 24 h exposure to the NO-donor

DETA NONOate was completely bacteriostatic at 5 mM while 0.5 and 0.05 mM had no effect

on bacterial growth. Thus, in paper IV we describe a novel concept, which allows for

intravesical delivery of antibacterial gases into the urinary bladder model via the catheter

retention balloon, resulting in bacteriostatic and bactericidal effects in the urine.

Fig 8. Effects of test (5 mM nitrite + 10 mM ascorbic acid) and control (ascorbic acid alone)

catheters on the growth of two strains of E coli in a model of urinary tract infection (pH 6.1).

log

CFU

/ml

1

2

3

4

5

6

7

8

9

Reference controlReference test Clinical isolate controlClinical isolate test

0 24h

38

GENERAL DISCUSSION

Acidified nitrite and antibacterial effects in urine Nitrite has been used in the meat curing process since the 1920s because it produces a

desirable bright pink colour and also enhances flavour. In addition, in 1934 it was shown by

Tanner and Evans that the growth of the anaerobic bacteria of the genus Clostridium was

inhibited by this process [195]. Since 1941, when Tarr showed that bacteria were susceptible

to low concentration of nitrite provided the meat was sufficiently acid, the antimicrobial

effects of acidified nitrite have been used to prevent from botulism [196]. Later it was shown

that addition of ascorbic acid enhanced the inhibition of Clostridium botulinum by nitrite and

that antibacterial effects of nitrite in meat involves generation of NO [197, 198]. However,

not until 1994 when Benjamin/Lundberg and co-workers demonstrated NO generation in the

stomach from acidified nitrite, it was appreciated that this chemical reaction was of

importance even in vivo [97, 98].

In this thesis we have shown (paper I) that large amounts of the antimicrobial gas NO is

formed in mildly acidified nitrite-containing human urine and this NO formation is greatly

enhanced by the addition of ascorbic acid. Furthermore, we have shown (paper I and II) that

mildly acidified nitrite-containing human urine has potent antimicrobial activity against three

of the most common urinary pathogens and this inhibitory effect is further increased by

ascorbic acid. We have also shown (paper III), that E. coli is effectively killed when it is first

allowed to generate nitrite and then transferred to mildly acidified urine. Finally we have

suggested (in paper IV) a new approach to prevent CAUTI by intravesical delivery of

antibacterial compounds generated from nitrite and ascorbic acid via the catheter retention

balloon.

What is the mechanism of action?

It is obvious from this thesis that acidified nitrite together with ascorbic acid shows

antimicrobial effects in urine. However, the exact chemical nature of the toxic nitrite-derived

compound is not known and neither is the exact mechanism by which bacterial killing occurs.

Dykhuizen and co-workers, showed bactericidal effects of acidified nitrite in the stomach

against several common gut pathogens. They claimed that the antimicrobial effect is due to an

additive contribution of reactive intermediates of nitrogen [199]. The chemistry of acidified

nitrite is very complex and a variety of reactive nitrogen intermediates (RNIs) and other

nitrogen oxides are generated directly or after reactions with other compounds.

39

PiliFlagella

Capsule

DNA

Cell wall

Cytoplasm

Cell-membrane

LIPIDS

SH

PROTEINS

RNI

Thiols

NH2

Fe-S

Heme-groups

Aromaticresidues

Amines

Iron-Sulfurcluster

Fig 9. Possible microbial cellular targets of reactive nitrogen intermediates (RNI). Figure

slightly modified from Fang (1997).

These include NO, N2O3, N2O4, NO+, HNO2, NO2, ONOO¯ and S-nitrosothiols, many of

which have antimicrobial activity. Other authors have shown that the sensitivity to different

RNIs differs greatly between microbial pathogens. NO has been shown to be microbicidal to

Mycobacterium tuberculosis and inhibition of NO production has been shown to induce

reactivation of the disease, but it has been suggested that NO has low antimicrobial activity

against E. coli [200, 201]. The question then is if the bactericidal effects of acidified nitrite

observed in this thesis are caused by NO. The fact that ascorbic acid potentiates bactericidal

effects of nitrite in urine suggests that the production of NO at some stage is important for the

antibacterial effects observed. Thus, ascorbic acid greatly increases the production of NO

from nitrite at the expense of most other nitrogen oxides mentioned above [100]. Another

fact, speaking in favour of NO, is that charged nitrogen oxides eg. NO+ or NO2- would not

pass the silicone membrane of the urinary catheter balloon, while NO being a small uncharged

molecule does. Indeed we did detect large amounts of NO outside of the membrane and we

40

did observe bactericidal effects in the urine outside of the catheter balloon. Yet another

observation favouring NO as a candidate of the nitrite effect is that the pure NO-donor DETA

NONOate, had antibacterial effects in the same system. In addition, the antibacterial effect

and the amount of NO produced from acidified nitrite when tested in a Mueller-Hinton

medium instead of urine, were clearly decreased (data not shown). The exact mechanism

whereby NO and other products from acidified nitrite inhibit bacteria is still not settled but

multiple cellular targets are most certainly involved (Fig 9) including DNA, surface proteins

and key enzymes in the respiratory chain [167]. The effects of RNIs on DNA could be

interaction with DNA repair system as well as direct inactivation of ribonucleotide reductase

by NO [202].

Bacterial defense systems

Some urinary pathogens e.g. Proteus species have the capacity of producing ammonia from

urea thereby increasing urinary pH to levels where nitrite is not converted to toxic nitrogen

oxides. Once the higher pH has been established acidification of urine is more difficult [203].

This seems to be an effective mechanism developed by some pathogenic bacteria to protect

against acid/nitrite induced damage. Helicobacter pylori for example, has the unique ability to

thrive within the human stomach and has a urea-specific channel that opens as the pH of the

medium drops and cytoplasmatic urease is able to gain access to urea [204]. Obviously, as

shown in this thesis, when pH falls to very low levels (4.5 and below) urinary bacterial

growth is retarded even in the absence of nitrite indicating also direct effects of acidity itself.

At the pH levels more commonly found in mildly acidified urine (5-5.5) however, the effect

of pH itself was only minor and required the presence of nitrite/ascorbic acid for more

pronounced antibacterial effects (paper II-III).

An other bacterial defense mechanism found in many bacteria, including E. coli, is the flavo

haemoglobins, an ancient family of proteins that have been shown to effectively bind NO

thereby protecting the bacteria from nitrosative stress [186, 205]. Studies in E coli have also

demonstrated specific antioxidant regulons (soxRS regulon), which may protect bacteria

against NO when induced [187]. Indeed, E. coli with deficient soxRS locus, was more

sensitive to NO-dependent killing [206]. Another important defense alternative for E coli,

especially under low oxygen tensions, is the cytochrom c nitrite reductase (NrfA), a bacterial

enzyme that may further reduce NO to other less toxic nitrogen metabolites [207]. In addition,

many denitrifying bacteria, including Pseudomonas species, possess NO reductase enzymes

(NOR), which convert NO to nitrous oxide and ultimately to nitrogen [208]. There are data

41

showing that a mutation of NO reductase gene was lethal to Pseudomonas stutzeri during

anaerobic growth in the presence of nitrate or nitrite [209]. The pH optimum for the nitrate

reductase is high (about pH 8) and it probably operates less effectively at a low pH [210].

Obviously, it is of value for the bacteria not to reduce nitrate to nitrite under acidic conditions,

as nitrite will automatically convert to toxic nitrogen oxides thereby leading to self-

destruction. Other compounds that can help bacteria to detoxify NO or related compounds

include glutathione, homocysteine and superoxide dismutase [167]. Taken together there are

many different possible bacterial defense mechanisms against RNIs, but apparently they were

all insufficient in our experiments. One can speculate that it is the rapid kinetics of NO release

from acidified nitrite described in this thesis, that prevent the bacteria from up regulating

these defensive pathways.

Possible clinical usefulness

Treatment of UTI

Acidification of urine e.g. with vitamin C has been used as a household remedy for urinary

tract infections although the mechanism of action remains uncertain. We here speculate that

some of these effects are related to generation of bactericidal nitrogen oxides from urinary

nitrite. In this process Vitamin C has a dual action; it helps to acidify the urine and in addition

its intrinsic reducing capacity greatly enhances nitrite reduction. The reported clinical effects

of Vitamin C are however not very consistent and there may be several reasons for this. First,

vitamin C itself does not seem to be a very effective urinary acidifier and sometimes it

completely fails to reduce urinary pH [57-59]. In addition, in cases where acidification of

urine might have been effective, the nitrite concentration in urine may have been to low. As

shown here vitamin C itself had poor antibacterial effect and acidity itself did not have a

major influence on bacterial growth in urine. However, with the further addition of nitrite

bacteria were effectively killed in this medium. Therefore, to refine and improve the clinical

effects of vitamin C we probably need to create conditions with sufficient concentrations of

nitrite in urine in combination with a consistent and effective acidification of the urine. Is this

then possible to achieve in vivo? The urinary ascorbic acid concentration (10mM) used in this

thesis is physiologically achievable and in fact, similar to those obtained after ingestion of 1-2

gram of vitamin C daily [211]. Urinary pH normally varies between 5 and 7 and urinary pH

levels as low as 4.6 have been reported after treatment with drugs used for urinary

acidification [212]. Urinary nitrite in patients with nitrite-positive urinary tract infection

42

varies between 10-700 μM [213]. Thus, in some cases these amounts would be sufficient

provided that the urine is made acidic and contain enough ascorbic acid. In addition, we could

supplement the diet with nitrate to further increase substrate supply to the bacteria in the urine

as shown here. Already one hour after ingestion of 10 mg/kg sodium-nitrate (which

corresponds to what is found in about 300g spinach), nitrate levels in urine increased to a

concentration that is well above what the bacteria’s need to generate enough nitrite.

Now, knowing that sufficient amounts of nitrite, ascorbic acid and low urinary pH is

physiologically achievable. How can we accomplish this in the clinical setting? For this

concept to work we need first to obtain high nitrite levels in urine. Then, we probably need to

lower the urinary pH quite rapidly in order to convert the nitrite into toxic RNI`s. Thus, a

sequential two-steps procedure may be preferable. A medicinal product resulting in sufficient

urinary nitrate levels would be useful to start with. The dose of nitrate ingested and the time

interval between nitrate intake and acidification of the urine will probably be critical. In this

thesis (paper III) we used a pre-incubation period of 20 h in nitrate-rich urine. With this long

incubation time maximum amounts of nitrite are generated. For practical reasons a shorter

incubation time will be necessary in a clinical setting which may result in less nitrite

generated. A potential problem is therefore that nitrite levels could be to low to effectively kill

the bacteria when the urine is acidified. There are several ways of acidifying the urine, but

even if pH can be sufficiently lowered it will likely be important that this process is rather

rapid so that bacteria will not have time to up regulate defense mechanisms. In fact, studies

with ammonium chloride (50 mg/kg) showed a significant decrease in urinary pH (4.7-5.4)

already about 4 hours after a single dose [214]. Various other agents for acidifying the urine

have been used, these include mandelic acid, gluconic acid and methionine [52-55].

Another potential problem is that infected urine often has higher pH and therefore

acidification is probably more difficult. On the other hand, considering the potent antibacterial

effect of acidified nitrite, a decrease in urinary pH below a critical level during a limited

period, could be sufficient to inhibit bacterial growth if nitrite levels are sufficiently high. In

this thesis we used pooled urine from many different volunteers on a normal western diet. It is

likely that the antimicrobial activity of acidified nitrite in urine will vary because of different

composition of urine e.g. in relation to diet. Also, urinary osmolality may influence the

antibacterial effects of nitrite in acidified urine. One great advantage with the sequential

procedure described here compared to many traditional antibiotics is that the bacteria in the

gastro-intestinal tract are probably much less, if at all affected. On the other hand, a

disadvantage with the concept is that it will only work in bacteria with nitrate reductases.

43

Bacteria that do not reduce nitrate e.g. Enterococcus faecalis will not be sensitive to this

treatment similarly to the E. coli nitrate reductase mutant used in paper III.

Taken together, we have suggested a new concept for treatment of urinary tract infections

caused by nitrate-reducing bacteria. This involves a two-step procedure were bacteria are first

fed with nitrate followed some hours later by acidification of the urine. According to this

theory the bacteria self-destruct by producing nitrite which is later turned against them in

form of toxic RNI`s when urine is acidified. Clearly, controlled in vivo studies are needed to

evaluate the clinical potential for this antimicrobial regime and to test the timing of the

therapeutic interventions (nitrate intake, ascorbic acid intake and urinary acidification).

Prevention of CAUTI

CAUTI continues to be large problem in modern medical care and this condition is associated

with significant higher costs, morbidity and even higher mortality. Immense amount of work

has been done trying to prevent these infections, for example the development of silver alloy

catheters, catheters with salicylic acid impregnation, immersion of catheters in antibiotic

solution, closed drainage systems and even electrification of catheters [70, 77, 78]. Despite

these efforts CAUTI continues to be a major problem in modern health care.

In this thesis (paper IV) we describe a new concept, which allows for direct intravesical

delivery of antibacterial gases into the urinary bladder via the catheter retention balloon. It is

tempting to speculate that this regime could be useful to treat and to prevent CAUTI`s. Is

then this concept applicable in vivo? Again a key question is if the in vitro concentrations of

nitrite, ascorbic acid and the pH used in this set up are effective in vivo also without causing

troublesome side-effects. The concentrations of ascorbic acid (10 mM) used here were the

same as described above. Natural acidification of salivary nitrite in the stomach, continuously

exposes the gastric mucosa to similar amounts of nitrogen oxides. Furthermore, a nitrite

concentration of about 3 mM, which is similar to what we used in this set up, is allowed as an

ingredient in meat curing. Thus, these antibacterial compounds are expected to have fairly

low acute toxicity to host cells in the concentrations used here. Whether a prolonged exposure

of the bladder to these RNSs could favour formation of nitrosamines, a group of potentially

carcinogenic chemicals, must indeed be further studied. However, the addition of ascorbic

acid as one component in the retention balloon likely inhibits the total formation of

nitrosamines [215]. The urinary pH in the bladder-model was not influenced by the acidic

solution in the intact catheter balloon. Even if the balloon broke by an accident, the small

amount (10ml) of solution with pH 2.5 would likely have low acute toxicity to the bladder

44

mucosa since probably at least some amount of residual urine is present thereby diluting and

buffering the acidic solution.

In paper IV we examined two different strains of bacteria. Although not studied here, it is

likely that also other urinary tract pathogens are sensitive to this procedure. Thus, from the

results presented in this thesis it is known that RNSs generated from acidified nitrite have

anti-bacterial effects against several of the most common urinary pathogens including also P.

aeruginosa and S. saprophyticus.

A potential advantage with this regime is that NO will likely diffuse easily and rapidly into

the biofilm generated by the bacteria. This biofilm normally provides a protective

environment for the microorganisms and thereby making them less susceptible to

antimicrobial agents. Naturally, this assumption has to be tested. Many other questions remain

to answer before we can consider using this method in the clinic. For example we need to

determine the durability of the antibacterial effect as well as, the exact dosing of nitrite and

ascorbic acid and the risk of bacterial resistance. Nevertheless, this simple concept of

substituting the content of the retention balloon of a urinary catheter with an active diffusible

drug instead of normal saline, is an attractive new alternative to the existing methods used to

treat and prevent CAUTI`s. With this novel method one could easily administrate repeated

individual doses of the active drug at chosen intervals without disturbing the integrity of the

closed drainage system. Considering the fact that CAUTIs are still very common and result in

substantially increased morbidity and health care costs, new approaches for their prevention

are highly warranted.

45

CONCLUSIONS

• Large amounts of NO were formed in mildly acidified nitrite-containing infected

human urine and in acidified control urine after addition of nitrite. This NO formation

was strongly pH dependent and was greatly increased by the addition of ascorbic acid.

• The growth of E. coli, P. aeruginosa and S. saprophyticus was dose-dependently

inhibited after two hours of incubation in mildly acidified nitrite-containing urine.

This bacteriostatic effect was greater at lower pH and was markedly potentiated by

ascorbic acid.

• Prolonged exposure of to physiological achievable concentrations of sodium nitrite

plus ascorbic acid resulted in bactericidal effects. In comparison with conventional

antibiotics tested in this same model nitrite seemed to be at least equally potent.

• A two-step procedure where E coli. was first fed with nitrate followed later by

acidification of the urine, resulted in effective bacterial killing. This killing was

dependent on the nitrite generated by the bacteria themselves.

• A novel concept for intravesical delivery of NO via a catheter retention balloon was

tested in vitro. The urinary pathogen E. coli was effectively killed by this procedure.

46

ACKNOWLEDGEMENTS

I wish to express my sincere gratitude to all those who have helped and supported me throughout these studies. I especially thank:

Professor Jon Lundberg, my brilliant tutor, for helping me during all phases of my work and for your countless numbers of inspiring and innovative “pilot-experiments”. For giving to me the huge responsibility to continue the “urine track” from your dissertation, for your excellent scientific guidance in combination with always being enthusiastic and encouraging. For your great patience when I during some periods did other important things in life such as being on paternity leave and practising robotic surgery. Professor Peter Wiklund, my co-tutor and chairman at my department, for introducing me to the fascinating field of nitric oxide research. For your unique triad of qualities; being an excellent scientist, an extraordinary talented surgeon and having a great personality. For always having answers and moving forwards. For encouraging me to give lectures in front of many people. For teaching me robotic surgery and for never switching off your mobile phone. My co-authors Mirco Govoni, Eddie Weitzberg Lars Engstrand, Edward Morcos for stimulating discussions and enjoyable collaboration. Peter Ekman, for supporting me to do science, for being an excellent speaker and for giving us at the Urology Department inspiring scientific summaries (veckobrev) from all over the world. Lars Norlen, for being such a great support in all kinds of clinical problems, for being a very skillful surgeon with an extraordinary talent for teaching this noble art and for employing me at the clinic. Ann-Charlotte Kinn and Jan Sehlin, for being my tutors during my residency at the Department of Urology and for your support and excellent clinical guidance. My lab-colleagues: Daniel Törnberg, Håkan Björne, Helena Marteus, Jörgen Palm, Max Herulf, Wilhelm Zetterquist, Emmelie Jansson, Claudia Reinders, Tanja Sobko, Helen Sylvin Roelinde Middelveld, Katarina Hallen, and Nasrin Bavand, for making work so enjoyable. My former and present colleagues at the Urology Department involved in the NO-research group, Ingrid Ehren, Abbi Hosseini, Olof Jansson and Lotta Renström-Koskela, for support and advice during international scientific conferences. Margareta Stensdotter and Carina Nihlen, for excellent technical assistance. Carina, I would never have been able to finish my thesis in 2005 without you! Torgny Kolmert, for taking special good care of me when I nine years ago did my first attempts in Urology procedures.

47

My colleagues and excellent robotic assistants Andreas Nilsson, Martin Jonsson, Andreas Thorstensson and Ramin Gaffarpour, for all the fun we have together, for taking very good care of my robotic-patients before and after surgery, making it possible for me to write this thesis. Guys, I will repay when you write your thesis! My colleagues in the prostate-cancer group, Stephan Brändstedt, Leif Haendler, Magnus Hellström and David Kudren and my other colleagues at the clinic Pelle Larsson, Elisabeth Farrelly, Daniela Volz, Marianne Brehmer and Eric Borgström, for support and understanding. Berit Björk and Annika Broström, for being helpful with administrative matters and support. Marie Dahlgren, for always helping me in the best way with administrative matters concerning salary and vacation (Marie, you know how much I hate filling in forms!) Carl-Eric and Sonja, my parents for your love and constant support in everything I have been doing in my life and for your indispensable help with the children. Sudha, my sister for being there and caring for every one around you and always supporting me and my family. Gunnar and Aase, for being the best parents-in-law you could ever dream of. Lars, my neighbour for “magic computer-skills” when my computer crashed, some weeks before printing deadline of my thesis. Finally, I would like to thank Anne, my wife and my “dream-team” friend for endless amounts of patience and love. Emma, Adam and Fabian, our wonderful children for being a constant source of joy in life.

48

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