Translational Science
Intravesical administration of green tea extract attenuates the inflammatory response of bacterial cystitis – a rat model Shilo Rosenberg, Ray Horowitz†, Shunit Coppenhagen-Glazer‡, Galina Pizov*, Anna Elia, Ofer N. Gofrit, Isaac Ginsburg‡ and Dov Pode Departments of Urology, *Pathology and †Clinical Microbiology and Infectious Diseases, Hadassah Medical Center, Hebrew University, and ‡Institute for Dental Sciences Hebrew University, Hadassah School of Dental Medicine, Jerusalem, Israel
Objective To explore the effect of intravesical instillation of green tea extract (GTE) on a rat model of bacterial cystitis.
Materials and Methods In vitro bactericidal properties of GTE were analysed by adding GTE to a suspension of uropathogenic E. coli (UPEC), streaking on MacConkey agar, and incubating overnight. In vivo effects of intravesical instillation of GTE on bacterial cystitis was analysed using a rat model of bacterial cystitis. In all, 42 female Sabra rats weighing 200–260 g were divided into five groups. Parameters measured were bladder weight (percentage of the total rat weight), dipstick urine analysis and histopathological changes in the bladder. Histological changes evaluated were degree of oedema, mixed inflammatory infiltration, urothelial epithelial invasion by neutrophils and reactive atypia.
Results No in vitro bactericidal activity was detected for GTE.
Introduction Uropathogenic Escherichia coli (UPEC) is the most common bacterial pathogen causing cystitis. Colonisation of the urinary bladder is mediated by its ability to adhere onto the urothelium by interaction between its fimbriae and the uroplakins on the urothelial surface membrane [1]. Women are more susceptible to UTIs due to anatomical, behavioural and physiological factors. Approximately half of all women have at least one episode of UTI during their lifetime and a recurrent episode occurring within half a year in 25–35% [2]. The most effective treatment for bacterial cystitis is antibiotics. Women having recurrent episodes are offered prophylactic antibacterial treatment. However, © 2013 The Authors BJU International © 2013 BJU International | doi:10.1111/bju.12544 Published by John Wiley & Sons Ltd. www.bjui.org
Intravesical instillation of GTE did not cause damage to the rat bladders. Intravesical instillation of GTE attenuated the inflammatory response to UPEC-SR71-induced bacterial cystitis in this rat model.
Conclusions Intravesical instillation of GTE attenuated the inflammatory response to UPEC-SR71-induced bacterial cystitis and is a novel approach to the treatment of bacterial cystitis. High concentrations of intravesical GTE did not cause histologically evident damage to the rat bladder. The results of this study are preliminary and further studies will be needed to explore the feasibility of using this approach in humans.
Keywords green tea extract, intravesical instillation, uropathogenic E. coli (UPEC), bacterial cystitis
antibiotic treatment is not without consequence. Two important disadvantages are emergence of resistant bacteria and drug-related side-effects. This has led clinicians to look for novel non-antibiotic therapies. Polyphenols constitute a large group of compounds present in coloured fruits vegetables and beverages, e.g. grapes, tea, coffee, pomegranate, cocoa and cinnamon [3]. The major polyphenols in green tea are the catechins: epicatechin, epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate. Understanding of the in vivo effects of green tea is incomplete but amounting data from both in vivo and in vitro studies suggests that catechins possess anti-adhesive, anti-neoplastic and anti-inflammatory traits. Oral green tea consumption inhibited bladder tumour growth in a rat model [4]. Green tea extract
BJU Int 2014; 114: 601–607 wileyonlinelibrary.com
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Blasting the resulting sequence against National Center for Biotechnology Information (NBCI) chromosomes revealed a close proximity with E. coli KTE203, which is a UTI isolate. The sequence was deposited in the gene bank (accession number: KF192074). This UPEC strain was named SR71. UPEC-SR71 was cultivated overnight in brain heart infusion broth (Difco), washed several times in normal saline, suspended in saline and adjusted spectrophotometrically at 540 nm to an O.D. of 15.0, which corresponds to ≈75 × 108 bacteria/mL verified by a standard colony forming unit (CFU) assay.
(GTE) consumed orally lowered plasma cholesterol and upregulated LDL receptors in a rabbit model [5]. In another study in a rat model of metabolic syndrome, GTE improved hypertension and insulin resistance [6]. In, in vitro studies, GTE was shown to possess antibacterial activity on skin pathogens and on multidrug resistant UPEC [7,8]. We have previously shown that various polyphenols, including catechins, that cover the membranes of several cell types, enhance its oxidant scavenging abilities [9]. The plasma bioavailability of polyphenols after oral intake of nutrients rich in polyphenols is extremely low (in the μM range) [10]. This is probably due to regulatory mechanisms preventing polyphenols from entering the blood circulation. Therefore urinary levels of polyphenols after oral intake are low and unpredictable. In the present study we explored the effect of intravesical instillation of GTE in a rat model of bacterial cystitis.
Materials and Methods UPEC was provided by the Microbiology Laboratory of the Hadassah Hospital, Jerusalem. The bacterium was isolated from a female patient with bacterial cystitis. This bacterial strain was analysed genetically and serologically by the E. coli Reference Center, at Pennsylvania State University (Table 1). In addition, the 16srRNA was amplified by PCR and sequenced.
To study the cytopathogical changes in the urothelium that occur after intravesical administration of UPEC-SR71, 42 Sabra female rats weighing 200–260 g were divided into five groups. Approval of the animal experiment was granted from the Animal Ethics Review Board (MD-09-11890-4). The rats received water and chow ad libidum with a 12:12 h light-dark cycle. Chinese green tea was purchased from a commercial source. GTE was prepared from green tea leaves grinded in a mortar, 200 mg portions were suspended in 2 mL saline and heated to boiling point. After centrifugation at 179 g, the liquid phase was removed and the concentration of total phenols in the extract was measured by the modified Folin-Ciocalteu’s reagent (FCR) assay (described below) and adjusted spectrophoptometrically at 760 nm to ≈40 mM of gallic acid equivalent (GAE) as a standard. The same concentrations of bacteria and catechins were used throughout our experiments.
Table 1 Genetic and serologic analysis of UPEC-SR71 strain. Performed at the E. coli reference center, at Pennsylvania State University. Gene LT STa STb STX1 STX2 EAE cnf1 cnf2 hlyD hlyA fimH sfa papGIII papGI papA papC foc fyuA iroN traT kii kpsII uidA PAI ompT usp O type antigen H type antigen
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Determination of Polyphenol Concentration by FCR Assay
Function LT (heat labile toxin) Heat stable toxin a Heat stable toxin b Shiga toxin types 1 Shiga toxin types 2 Intimin- gamma Cytotoxic necrotizing factors 1 Cytotoxic necrotizing factors 2 α-hemolysin α-hemolysin D-mannose-specific adhesin Central region of sfa (S fimbriae) allele III cystitis-associated allele I, J96-associated papG variant Major structural subunit of P fimbrial shaft; defines F antigen Pilus assembly, central region of pap operon Pilus tip molecule, F1C fimbriae (sialic acid-specific) Yersinia siderophore receptor (ferric yersiniabactin uptake) Novel catecholate siderophore receptor Serum-resistance associated Group II capsular polysaccharide synthesis (e.g. K1, K5, K12) Group II capsular polysaccharide synthesis (e.g. K1, K5, K12) Beta-D-glucuronidase Pathogenicity island Outer membrane protein (protease) T Uropathogenic-specific protein (bacteriocin)
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– – – – – – + – + – + + – – + + + + + + – + + + – + 4 5
FCR is a mixture of phosphomolybade and phosphotungstate used for the colorimetric measurements of polyphenols present in the GTE. Briefly, 5 μL GTE was added to 800 μL normal saline followed by addition of 50 μL Folin reagent and then incubated for 5 min. Finally, 150 μL of a saturated sodium carbonate solution (25% w/v) were added. The optical densities in the supernatant fluids of the GTE were determined spectophotometrically at 760 nm. The amounts of polyphenols were expressed as GAE [11]. In vitro Evaluation of Bactericidal Activity of the GTE Two rows of 10 test tubes, each containing 900 μL saline were prepared. Bacterial suspension (100 μL) at 75 × 108 bacteria/mL, was added to the first test tube in each row and a series of 10-fold dilutions were prepared in both rows to the tenth order (logarithmic dilutions). Then 300 μL GTE was added to each test tube in one row. After 30 min incubation at room temperature, 100 μL from each test tube were streaked on MacConkey agar plates, incubated overnight at 37 °C and the numbers of colonies were counted.
Green tea extract attenuates inflammatory response of bacterial cystitis
Bacterial Cystitis Model The rats were anaesthetised with an i.p. injection of 6 mg/100 g ketamine HCl and 2.5 mg/100 g xylazine. A 22 G venflon was inserted transurethrally and UPEC-SR71 suspended in 500 μL saline at 75 × 108 bacteria/mL was instilled. The urethral orifice was then tied for 30 min with a silk 2/0 suture. In pilot studies, maximum inflammatory response was seen 3 days after inoculation.
in the urine was determined using the standard CFU method streaked and grown on MacConkey agar. Histological Analysis Four parameters were considered for grading the inflammatory response to infection (Fig. 1). The histological sections were cut uniformly coronal in the middle of the bladder. One pathologist, whom was ‘blinded’ to the origin of
Study Groups Five groups of female rats were used. Instillation of saline, GTE and UPEC-SR71 were carried out under anaesthesia with an i.p. injection of 6 mg/100 g ketamine HCl and 2.5 mg/100 g xylazine. Group A: six rats that did not receive intravesical instillation (controls). Group B: six rats to which 300 μL GTE at 40 mM GAE was instilled transurethrally using a 22 G venflon. After instillation, the venflon was plugged for 15 min. Subsequently the venflon was unplugged and the bladder drained by gentle suprapubic massage. A second bolus of GTE, of the same volume, was instilled and the urethral orifice blocked for 30 min with a silk 2/0 suture. Group C: six rats to which UPEC-SR71 was administered intravesically in 500 μL saline at a concentration of 75 × 108 bacteria/mL. The urethral orifice was blocked for 30 min with 2/0 silk suture. Group D: 12 rats which received transurethral instillation of saline and UPEC-SR71. First, 300 μL saline was instilled transurethrally and the venflon plugged for 15 min. Then the venflon was unplugged and bladder emptied by gentle suprapubic massage. UPEC-SR71 (500 μL) was instilled through a 22-G venflon followed by 300 μL saline giving a total volume of 800 μL. The urethral orifice was blocked with a 2/0 silk suture for 30 min. Group E: 12 rats which received GTE and UPEC-SR71. The procedure was identical as in group D except that GTE was instilled instead of saline. At 3 days after the transurethral procedures the rats were anaesthetised and weighed, after death (with a lethal dose of pentothal) the bladders were removed, weighed and recorded as a fraction of the rat’s weight (Wt%). The bladders were then fixed in 4% neutral formaldehyde, embedded in paraffin blocks and 7-μm sections were stained with haematoxylin and eosin. Urine samples for urine analysis and culture were obtained by puncture of the bladder dome with a 25-G needle, before removing the bladders. Pyuria was estimated using a urinary test strip (Multistix 10 SG, Bayer) and regarded positive if the strip indicated ≥25 white blood cells per high power field. The in vivo bactericidal properties of GTE were determined by obtaining urine for culture from the rats of each group immediately before death. The concentration of UPEC-SR71
Fig. 1 (a) Normal rat bladder wall (haematoxylin and eosin [H&E], ×10). (b) White arrow pointing at inflammatory infiltrate in the lamina propria (H&E, ×10). (c) Neutrophilic invasion of the urothelium (yellow arrow) and reactive atypia of the urothelium (black arrow; H&E, ×40).
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each slide, graded each slide according to the grading score described below. Oedema (score 0–4): 0, No oedema; 1, Oedema seen in the lamina propria in a quarter of the bladder circumference; 2, Oedema seen in the lamina propria in a half of the bladder circumference; 3, Oedema seen in the lamina propria in three quarters of the bladder circumference; and 4, Severe oedema in the lamina propria of the whole bladder circumference. Mixed inflammatory infiltrate (score 0–3): 0, Absence of inflammatory cell infiltration seen; 1, Focal superficial infiltration present in the lamina propria of one urothelial fold; 2, Infiltration of the lamina propria of half the bladder circumference; and 3, Transmural infiltration of the detrusor by inflammatory cells. Urothelial epithelial invasion (present or absent): At least one urothelial fold invaded by neutrophils.
Reactive atypia (present or absent): considered positive if reactive atypia was present in more than a quarter of the bladders’ urothelial lining.
Results Bladder Weight and Urine Analysis The bladders were harvested 3 days after inoculation, weighed and presented as a percentage of the total rat weight. The variances of relative weight among the five groups are presented by ANOVA statistical analysis (Table 2). The relative bladder weight of group E rats (UPEC-SR71+GTE) was significantly lower than groups C (cystitis model) and D (UPEC-SR71+saline). There was no significant difference in the relative bladder weight among groups A (untreated rats) and B (GTE instillation alone). In comparing pyuria, groups C and D urine had significantly more pyuria than urine of group E. Results compared by chi square analysis (Table 2).
Table 2 Relative rat bladder weight shows similar relative weights in groups A, B and C, D. Group E rat bladders have reduced relative weight indicating a protective role for GTE. Pyuria among the groups shows the same trend. Results are statistically significant. Group A B C D E
Treatment
No. of rats
Relative weight %*
Pyuria in urine**, %
Normal bladders (controls) Intravesical GTE UPEC-SR71 cystitis UPEC-SR71 + saline UPEC-SR71 + GTE
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0.031483 0.032867 0.063683 0.064692 0.040350
0 16.6 100 83.33 25
*ANOVA analysis of relative bladder weight (P < 0.001); **Chi square analysis of pyuria in urine of bladders among groups (P < 0.001).
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Fig. 2 In vitro bactericidal analysis: (A) Positive (left) and negative (right) controls. (B,C,D) Logarithmic dilutions of UPEC-SR71 concentration 101, 105, 1010, respectively. Left petri dish with UPEC-SR71, right petri dish UPEC-SR71+GTE. No bactericidal effect detected.
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Green tea extract attenuates inflammatory response of bacterial cystitis
Fig. 3 (a)
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analysis of bladder oedema among groups. Oedema in group E shows a statistically significant decrease vs C and D. (b)
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inflammatory infiltrate among groups. Group E shows a statistically significant decrease vs group C and D bladders. (c) The difference in reactive atypia among groups (chi square analysis P < 0.01). (d) The difference in urothelial neutrophilic invasion among groups (chi square analysis P < 0.01).
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In Vitro and in Vivo Bactericidal Analysis GTE at the concentrations tested (40 mM GAE) had no bactericidal effect in vitro as compared with controls (Fig. 2). The in vitro experiment was repeated three times and the same results obtained. There was no statistically significant difference in colony count on MacConkey agar between the rows. In addition, urine cultures collected from group D and E rats before death showed >105 CFU/mL of viable UPEC-SR71. Histological Analysis Each of the four inflammatory parameters was evaluated separately. The variances in oedema and inflammatory infiltrates among the groups were analysed using ANOVA. Group C (cystitis model) and D (UPEC-SR71+saline) bladders showed a statistically significant increase in both oedema and
inflammatory infiltrate vs group E (UPEC-SR71+GTE) as shown in Fig. 3a,b. Reactive atypia and urothelial neutrophilic invasion were significantly more prominent in groups C and D vs group E by chi square analysis. The administration of GTE (group E) decreased reactive atypia and urothelial neutrophilic invasion by 75% and 66% respectively (Fig. 3c,d). Histologically, GTE attenuated the inflammatory response of UPEC-SR71 cystitis in the rat bladder (Fig. 4).
Discussion The present study shows that intravesical GTE instillation attenuates the inflammatory response of UPEC-SR71-induced bacterial cystitis. Furthermore, intravesical GTE did not cause histological damage to the rat bladder. In addition, GTE did not prove bactericidal neither in vivo or in vitro at the concentrations used in the present experiments. © 2013 The Authors BJU International © 2013 BJU International
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Group A
Group D
Group E
Fig. 4 Histological evidence to the attenuating qualities of green tea catechins on UPEC-SR71 cystitis. Upper, middle and lower row magnification ×10, ×20, and ×40, respectively. Group A (controls), Group D (UPEC-SR71+saline), Group E (UPEC-SR71+GTE).
The relative bladder weight (Table 2) and histology (Fig. 3) were similar between group A (untreated rats) and B rats (GTE only) suggesting that in our experimental setting intravesival instillation did not harm the rats’ urinary bladder. Instillation of GTE caused a decrease in bladder inflammation (Fig. 4) All four histological parameters used in measuring inflammation: oedema, mixed inflammatory infiltrate, reactive atypia and urothelial neutrophilic invasion, showed a statistically significant decrease when comparing groups D (UPEC-SR71+saline) with E (UPEC-SR71+GTE) rats (Fig. 3). In addition, when comparing relative bladder weight and pyuria among the groups, the results clearly show that intravesical instillation of GTE had a protective effect on the infected bladders (Table 2). Application of intravesical GTE may prove advantageous in situations where a high bacterial load exists in the urinary bladder. Examples are patients on clean intermittent catheterisation, patients with persistent bacteriuria with or without a resistant strain and female patients with multiple antibiotic allergies and recurrent lower UTI. GTE did not show bactericidal activity in vitro or in vivo. In vitro, logarithmic dilutions to the tenth order of UPECSR71 concentration were carried out. This experiment was repeated three times. Overnight growth of UPEC-SR71 incubated with GTE was identical to the matched controls with no statistical difference in bacterial colony growth on MacConkey agar (Fig. 2). Before death group D and E rats (data not shown) urine was sent for culture. All cultures
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were positive for UPEC-SR71 growth with >105 CFU/mL. Some evidence for the antimicrobial qualities of green tea has been reported in vitro [12]. However, in the present study we did not find conclusive evidence for such activity of UPEC-SR71. This may be due to high concentration of bacterial inoculum, relatively low concentrations of GTE or a resistant bacterial strain. Furthermore, we did not measure the specific concentrations of polyphenols in our GTE, as EGCG and EGC are known to have a higher antimicrobial activity. The most investigated polyphenol as a protector against UTI is proanthocyanidin present in cranberry juice. It endows its protective effect by inhibiting adhesion of UPEC to the surface urothelial cells [13]. It has been shown that this anti-adhesive quality is maintained against antibiotic-resistant bacteria [14]. Proantocyanidin is a catechin oligomer; however, we did not find studies exploring the use of aqueous GTE in the prevention of UTI. The results of clinical studies evaluating the protective role of cranberry juice on recurrent UTI have been ambiguous. In these clinical studies, cranberry juice consumption is always administered per os and urinary levels of polyphenols after consumption are never measured. Urine concentration of polyphenols is unpredictable after oral consumption due to their poor bioavailability. It has been shown that although animals might consume millimolar amounts of polypohenols only micromolar amounts of polyphenols reach the plasma or urine [10]. The present findings that intravesical instillation of GTE, at the time of
Green tea extract attenuates inflammatory response of bacterial cystitis
bladder inoculation with UPEC-SR71, attenuates the inflammatory response against UTI is a novel finding. An in vivo model of intravesical catechin instillation has been described in the literature. In that study EGCG was compared with mitomycin C as an agent for the prevention of tumour recurrence after transurethral bladder tumour resection [15]. Intravesical treatments exist and have been shown to protect against inflammatory conditions of the urinary bladder. However, we did not find studies using GTE for this purpose [16].
3
The present study has several limitations. First, we have not provided a mechanism of action for the observations reported herein. In a pilot study (data not shown) intravesical instillation of GTE a day after inducing cystitis had no histological anti-inflammatory effect on rat urinary bladder when the rats were killed on the third day after inoculation. Taken together the data presented suggests that attenuation of the inflammatory response is probably the result of an anti-adhesive effect of GTE. Second, although no histological damage was seen after intravesical instillation of a large concentration of polyphenols, the influence on the functional status of the urinary bladder is unknown. Finally, further studies will be needed to understand the consequences of long-term intravesical polyphenol instillation to the bladder and the possible side-effects to the host.
7
In conclusion, intravesical GTE for the prevention of UTI is a novel concept that to our knowledge has not been previously described. In this preliminary study of an in vivo rat model, intravesical GTE attenuated the inflammatory response to UPEC-SR71 cystitis in a rat bladder. Intravesical instillation of GTE did not induce evident tissue damage to the rat bladder and was not found to be bactericidal. This route of administration of polyphenols bypasses the gastrointestinal tract and liver metabolism maximising levels in the target organ. Further studies are needed to explore the feasibility of using this in humans.
13
Conflict of Interest None declared.
References 1
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Hannan TJ, Totsika M, Mansfield KJ, Moore KH, Schembri MA, Hultgren SJ. Host-pathogen checkpoints and population bottlenecks in persistent and intracellular uropathogenic Escherichia coli bladder infection. FEMS Microbiol Rev 2012; 36: 616–48 Dielubanza EJ, Schaeffer AJ. Urinary tract infections in women. Med Clin North Am 2011; 95: 27–41
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Ginsburg I, Kohen R, Koren E. Microbial and host cells acquire enhanced oxidant-scavenging abilities by binding polyphenols. Arch Biochem Biophys 2011; 506: 12–23 Sato D. Inhibition of urinary bladder tumors induced by N-butyl-N(4-hydroxybutyl)-nitrosamine in rats by green tea. Int J Urol 1999; 6: 93–9 Bursill CA, Abbey M, Roach PD. A green tea extract lowers plasma cholesterol by inhibiting cholesterol synthesis and upregulating the LDL receptor in the cholesterol-fed rabbit. Atherosclerosis 2007; 193: 86–93 Ihm SH, Jang SW, Kim OR et al. Decaffeinated green tea extract improves hypertension and insulin resistance in a rat model of metabolic syndrome. Atherosclerosis 2012; 224: 377–83 Sharma A, Gupta S, Sarethy IP, Dang S, Gabrani R. Green tea extract: possible mechanism and antibacterial activity on skin pathogens. Food Chem 2012; 135: 672–5 Reygaert W, Jusufi I. Green tea as an effective antimicrobial for urinary tract infections caused by Escherichia coli. Front Microbiol 2013; doi: 10.3389/fmicb.2013.00162 Koren E, Kohen R, Ginsburg I. Polyphenols enhance total oxidant-scavenging capacities of human blood by binding to red blood cells. Exp Biol Med (Maywood) 2010; 235: 689–99 Fung ST, Ho CK, Choi SW, Chung WY, Benzie IF. Comparison of catechin profiles in human plasma and urine after single dosing and regular intake of green tea (Camellia sinensis). Br J Nutr 2013; 109: 2199–207 Singleton V, Orthofer R, Lamuela-Raventos R. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Method Enzymol 1999; 299: 152–78 Taylor PW, Hamilton-Miller JM, Stapleton PD. Antimicrobial properties of green tea catechins. Food Sci Technol Bull 2005; 2: 71–81 Zafriri D, Ofek I, Adar R, Pocino M, Sharon N. Inhibitory activity of cranberry juice on adherence of type 1 and type P fimbriated Escherichia coli to eucaryotic cells. Antimicrob Agents Chemother 1989; 33: 92–8 Gupta A, Dwivedi M, Mahdi AA, Nagana Gowda GA, Khetrapal CL, Bhandari M. Inhibition of adherence of multi-drug resistant E. coli by proanthocyanidin. Urol Res 2012; 40: 143–50 Selman SH, Keck RW. A comparative study of the inhibiting effects of mitomycin C and polyphenolic catechins on tumor cell implantation/growth in a rat bladder tumor model. J Urol 2011; 186: 702–6 Constantinides C, Manousakas T, Nikolopoulos P, Stanitsas A, Haritopoulos K, Giannopoulos A. Prevention of recurrent bacterial cystitis by intravesical administration of hyaluronic acid: a pilot study. BJU Int 2004; 93: 1262–6
Correspondence: Shilo Rosenberg, Department of Urology, Hadassah-Hebrew University Medical Center, Jerusalem 60850, Israel. e-mail: [email protected] Abbreviations: CFU, colony forming unit; EGCG, epigallocatechin-3-gallate; FCR, Folin-Ciocalteu’s reagent; GAE, gallic acid equivalent; GTE, green tea extract; UPEC, uropathogenic Escherichia coli.
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Intravesical administration of green tea extract attenuates the inflammatory response of bacterial cystitis – a rat model Shilo Rosenberg, Ray Horowitz†, Shunit Coppenhagen-Glazer‡, Galina Pizov*, Anna Elia, Ofer N. Gofrit, Isaac Ginsburg‡ and Dov Pode Departments of Urology, *Pathology and †Clinical Microbiology and Infectious Diseases, Hadassah Medical Center, Hebrew University, and ‡Institute for Dental Sciences Hebrew University, Hadassah School of Dental Medicine, Jerusalem, Israel
Objective To explore the effect of intravesical instillation of green tea extract (GTE) on a rat model of bacterial cystitis.
Materials and Methods In vitro bactericidal properties of GTE were analysed by adding GTE to a suspension of uropathogenic E. coli (UPEC), streaking on MacConkey agar, and incubating overnight. In vivo effects of intravesical instillation of GTE on bacterial cystitis was analysed using a rat model of bacterial cystitis. In all, 42 female Sabra rats weighing 200–260 g were divided into five groups. Parameters measured were bladder weight (percentage of the total rat weight), dipstick urine analysis and histopathological changes in the bladder. Histological changes evaluated were degree of oedema, mixed inflammatory infiltration, urothelial epithelial invasion by neutrophils and reactive atypia.
Results No in vitro bactericidal activity was detected for GTE.
Introduction Uropathogenic Escherichia coli (UPEC) is the most common bacterial pathogen causing cystitis. Colonisation of the urinary bladder is mediated by its ability to adhere onto the urothelium by interaction between its fimbriae and the uroplakins on the urothelial surface membrane [1]. Women are more susceptible to UTIs due to anatomical, behavioural and physiological factors. Approximately half of all women have at least one episode of UTI during their lifetime and a recurrent episode occurring within half a year in 25–35% [2]. The most effective treatment for bacterial cystitis is antibiotics. Women having recurrent episodes are offered prophylactic antibacterial treatment. However, © 2013 The Authors BJU International © 2013 BJU International | doi:10.1111/bju.12544 Published by John Wiley & Sons Ltd. www.bjui.org
Intravesical instillation of GTE did not cause damage to the rat bladders. Intravesical instillation of GTE attenuated the inflammatory response to UPEC-SR71-induced bacterial cystitis in this rat model.
Conclusions Intravesical instillation of GTE attenuated the inflammatory response to UPEC-SR71-induced bacterial cystitis and is a novel approach to the treatment of bacterial cystitis. High concentrations of intravesical GTE did not cause histologically evident damage to the rat bladder. The results of this study are preliminary and further studies will be needed to explore the feasibility of using this approach in humans.
Keywords green tea extract, intravesical instillation, uropathogenic E. coli (UPEC), bacterial cystitis
antibiotic treatment is not without consequence. Two important disadvantages are emergence of resistant bacteria and drug-related side-effects. This has led clinicians to look for novel non-antibiotic therapies. Polyphenols constitute a large group of compounds present in coloured fruits vegetables and beverages, e.g. grapes, tea, coffee, pomegranate, cocoa and cinnamon [3]. The major polyphenols in green tea are the catechins: epicatechin, epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate. Understanding of the in vivo effects of green tea is incomplete but amounting data from both in vivo and in vitro studies suggests that catechins possess anti-adhesive, anti-neoplastic and anti-inflammatory traits. Oral green tea consumption inhibited bladder tumour growth in a rat model [4]. Green tea extract
BJU Int 2014; 114: 601–607 wileyonlinelibrary.com
Rosenberg et al.
Blasting the resulting sequence against National Center for Biotechnology Information (NBCI) chromosomes revealed a close proximity with E. coli KTE203, which is a UTI isolate. The sequence was deposited in the gene bank (accession number: KF192074). This UPEC strain was named SR71. UPEC-SR71 was cultivated overnight in brain heart infusion broth (Difco), washed several times in normal saline, suspended in saline and adjusted spectrophotometrically at 540 nm to an O.D. of 15.0, which corresponds to ≈75 × 108 bacteria/mL verified by a standard colony forming unit (CFU) assay.
(GTE) consumed orally lowered plasma cholesterol and upregulated LDL receptors in a rabbit model [5]. In another study in a rat model of metabolic syndrome, GTE improved hypertension and insulin resistance [6]. In, in vitro studies, GTE was shown to possess antibacterial activity on skin pathogens and on multidrug resistant UPEC [7,8]. We have previously shown that various polyphenols, including catechins, that cover the membranes of several cell types, enhance its oxidant scavenging abilities [9]. The plasma bioavailability of polyphenols after oral intake of nutrients rich in polyphenols is extremely low (in the μM range) [10]. This is probably due to regulatory mechanisms preventing polyphenols from entering the blood circulation. Therefore urinary levels of polyphenols after oral intake are low and unpredictable. In the present study we explored the effect of intravesical instillation of GTE in a rat model of bacterial cystitis.
Materials and Methods UPEC was provided by the Microbiology Laboratory of the Hadassah Hospital, Jerusalem. The bacterium was isolated from a female patient with bacterial cystitis. This bacterial strain was analysed genetically and serologically by the E. coli Reference Center, at Pennsylvania State University (Table 1). In addition, the 16srRNA was amplified by PCR and sequenced.
To study the cytopathogical changes in the urothelium that occur after intravesical administration of UPEC-SR71, 42 Sabra female rats weighing 200–260 g were divided into five groups. Approval of the animal experiment was granted from the Animal Ethics Review Board (MD-09-11890-4). The rats received water and chow ad libidum with a 12:12 h light-dark cycle. Chinese green tea was purchased from a commercial source. GTE was prepared from green tea leaves grinded in a mortar, 200 mg portions were suspended in 2 mL saline and heated to boiling point. After centrifugation at 179 g, the liquid phase was removed and the concentration of total phenols in the extract was measured by the modified Folin-Ciocalteu’s reagent (FCR) assay (described below) and adjusted spectrophoptometrically at 760 nm to ≈40 mM of gallic acid equivalent (GAE) as a standard. The same concentrations of bacteria and catechins were used throughout our experiments.
Table 1 Genetic and serologic analysis of UPEC-SR71 strain. Performed at the E. coli reference center, at Pennsylvania State University. Gene LT STa STb STX1 STX2 EAE cnf1 cnf2 hlyD hlyA fimH sfa papGIII papGI papA papC foc fyuA iroN traT kii kpsII uidA PAI ompT usp O type antigen H type antigen
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Determination of Polyphenol Concentration by FCR Assay
Function LT (heat labile toxin) Heat stable toxin a Heat stable toxin b Shiga toxin types 1 Shiga toxin types 2 Intimin- gamma Cytotoxic necrotizing factors 1 Cytotoxic necrotizing factors 2 α-hemolysin α-hemolysin D-mannose-specific adhesin Central region of sfa (S fimbriae) allele III cystitis-associated allele I, J96-associated papG variant Major structural subunit of P fimbrial shaft; defines F antigen Pilus assembly, central region of pap operon Pilus tip molecule, F1C fimbriae (sialic acid-specific) Yersinia siderophore receptor (ferric yersiniabactin uptake) Novel catecholate siderophore receptor Serum-resistance associated Group II capsular polysaccharide synthesis (e.g. K1, K5, K12) Group II capsular polysaccharide synthesis (e.g. K1, K5, K12) Beta-D-glucuronidase Pathogenicity island Outer membrane protein (protease) T Uropathogenic-specific protein (bacteriocin)
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– – – – – – + – + – + + – – + + + + + + – + + + – + 4 5
FCR is a mixture of phosphomolybade and phosphotungstate used for the colorimetric measurements of polyphenols present in the GTE. Briefly, 5 μL GTE was added to 800 μL normal saline followed by addition of 50 μL Folin reagent and then incubated for 5 min. Finally, 150 μL of a saturated sodium carbonate solution (25% w/v) were added. The optical densities in the supernatant fluids of the GTE were determined spectophotometrically at 760 nm. The amounts of polyphenols were expressed as GAE [11]. In vitro Evaluation of Bactericidal Activity of the GTE Two rows of 10 test tubes, each containing 900 μL saline were prepared. Bacterial suspension (100 μL) at 75 × 108 bacteria/mL, was added to the first test tube in each row and a series of 10-fold dilutions were prepared in both rows to the tenth order (logarithmic dilutions). Then 300 μL GTE was added to each test tube in one row. After 30 min incubation at room temperature, 100 μL from each test tube were streaked on MacConkey agar plates, incubated overnight at 37 °C and the numbers of colonies were counted.
Green tea extract attenuates inflammatory response of bacterial cystitis
Bacterial Cystitis Model The rats were anaesthetised with an i.p. injection of 6 mg/100 g ketamine HCl and 2.5 mg/100 g xylazine. A 22 G venflon was inserted transurethrally and UPEC-SR71 suspended in 500 μL saline at 75 × 108 bacteria/mL was instilled. The urethral orifice was then tied for 30 min with a silk 2/0 suture. In pilot studies, maximum inflammatory response was seen 3 days after inoculation.
in the urine was determined using the standard CFU method streaked and grown on MacConkey agar. Histological Analysis Four parameters were considered for grading the inflammatory response to infection (Fig. 1). The histological sections were cut uniformly coronal in the middle of the bladder. One pathologist, whom was ‘blinded’ to the origin of
Study Groups Five groups of female rats were used. Instillation of saline, GTE and UPEC-SR71 were carried out under anaesthesia with an i.p. injection of 6 mg/100 g ketamine HCl and 2.5 mg/100 g xylazine. Group A: six rats that did not receive intravesical instillation (controls). Group B: six rats to which 300 μL GTE at 40 mM GAE was instilled transurethrally using a 22 G venflon. After instillation, the venflon was plugged for 15 min. Subsequently the venflon was unplugged and the bladder drained by gentle suprapubic massage. A second bolus of GTE, of the same volume, was instilled and the urethral orifice blocked for 30 min with a silk 2/0 suture. Group C: six rats to which UPEC-SR71 was administered intravesically in 500 μL saline at a concentration of 75 × 108 bacteria/mL. The urethral orifice was blocked for 30 min with 2/0 silk suture. Group D: 12 rats which received transurethral instillation of saline and UPEC-SR71. First, 300 μL saline was instilled transurethrally and the venflon plugged for 15 min. Then the venflon was unplugged and bladder emptied by gentle suprapubic massage. UPEC-SR71 (500 μL) was instilled through a 22-G venflon followed by 300 μL saline giving a total volume of 800 μL. The urethral orifice was blocked with a 2/0 silk suture for 30 min. Group E: 12 rats which received GTE and UPEC-SR71. The procedure was identical as in group D except that GTE was instilled instead of saline. At 3 days after the transurethral procedures the rats were anaesthetised and weighed, after death (with a lethal dose of pentothal) the bladders were removed, weighed and recorded as a fraction of the rat’s weight (Wt%). The bladders were then fixed in 4% neutral formaldehyde, embedded in paraffin blocks and 7-μm sections were stained with haematoxylin and eosin. Urine samples for urine analysis and culture were obtained by puncture of the bladder dome with a 25-G needle, before removing the bladders. Pyuria was estimated using a urinary test strip (Multistix 10 SG, Bayer) and regarded positive if the strip indicated ≥25 white blood cells per high power field. The in vivo bactericidal properties of GTE were determined by obtaining urine for culture from the rats of each group immediately before death. The concentration of UPEC-SR71
Fig. 1 (a) Normal rat bladder wall (haematoxylin and eosin [H&E], ×10). (b) White arrow pointing at inflammatory infiltrate in the lamina propria (H&E, ×10). (c) Neutrophilic invasion of the urothelium (yellow arrow) and reactive atypia of the urothelium (black arrow; H&E, ×40).
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each slide, graded each slide according to the grading score described below. Oedema (score 0–4): 0, No oedema; 1, Oedema seen in the lamina propria in a quarter of the bladder circumference; 2, Oedema seen in the lamina propria in a half of the bladder circumference; 3, Oedema seen in the lamina propria in three quarters of the bladder circumference; and 4, Severe oedema in the lamina propria of the whole bladder circumference. Mixed inflammatory infiltrate (score 0–3): 0, Absence of inflammatory cell infiltration seen; 1, Focal superficial infiltration present in the lamina propria of one urothelial fold; 2, Infiltration of the lamina propria of half the bladder circumference; and 3, Transmural infiltration of the detrusor by inflammatory cells. Urothelial epithelial invasion (present or absent): At least one urothelial fold invaded by neutrophils.
Reactive atypia (present or absent): considered positive if reactive atypia was present in more than a quarter of the bladders’ urothelial lining.
Results Bladder Weight and Urine Analysis The bladders were harvested 3 days after inoculation, weighed and presented as a percentage of the total rat weight. The variances of relative weight among the five groups are presented by ANOVA statistical analysis (Table 2). The relative bladder weight of group E rats (UPEC-SR71+GTE) was significantly lower than groups C (cystitis model) and D (UPEC-SR71+saline). There was no significant difference in the relative bladder weight among groups A (untreated rats) and B (GTE instillation alone). In comparing pyuria, groups C and D urine had significantly more pyuria than urine of group E. Results compared by chi square analysis (Table 2).
Table 2 Relative rat bladder weight shows similar relative weights in groups A, B and C, D. Group E rat bladders have reduced relative weight indicating a protective role for GTE. Pyuria among the groups shows the same trend. Results are statistically significant. Group A B C D E
Treatment
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0 16.6 100 83.33 25
*ANOVA analysis of relative bladder weight (P < 0.001); **Chi square analysis of pyuria in urine of bladders among groups (P < 0.001).
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Fig. 2 In vitro bactericidal analysis: (A) Positive (left) and negative (right) controls. (B,C,D) Logarithmic dilutions of UPEC-SR71 concentration 101, 105, 1010, respectively. Left petri dish with UPEC-SR71, right petri dish UPEC-SR71+GTE. No bactericidal effect detected.
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Green tea extract attenuates inflammatory response of bacterial cystitis
Fig. 3 (a)
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inflammatory infiltrate among groups. Group E shows a statistically significant decrease vs group C and D bladders. (c) The difference in reactive atypia among groups (chi square analysis P < 0.01). (d) The difference in urothelial neutrophilic invasion among groups (chi square analysis P < 0.01).
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In Vitro and in Vivo Bactericidal Analysis GTE at the concentrations tested (40 mM GAE) had no bactericidal effect in vitro as compared with controls (Fig. 2). The in vitro experiment was repeated three times and the same results obtained. There was no statistically significant difference in colony count on MacConkey agar between the rows. In addition, urine cultures collected from group D and E rats before death showed >105 CFU/mL of viable UPEC-SR71. Histological Analysis Each of the four inflammatory parameters was evaluated separately. The variances in oedema and inflammatory infiltrates among the groups were analysed using ANOVA. Group C (cystitis model) and D (UPEC-SR71+saline) bladders showed a statistically significant increase in both oedema and
inflammatory infiltrate vs group E (UPEC-SR71+GTE) as shown in Fig. 3a,b. Reactive atypia and urothelial neutrophilic invasion were significantly more prominent in groups C and D vs group E by chi square analysis. The administration of GTE (group E) decreased reactive atypia and urothelial neutrophilic invasion by 75% and 66% respectively (Fig. 3c,d). Histologically, GTE attenuated the inflammatory response of UPEC-SR71 cystitis in the rat bladder (Fig. 4).
Discussion The present study shows that intravesical GTE instillation attenuates the inflammatory response of UPEC-SR71-induced bacterial cystitis. Furthermore, intravesical GTE did not cause histological damage to the rat bladder. In addition, GTE did not prove bactericidal neither in vivo or in vitro at the concentrations used in the present experiments. © 2013 The Authors BJU International © 2013 BJU International
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Group A
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Fig. 4 Histological evidence to the attenuating qualities of green tea catechins on UPEC-SR71 cystitis. Upper, middle and lower row magnification ×10, ×20, and ×40, respectively. Group A (controls), Group D (UPEC-SR71+saline), Group E (UPEC-SR71+GTE).
The relative bladder weight (Table 2) and histology (Fig. 3) were similar between group A (untreated rats) and B rats (GTE only) suggesting that in our experimental setting intravesival instillation did not harm the rats’ urinary bladder. Instillation of GTE caused a decrease in bladder inflammation (Fig. 4) All four histological parameters used in measuring inflammation: oedema, mixed inflammatory infiltrate, reactive atypia and urothelial neutrophilic invasion, showed a statistically significant decrease when comparing groups D (UPEC-SR71+saline) with E (UPEC-SR71+GTE) rats (Fig. 3). In addition, when comparing relative bladder weight and pyuria among the groups, the results clearly show that intravesical instillation of GTE had a protective effect on the infected bladders (Table 2). Application of intravesical GTE may prove advantageous in situations where a high bacterial load exists in the urinary bladder. Examples are patients on clean intermittent catheterisation, patients with persistent bacteriuria with or without a resistant strain and female patients with multiple antibiotic allergies and recurrent lower UTI. GTE did not show bactericidal activity in vitro or in vivo. In vitro, logarithmic dilutions to the tenth order of UPECSR71 concentration were carried out. This experiment was repeated three times. Overnight growth of UPEC-SR71 incubated with GTE was identical to the matched controls with no statistical difference in bacterial colony growth on MacConkey agar (Fig. 2). Before death group D and E rats (data not shown) urine was sent for culture. All cultures
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were positive for UPEC-SR71 growth with >105 CFU/mL. Some evidence for the antimicrobial qualities of green tea has been reported in vitro [12]. However, in the present study we did not find conclusive evidence for such activity of UPEC-SR71. This may be due to high concentration of bacterial inoculum, relatively low concentrations of GTE or a resistant bacterial strain. Furthermore, we did not measure the specific concentrations of polyphenols in our GTE, as EGCG and EGC are known to have a higher antimicrobial activity. The most investigated polyphenol as a protector against UTI is proanthocyanidin present in cranberry juice. It endows its protective effect by inhibiting adhesion of UPEC to the surface urothelial cells [13]. It has been shown that this anti-adhesive quality is maintained against antibiotic-resistant bacteria [14]. Proantocyanidin is a catechin oligomer; however, we did not find studies exploring the use of aqueous GTE in the prevention of UTI. The results of clinical studies evaluating the protective role of cranberry juice on recurrent UTI have been ambiguous. In these clinical studies, cranberry juice consumption is always administered per os and urinary levels of polyphenols after consumption are never measured. Urine concentration of polyphenols is unpredictable after oral consumption due to their poor bioavailability. It has been shown that although animals might consume millimolar amounts of polypohenols only micromolar amounts of polyphenols reach the plasma or urine [10]. The present findings that intravesical instillation of GTE, at the time of
Green tea extract attenuates inflammatory response of bacterial cystitis
bladder inoculation with UPEC-SR71, attenuates the inflammatory response against UTI is a novel finding. An in vivo model of intravesical catechin instillation has been described in the literature. In that study EGCG was compared with mitomycin C as an agent for the prevention of tumour recurrence after transurethral bladder tumour resection [15]. Intravesical treatments exist and have been shown to protect against inflammatory conditions of the urinary bladder. However, we did not find studies using GTE for this purpose [16].
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The present study has several limitations. First, we have not provided a mechanism of action for the observations reported herein. In a pilot study (data not shown) intravesical instillation of GTE a day after inducing cystitis had no histological anti-inflammatory effect on rat urinary bladder when the rats were killed on the third day after inoculation. Taken together the data presented suggests that attenuation of the inflammatory response is probably the result of an anti-adhesive effect of GTE. Second, although no histological damage was seen after intravesical instillation of a large concentration of polyphenols, the influence on the functional status of the urinary bladder is unknown. Finally, further studies will be needed to understand the consequences of long-term intravesical polyphenol instillation to the bladder and the possible side-effects to the host.
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In conclusion, intravesical GTE for the prevention of UTI is a novel concept that to our knowledge has not been previously described. In this preliminary study of an in vivo rat model, intravesical GTE attenuated the inflammatory response to UPEC-SR71 cystitis in a rat bladder. Intravesical instillation of GTE did not induce evident tissue damage to the rat bladder and was not found to be bactericidal. This route of administration of polyphenols bypasses the gastrointestinal tract and liver metabolism maximising levels in the target organ. Further studies are needed to explore the feasibility of using this in humans.
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Conflict of Interest None declared.
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Correspondence: Shilo Rosenberg, Department of Urology, Hadassah-Hebrew University Medical Center, Jerusalem 60850, Israel. e-mail: [email protected] Abbreviations: CFU, colony forming unit; EGCG, epigallocatechin-3-gallate; FCR, Folin-Ciocalteu’s reagent; GAE, gallic acid equivalent; GTE, green tea extract; UPEC, uropathogenic Escherichia coli.
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