Application of a nanotechnology antimicrobial spray to prevent lower urinary tract infection: a multicenter urology trial

J Transl Med. 2012 Sep 19;10 Suppl 1(Suppl 1):S14. doi: 10.1186/1479-5876-10-S1-S14

Wei He¹, Dongmin Wang², Zhangqun Ye¹, Weihong Qian³, Yan Tao³, Xiaofeng Shi³, Ling Liu⁴, Jin Chen⁵,

Ling Qiu⁶, Peng Wan⁷, Xiaojun Jia⁸, Xia Li⁹, Caixia Gao¹⁰, Xuexia Ma¹¹, Biyan Wen⁹, Nianzhen Chen¹², Ping Li¹³, Zhengzheng Ren¹⁴, Li Lan¹⁵, Siyi Li¹⁶, Yi Zuo¹⁷, Hua Zhang¹⁸, Liming Ma¹⁸, Yueping Zhang¹⁸, Zhicong Li¹⁹, Weiping Su²⁰, Qing Yang²¹, Qingli Chen²², Xuejing Wang²², Zhenni Ye²³, JP Chen²⁴, Wings TY Loo^24,25*,

Louis WC Chow²⁵, Adrian YS Yip²⁵, Elizabeth LY Ng²⁵, Mary NB Cheung²⁵, Zhiping Wang^2†

From Organisation for Oncology and Translational Research (OOTR) 7th Annual Conference Hong Kong. 13-14 May 2011

Abstract

Background: Catheter-associated urinary tract infection (CAUTI) is a common nosocomial device-associated infection. It is now recognized that the high infection rates were caused by the formation of biofilm on the surface of the catheters that decreases the susceptibility to antibiotics and results in anti-microbial resistance.

In this study, we performed an in vitro test to explore the mechanism of biofilm formation and subsequently conducted a multi-center clinical trial to investigate the efficacy of CAUTI prevention with the application of JUC, a nanotechnology antimicrobial spray.

Methods: Siliconized latex urinary catheters were cut into fragments and sterilized by autoclaving. The sterilized sample fragments were randomly divided into the therapy and control group, whereby they were sprayed with JUC and distilled water respectively and dried before use.

The experimental standard strains of Escherichia coli (E. coli) were isolated from the urine samples of patients. At 16 hours and 7 days of incubation, the samples were extracted for confocal laser scanning microscopy.

A total of 1,150 patients were accrued in the clinical study. Patients were randomized according to the order of surgical treatment. The odd array of patients was assigned as the therapy group (JUC), and the even array of patients was assigned as the control group (normal saline).

Results: After 16 hours of culture, bacterial biofilm formed on the surface of sample fragments from the control group. In the therapy group, no bacterial biofilm formation was observed on the sample fragments. No significant increase in bacterial colony count was observed in the therapy group after 7 days of incubation.

On the 7th day of catheterization, urine samples were collected for bacterial culture before extubation. Significant difference was observed in the incidence of bacteriuria between the therapy group and control group (4.52% vs. 13.04%, p < 0.001).

Conclusions: In this study, the effectiveness of JUC in preventing CAUTI in a hospital setting was demonstrated in both in vitro and clinical studies.

Background

Catheter-associated urinary tract infection (CAUTI) is a common nosocomial device-associated infection. Urinary tract infection (UTI) accounts for up to 40% of nosoco- mial infections and is one of the main types of health- care-associated infections (HAI). About 80% of UTIs are catheter-associated [1,2]. In the United States, approxi- mately 95% of UTIs were associated with the indwelling catheters [3], and interestingly, 15-25% of patients in short-term hospital care need to be inserted with indwel- ling urinary catheters [4]. Every year, there are more than 5 million patients necessitating catheterization therapy [5] and approximately 1 million patients suffering from CAUTI [6]. The findings of a European study indicated that 5.4% of patients aged 65 or above required the use of an indwelling urinary catheter [7].

CAUTI is a highly common infection and comes with considerable risk. The duration of hospitalization owing to CAUTI increased from 2.4 to 5.4 days in the United States [8]. On average, the costs of diagnosing and treating CAUTI is US$ 589, excluding extension of hospital costs [9]. Taking into account the expenses of hospitalization, the average cost increases from US$ 2,836 to 3,803 [10,11]. The Centers for Disease Control and Prevention (CDC) pointed out that UTI leads to deaths of over 13,000 patients every year in the United States [12], indicating a growing medical problem.

It is now recognized that the high infection rates were caused by the formation of biofilm on the surface of the catheters that decreases the susceptibility to  antibiotics and results in anti-microbial resistance [8,13,14]. The for- mation of biofilm as a result of extracellular polysacchar- ide matrix secretions from microorganisms has been demonstrated in clinical studies. Bacterial biofilm is a spe- cial honeycomb-shaped structure that forms a very com- plex ecosystem; magnification of biofilm will reveal microcolonies under the microscope [15-18]. Organisms with biofilm can withstand shear force, pH changes, and antimicrobial agents, and prevent macrophage phagocyto- sis [13,19]. The proximity of cells allows more frequent genetic information exchange than other free cells [20]. Therefore, antimicrobial resistance genes and strains can be spread easily. With respect to catheters, the formation of biofilm will protect the pathogenic bacteria residing at the urinary tract from antimicrobial medicine and host immune response [15]. It will then facilitate the growth of bacteria which further complicates the problem of CAUTI [13].

Recent research focused on the development of preven- tive methods for biofilm formation and changes, includ- ing furanone, furacilinum, silver-coated catheters, in addition to other techniques. [21-24]. Johnson  et  al. [21,2] discovered that catheters containing silver hydrogel and nitrofurazone coating have excellent effects of

inhibiting biofilm formation, but no inhibitory effects for Pseudomonas aeruginosa. According to the study con- ducted by CDC, the results of a comparison of patients inserted with silver-coated catheters and standard cathe- ters for one week revealed no difference in bacteriuria prevention [25]. Silver-bearing catheters can decrease the effect of bacteriuria in a week after indwelling. Burton et al.[8] discovered the new oPDM-plus-PS (N, N’-(1,2- phenylene) dimaleimide [oPDM]-plus-protamine sulfate [PS]) coating can inhibit Pseudomonas aeruginosa and Staphylococcus epidermidis adhering to the catheters, but now these coated catheters can only provide short-term CAUTI prevention upon urinary catheter insertion [13]. Recently, Stickler et al.[26] revealed that bacteria on the biofilm of catheters produced quorum-sensing signal that can control the genetic expression of forming biofilm. If the signal is blocked, the formation of biofilm can be impeded. For example, the mutant Pseudomonas aerugi- nosa in the absence of quorum-sensing signal was unable to produce a three-dimensional biofilm [27]. An impor- tant finding was established regarding iron and the for- mation of biofilm. Clinical investigations have detected that elements such as iron are necessary nutrients for biofilm formation. The production of catheters without iron is a new development, but it has not been tested in clinical trials [13]. The use of probiotics can also be con- sidered. Trautner et al.[28,29] observed that the rates of pathogenic bacterial infection and CAUTI were reduced if the catheters were inoculated with the non-pathogenic Escherichia coli (E. coli). Although these methods are considerable, there is no conclusive evidence and the cost-effectiveness remains unclear.

The traditional use of JUC applied to the wounds of post-surgery patients has proven to be effective in the hos- pital and out-patient setting: application of JUC did not result in drug resistance, nor stimulate serious adverse reactions and reduced the average wound healing time of patients [30]. JUC is composed by nano-manufacture tech- nology, with nano-cations on the nano-scale molecular structure produced and then prepared in water-soluble spray [31]. JUC achieves antibacterial action on skin and wound surface by physical mechanisms and can therefore be regarded as a physical antimicrobial agent [31]. Upon application, JUC prevents bacterial growth by forming an invisible, positively charged protective film on the sprayed surface, isolating and eradicating negatively charged patho- genic micro-organisms including bacteria, fungi and viruses [31,32].

There is no effective way to prohibit biofilm formation clinically; therefore, there is still an unmet need for the establishment of a new clinical application. In this study, we performed an in vitro test to explore the mechanism of biofilm formation and subsequently conducted a mul- ticenter clinical trial to investigate the efficacy of CAUTI prevention with the application of JUC, a nanotechnology antimicrobial spray.

Methods

In vitro testing

Bacteria

The experimental standard strains of E. coli were  iso- lated from the urine samples of UTI patients  at  the Second Hospital of Lanzhou University. Bacteria were cultured in Luria-Bertaini broth [33]. The bacterial sus- pension was prepared and  the  bacterial  concentration was adjusted to 7.4 × 10⁹CFU/ml.

Preparation of sample fragment

Siliconized latex urinary catheters were cut into sample fragments and sterilized by autoclaving. The sterilized sample fragments were randomly divided between the therapy and control group, with 8 pieces of fragments in each group. The sample fragments were respectively sprayed with JUC and distilled water, and dried before use. The E. coli suspension was injected into 24 well plates in which the sample fragments were placed. The plates were then incubated at 37 °C and washed with PBS solution every 48 hours [34]. At 16 hours and 7 days of incubation, the samples were extracted for confocal laser scanning microscopy.

Confocal laser scanning microscopy

The cultured samples were soaked in 1 ml PBS solution. After 50 μg/ml propidium iodide was added, the sam- ples were left for  dyeing  in  a  dark  area  at  4°C  for 15 minutes or at room temperature for 30 minutes. The samples were then placed upside down on a glass slide for observing the biofilm formation using laser scanning microscopy [35,36].

Clinical trial

The clinical study commenced in March 2010 and was completed in December 2011. Patients undergoing urolo- gical surgery in need of indwelling urethral catheter and more than 7 days of hospitalization were  recruited.  A total of 1,150 patients (869 male and 281 female), aged from 2 to 82 years, were accrued. Twenty-three hospitals participated in this clinical trial, and every hospital accrued 25 patients each to the control group and ther- apy group. All patients were operated due to urological diseases, including but not limited to urinary tract stones, tumors, prostatic hyperplasia, ureteral stenosis and hydronephrosis. Indwelling urethral catheter was neces- sary for patients requiring over 7 days of hospital stay. The midstream urine bacterial culture [15-17] was nega- tive at the time of inclusion in the study. Exclusion cri- teria of the study included patients with a long-term use of balloon catheter, intermittent self-catheterization, pre- vious treatment of percutaneous paracentetic suprapubic cyctostomy and UTI patients. Patients were randomized according to the order of surgical treatment. The odd

array of patients (575 cases) was assigned as the therapy group (the JUC), and the even array of patients  (575 cases) was assigned as the control group (normal saline). Patients who were eligible for the trial were explained the nature and purpose of the trial by the investigator, and informed consent was obtained for inclusion in the trial. The Ethics Committee of Tongji Hospital approved the clinical study (Approval Number: 2010006D).

Study design

Therapy group

Prior to the insertion of the catheter into the ureter of the patient at the time of surgery, JUC was sprayed on the sur- face of the catheter to allow formation of a physical anti- microbial membrane. After surgery, in addition to tradi- tional nursing care, JUC was sprayed onto the skin and mucous membrane around the urethral orifice, the cathe- ter and the drainage tube attachment point. This was done twice a day with 1 ml per spray (approximately 10 sprays) until the catheter was removed on the 7th day.

Control group

The catheter was inserted during surgery. After surgery, conventional nursing care with normal saline was per- formed until the catheter was removed on the 7th day.

During the study, and according to routine clinical practice, antibiotics were prescribed to patients after surgery. The types, dosage and route of antibiotics pre- scribed to the patients were carefully and strictly recorded according to the class of antibiotics per institu- tional guidelines (Table 1).

Class one, class two and class three antibiotics were cumulatively given 521 (40.08%), 572 (44%) and 207 (15.92%) treatment times respectively. There were no restrictions of use for class one antibiotics: they were pro- ven to be safe and effective for long-term clinical applica- tion with minimal effects  on  antimicrobial  resistance. The drugs belonging to class one are considered rela- tively inexpensive antimicrobial agents. Class two anti- biotics demonstrated properties of restricted use, with concerned safety, efficacy, and antimicrobial resistance in humans. In comparison, class two drugs were relatively more expensive than the drugs of class  one,  non- restricted use antibiotics. Class  three  antibiotics  are newly approved, antimicrobial agents with limited safety and efficacy information. There were reported adverse reactions with the use of class three antibiotics. Owing to the concerned safety of class three drugs, they are not recommended for use. Special attention should be made for clinical use to avoid bacterial resistance to antimicro- bial agents. Amongst the three classes of antibiotics, class three are relatively more expensive in nature.

The clinical practice on the use of antibiotics differed between all hospitals. A total of 150 patients required the combination use of antibiotics, which were prescribed for0 to 7 days according to the condition of the patient. The percentage use of class one antibiotics  in  Guangzhou First Municipal People’s Hospital and The First Affiliated Hospital of the Sun Yat-sen University was 97.92% and 94.12% respectively, but the use of class two antibiotics was 97.40% in Daping Hospital of the Third Military Medical University. No significant difference was observed in the use of antibiotics between therapy and control groups in each hospital. For example, in the Second Hospital of Xi’an Jiaotong University, class one antibiotics were given to 10 cases in both therapy and control groups, class two antibiotics were given to 12 and 18 cases in treatment and  control  groups  respectively, and class three antibiotics were given to 8 and 6 cases in treatment and control groups respectively. In the General Hospital of Guangzhou Military Command of PLA, class one antibiotics were given to 10 cases in both the therapy and control groups, class two antibiotics were given to 13 and 16 cases in the treatment and control groups respec- tively, and class three antibiotics were given to 4 and 3 cases in treatment and control groups respectively. Despite the difference in the clinical practice on the use of antibiotics between hospitals, the results were statisti- cally meaningful.

After surgery, the body temperature and UTI symptoms were recorded every day. After 7 days of catheterization, urine samples were collected under aseptic condition for bacterial culture before extubation [37].

Outcome assessment

The collected urine samples with colony count ≥ 10³ CFU/ml was considered as CAUTI, based on the quanti- tative urine culture. [9,38,39].

Statistical analysis

Parameters were compared using SPSS version 14.0. The T-test was used to compare the incidence of CAUTI between groups, where P < 0.05 was considered as sta- tistically significant.

Table 1 Classification of antibiotics used in the clinical trial

Results

In vitro test results

After 16 hours of culture,  bacterial  biofilm  formed  on the surface of sample  fragment  in  the  control  group. The bacterial biofilm was dyed red by propidium iodide fluorescent dye (Figure 1A). In the therapy group, no bacterial biofilm formation was observed on the sample fragments. Only small red dots representing a very small number of free bacteria were observed  under  micro- scope (Figure 1B).

After 7 days of culture, in the control group with dis- tilled water, the surface of the sample fragments formed a thick, uniform color, dense and darkly stained layer of bacterial biofilm. Due to bacterial overgrowth, the bio- film was cross-linked to  form  clumps  of  bacteria  and the surface of the sample fragments was  rough  and uneven (Figure 2A). The surface of sample fragments in the therapy group formed only a small amount of thin membranous structure with smooth surface and  light color. No other abnormality was observed (Figure 2B).

Clinical trial results

Significant difference was not observed in demographics including age, gender, etiology, and geographical distri- bution between the two groups. On the 7th day of cathe- terization, urine samples were collected for bacterial culture before extubation. In the therapy group, positive bacterial culture was detected in 26 (4.52%) cases, of which 24 cases were E. coli, 1 case was Enterococcus fae- calis and 1 case was smooth Candida. In the  control group, bacteriuria was detected in 75 (13.04%) cases, of which 69 cases were E. coli, 2 cases were Enterococcus faecalis, 2 cases were Enterococcus cloacae, 1 case was Candida albicans and 1 case was Pseudomonas aerugi- nosa. Detailed results were shown in Table 2. Among all 101 cases of infections, 93 (92.08%) cases were E. coli infections, 3 (2.97%) cases were Enterococcus faecalis, and 2 (1.98%) cases were Enterococcus cloacae. Signifi- cant difference was observed in the incidence of bacteriuria between the control group and control group (4.52% vs. 13.04%, p < 0.001).

Discussion

Types of bacteria

UTI is a major nosocomial infection. CAUTI is one of the most common types of bacterial infections  [1,2]; ample intestinal bacteria cultivates around the urethra [3,38]. A majority of the short-term  CAUTIs  were caused by a single strain of bacteria, such as E. coli, Pro- teus mirabilis and Klebsiella pneumoniae, whereas long- term CAUTI was caused by multiple microorganisms [3,40,41]. Urethra pathogenic E. coli is the most com- mon cause of CAUTI which constitutes 50% of hospital- acquired UTIs [3,42]. In our study, similar results were observed. E. coli infection was dominated by 92.08%, while other bacteria such as Enterococcus faecalis and Enterococcus cloacae constituted a minute proportion of UTIs.

Prevention of catheter-associated infection

Twenty years ago, the U.S. CDC clearly emphasized that hand hygiene, sterile catheterization and closed drainage systems were the necessary elements in preventing CAUTI [43,44]. Recently, the Healthcare-Associated Infections Allied Task Force proposed several frame- works, including infection surveillance, enhancement of education and training  in the prevention of CAUTI, the use of appropriate technology for catheter insertion, replacement of indwelling urinary catheter by condoms

and intermittent catheterization, immediate  removal  of the catheter, and other frameworks to prevent the occurrence of CAUTI [43,45,46].

The World Health Organization claimed systemic pro- phylactic antibiotic, irrigation of bladder, instilling normal saline or antibiotics, sterile drainage bag and other mea- sures are ineffective in preventing the occurrence of CAUTI [1]. The use of anti-microbial drugs, anti-microbial drainage bag and irrigation of bladder can only temporarily reduce the chance of bacteriuria [13]. Furthermore, some studies have shown that the use of soap, skin cleansing foam, povidone iodine or saline in perineal care do not affect the incidence of CAUTI [47]. As for materials of the catheter, the single biological surface coated with silicon, polyurethane, synthetic biomaterials, or hydrogel material, were not proven effective in the prevention of bacterial colonization [5,16].

The formation of biofilm is regarded as one of the major causes of anti-microbial resistance and refractory CAUTI [13]. Therefore, the prevention of biofilm formation has been the research focus toward reducing the incidence of CAUTI. In this study, we investigated the use of new nanotechnology anti-microbial spray JUC, composed of organic silicon quaternary ammonium salt. JUC forms a positively charged film which isolates and kills negatively charged pathogenic micro-organisms including bacteria, fungi and viruses. The physical attraction between the film and the micro-organism would not lead to drug resistance [30].

In the laboratory, the formation of biofilm can be initiated by a small amount of bacteria. The  bacteria clump together and form bacterial  colonies. It then starts to form a biofilm. When the biofilm matures, it begins to shrink and collapse [48,49]. In the in vitro study, the initial stage of biofilm formation was observed at 16 hours of bacterial culture in the control group. After 7 days of incu- bation, aggregation of bacterial clumps was observed and the surface of sample fragments was unevenly rough, which illustrated the formation of mature biofilm. How- ever, only few free bacteria, represented by red dots under microscope, were also observed in the therapy group. A thin membranous structure was observed which was at a stage between the bacterial colony formation and  the initial formation of biofilm. The in vitro study clearly demonstrated that the physical anti-microbial film formed by JUC could prevent biofilm formation for 7 days upon application, which was validated in the clinical study. A significantly lower incidence of CAUTI was observed clini- cally in the therapy group (4.52%) than in the control group (13.04%), which further confirmed the effectiveness of JUC in the prevention of CAUTI.

Comparison between clinical trials

The pre-operative use of anti-microbial drugs as an effec- tive way for the prevention of bacterial infection is widely accepted [50]. In many clinical trials, prophylactic anti- biotics were commonly prescribed for the prevention of infection  in  catheterized patients [5].  The UTI rate did not exclude the factor of antibiotics use. In Tambyah’s study [6], the mean antibiotics use was 1.6 ± 1.7 per catheter-day, and the incidence of CAUTI was 14.9% in the urology department. In the surgical unit,  1,162 patients were catheterized patients after  surgery.  The onset of CAUTI was 6.4 ± 6.1 catheterized days, and the incidence was 11.9% [6]. In Darouiche’s study [51], 124 patients were catheterized in place for 14 days with regu- lar silicone bladder catheters or silicone bladder catheters impregnated with minocycline and rifampin after radical prostatectomy. All patients were given a single parental dose of 1g cefazolin as prophylactic antibiotic before anesthesia. The UTI rates measured 7 days after surgery were 15.2% and 39.7% in patients with regular catheters and medicated catheters respectively. The incidence was much higher than the therapy or control group patients of our study.

Compared to the study of 1,497 patients with an overall incidence of CAUTI of 14.9% by Tambyah et al. [6], the incidence of CAUTI was higher than the therapy group (4.52%) and slightly higher than the control group (13.04%) of our study. In Tambyah’s study, the patients were catheterized with nitrofurazone-impregnated sili- cone catheters, silver-polyurethane hydrogel catheters or control catheters and obvious differences were not observed in the incidence of UTI between medicated catheters and control catheters. Despite the different practices of the use of antibiotics between Tambyah’s study and our study, it seems the duration and types of post-operative antibiotics were not associated with the incidence of CAUTI. However, a significantly reduced incidence of CAUTI was observed in the therapy group of our study, indicating that the use of JUC, which was effective in preventing the biofilm formation, could be vital to lowering the incidence of CAUTI.

Table 2 Comparison of post-operative urinary bacterial culture between the control and therapy group

* P<0.001, statistically significant

Conclusions

In the clinical trial, only 4.52% of the patients from the therapy group were diagnosed with CAUTI, compared to 13.04% from the control group. In vitro testing also showed no obvious biofilm formation in the therapy group sprayed with JUC after 7 days of bacterial incuba- tion. Biofilm began forming after 16 hours of incubation in the control group. The results from the clinical trial and in vitro test demonstrated the effectiveness of JUC in the prevention of CAUTI and formation of biofilm.

Acknowledgements

We express our heartfelt thanks for the strong support of Chinese Medical Association Society of Urology. We are also thankful for the close collaborations with Tongji Affiliated Hospital of Tongji Medical College of the Huazhong University of Science & Technology, the Second Hospital of Lanzhou University, Peking University People’s Hospital, the Second Military Medical University (Shanghai Changhai Hospital), the First Affiliated Hospital of the Sun Yat-sen University, the Second Affiliated Hospital of the Sun Yat- sen University, the Third Affiliated Hospital of the Sun Yat-sen University, the Second Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, the First Affiliated Hospital of Southern Medical University (Nanfang Hospital), the First Affiliated Hospital of Guangzhou Medical University, General Hospital of Guangzhou Military Command of PLA, Wuhan General Hospital of Guangzhou Military Region, Guangzhou First Municipal People’s Hospital, Foshan Hospital of Traditional Chinese Medicine, West China Hospital of the Sichuan University, Daping Hospital of the Third Military Medical University, Xiangya Hospital of the Central-south University, the Second Hospital of Xi’an Jiaotong University, the First Affiliated Hospital of Nanjing Medical University, Nanjing Drum Tower Hospital Affiliated to the Nanjing University Medical School, the Second Affiliated Hospital of Kunming Medical College, Huai’an First Hospital Affiliated to Nanjing Medical University and the Affiliated Hospital of Nantong University.

This article has been published as part of Journal of Translational Medicine Volume 10 Supplement 1, 2012: Selected articles from the Organisation for Oncology and Translational Research (OOTR) 7th Annual Conference. The full contents of the supplement are available online at http://www.translational- medicine.com/supplements/10/S1.

Author details

¹Department of Urology, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science & Technology, Hubei PRC. ²Institute of Urology, the Second Hospital of Lanzhou University, Lanzhou, PRC. ³Wuhan General Hospital of Guangzhou Military Region, Guangzhou, PRC. ⁴West China Hospital, Sichuan University, Sichuan, PRC. ⁵Daping Hospital, Third Military Medical University, Chongqing, PRC. ⁶The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, PRC. ⁷The Second Military Medical University, Changhai Hospital, Shanghai, PRC. ⁸Peking University People’s Hospital, Beijing, PRC. ⁹The Third Affiliated Hospital, Sun Yat-sen University, Guangdong, PRC. ¹⁰The Second Hospital of Xi’an Jiaotong University, Xi’an, PRC. ¹¹The Second Affiliated Hospital, Sun Yat-sen University, Guangzhou, PRC. ¹²Xiangya Hospital of Central-south University, Changsha, PRC. ¹³Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, PRC. ¹⁴Huai’an First Hospital Affiliated to Nanjing Medical University, Jiangsu, PRC. ¹⁵The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, PRC. ¹⁶The Second Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, Guangzhou, PRC. ¹⁷The First Affiliated Hospital of Southern Medical University (Nanfang Hospital), Guangzhou, PRC. ¹⁸Affiliated Hospital of

Nantong University, Jiangsu, PRC. ¹⁹Foshan Hospital of Traditional Chinese Medicine, Guangdong, PRC. ²⁰Guangzhou First Municipal People’s Hospital, Guangzhou, PRC. ²¹General Hospital of Guangzhou Military Command of PLA, Guangzhou, PRC. ²²The First Affiliated Hospital of Nanjing Medical University, Jiangsu, PRC. ²³The Second Affiliated Hospital of Kunming Medical College, Yunnan, PRC. ²⁴School of Chinese Medicine, The University of Hong Kong, Hong Kong SAR. ²⁵UNIMED Medical Institute, Hong Kong SAR.

Authors’ contributions

QC, NC, JC, JPC, CG, WH, XJ, LL, ZL, SL, XL, PL, LL, XM, LM, WQ, LQ, ZR, XS, WS, YT, PW, XW, DW, ZW, BW, QY, ZY, ZY, YZ, HZ, YZ equally conducted

clinical test planning and performance. LWCC, WTYL, MNBC, AYSY and ELYN participated in the writing of the manuscript.

Competing interests

The authors state they have no competing interests to declare.

Published: 19 September 2012

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1.Application of a nanotechnology antimicrobial spray to prevent lower urinary tract infection a multicenter urology trial.pdf