3% hydrogen peroxide to disinfect urine-contaminated surfaces

Summary

Purpose

Disinfecting urine-contaminated floors, surfaces and objects is a persistent challenge in healthcare. While chlorine-based compounds such as bleach are often used to decontaminate surfaces, they are known to degrade plastics and may leave harmful residues and release potentially irritant vapors making them unsuitable disinfectants for materials that come in direct contact with humans. The objective of this study was to evaluate an alternative urine disinfection procedure. Treating urine-contaminated surfaces with 3% hydrogen peroxide (H₂O₂) was hypothesized to remove bacteria. Furthermore, when applicable, the efficacy of the same H₂O₂ stock solution for its repeated use over time was assessed further increasing simplicity and accessibility.

Materials and methods

The effectiveness of disinfecting two materials, a flat plastic surface and a long lumen representing a more challenging surface to clean, was evaluated with a commonly used method of water and soap versus using a 3% H₂O₂ solution.

Results

Contamination persisted when washing with soap and water but was effectively removed after one hour of H₂O₂ storage for flat plastic surfaces and after 3 hours for lumen surfaces. The same stock of H₂O₂ solution could be reused for up to three weeks with no colony formation.

Conclusions

The results show that bacteria can be removed from a urine-contaminated surface by being soaked in 3% H₂O₂ for one to three hours based on the surface type without the need for scrubbing or rinsing. The same stock solution can be used for repeated washes for up to three weeks to expand its sustainability and accessibility.

Introduction

Disinfecting urine-contaminated surfaces poses a persistent challenge to the health care system. Urine, unlike other biological substances, contains a host of different urobacteria, such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) [1]. Therefore, surfaces and devices contaminated with urine require effective decontamination to prevent pathogen transmission. For simple-to-clean surfaces such as hospital floors, walls and bathrooms, chemical compounds namely chlorine or quaternary ammonia are commonly used [2,3]. However, these cleaning agents are known to degrade, stiffen, and eventually crack plastics and may have unpleasant and irritating odors and leave harmful residues on devices that come into direct contact with the human body. In addition, the contact of such cleaning agents with biological materials may not be safe. For example, devices such as intermittent urinary catheters directly contact the human body and urine. These catheters are manufactured for single use [[4], [5], [6]], and it is highly recommended by the Food and Drug Administration (FDA) and the Canadian Urological Association (CUA) to follow manufacturer instructions, disposing of catheters after one use. Despite these recommendations, for reasons such as cost and accessibility, users may resort to reusing catheters. Catheter reuse in North America alone remains around 35% [4]. Acknowledging cases where single use is not possible, the Canadian Urological Association best practice report states that catheter reuse may be considered yet warns that individuals should be informed about concerns regarding its effectiveness and the lack of strong evidence supporting cleaning methods [7].

The recommendations on health websites and blog posts by users and physicians vary from washing a used catheter with soap and water, vinegar, or bleach to microwaving or placing it in boiling water. However, the efficacy of these methods in removing bacteria has not been thoroughly studied, and the most recommended procedure of washing with warm water and soap is not confirmed to decontaminate the catheter completely and may cause urinary tract infections (UTIs) [4,8]. The urine culture rates in clinical studies of catheter cleaning via other less commonly available and used disinfectants, such as different percentages of chlorhexidine or benzalkonium bromide, is summarized by Sun et al. indicating 0.5% chlorhexidine might cause less UTIs [9]. However, the routine use of these agents is not commonly accessible or affordable for the users. Overall, a sustainable, low-cost cleaning protocol can significantly reduce the challenges of cleaning various urine-contaminated surfaces.

Herein, a simple and accessible disinfection protocol for urine-contaminated surfaces was sought after specifically using a commercial grade 3% hydrogen peroxide (H₂O₂). The household agent is used to eliminate bacteria and viruses on various surfaces [10,11]. Using H₂O₂ for disinfecting materials that contact the human body is an established practice. For example, it is used as a contact lens and an orthodontic retainer cleaner and even as an intraoperative irrigation solution [[12], [13], [14]]. It also reduces pathogens on materials without harming live tissues or compromising polymers durability [12,15,16]. Further, unlike other chemical disinfectants, H₂O₂ does not leave chemical residues over time [12,15,16].

The disinfectant efficacy of H₂O₂ on urine-contaminated surfaces remains largely unassessed. This study evaluates the comparative effectiveness of standard soap and water washing versus soaking in a 3% H₂O₂ solution. Two surface types were examined: a flat plastic surface and a long lumen, the latter representing the most common urine-contaminated surface, a catheter, whose narrow lumen geometry is particularly challenging to disinfect. To access efficacy, surfaces were contaminated with E. coli and S. aureus, two common bacteria found in urine. Subsequent tests evaluated disinfection on surfaces contaminated with human urine. The efficacy of H₂O₂ was then further challenged with a higher concentration of urobacteria, representative of individuals experiencing a urinary tract infection (UTI) [17]. The bacteria colonies were completely removed after being stored in H₂O₂ for one and three hours for flat and long lumen samples, respectively. In addition, the same H₂O₂ solution could be reused for up to three weeks for continuous catheter cleaning, which significantly increases the convenience, accessibility, cost and sustainability of the presented protocol. An overview of the study and the outcomes is provided in Figure 1.

Figure 1.

Summary of the study and the outcome.

Methods

Study design

An in vitro experimental study was developed to assess the efficacy of disinfection methods of soap and water versus hydrogen peroxide in removing bacterial contamination from plastics under controlled laboratory conditions.

Study samples

A simple plastic surface and a long lumen sample (Medline Vinyl Intermittent Catheters, Canada) were contaminated with liquid cultures of E. coli and/or S. aureus in different concentrations of colony forming units (CFU) to represent bacterial levels found in a healthy sample of urine. A healthy sample of urine contains anywhere from 0-10,000 CFU/ml [18]. Therefore, in this study, a healthy urine concentration culture was referred to as containing 10,000 CFU/ml of E. coli and S. aureus in a ratio 95:5 [19]. Another set of samples were contaminated with a more challenging form of urine, specifically urine with a high urobacterial load measured in some individuals experiencing a UTI. Urine from a UTI case generally contains 10–100 times more bacteria than a healthy sample. Accordingly, a high urobacterial load was defined to contain 10^{^}5 CFU/ml of E. coli [20]. All bacteria cultures were made in lysogeny broth (Sigma-Aldrich, Missouri, USA) and the culture concentrations were quantified through a serial dilution plating procedure described elsewhere [21]. A third set of samples were contaminated with human urine purchased from Innovative Research (Toronto, Canada). Biofilm formation and consequently surface contamination were established by incubation in each of these solutions for two hours at 37°C [22,23]. Following contamination, the surfaces were treated using various solutions, as described in the subsequent sections.

Sampling technique

After disinfection, to detect any remaining bacterial contamination, a variation of the Maki et al. Semiquantitative Roll-Plate Method was used during which the plastic surfaces were rolled on nutrient agar dishes and incubated for 72 hours at 37°C [22]. To prepare the agar dishes, 1.5 w% of agarose (VWR Chemicals, Ohio, USA) was melted in LB broth solution, poured on a Petri dish, and let cool down at room temperature to form a gel. The outer lumen surface was first rolled on the agar plate to capture the outer bacteria, then cut in the middle to roll and culture the interior. After 72 hours, images of the plates were taken to detect and count any CFU.

Experimental conditions

Soap and water cleaning efficacy

First, bacteria removal efficacy via the common cleaning procedure of warm water and soap was investigated on long lumen samples. The samples were contaminated with a healthy urine concentration culture of E. coli and S. aureus. The catheters were either rinsed by holding their tip under running warm water after adding drops of liquid soap (Soft-soap®) or were soaked in 100 ml of water and three drops of soap for 3, 8, or 24 hours.

Hydrogen peroxide cleaning efficacy

To access surface-level disinfection, the efficacy of H₂O₂ was first tested on flat plastic surfaces to remove any consideration of the fluid penetration. The flat plastic pieces were then contaminated with healthy or a high urobacterial load of E. coli and S. aureus or healthy human urine. The plastic pieces (n = 5) were placed in 100 ml of 3% H₂O₂ (Life BRAND™, MB, Canada) or in soap and water for one hour. The H₂O₂ container was kept with a closed cap and covered with aluminum foil to reduce air and light exposure, respectively.

Reuse of hydrogen peroxide stock solution

Finally, the efficacy of reusing the same stock of H₂O₂ to disinfect long lumen plastic without its replenishment as it gets exposed to light and oxygen over time was investigated. E. coli and S. aureus at high concentrations representative of high urobacterial load contamination [17,19,24] were added to the stock of H₂O₂. In addition, the container was left exposed to air and ambient light for 8 hours per week to simulate repeated opening during routine cleaning in practical settings.

Over the month-long study, E. coli and S. aureus contaminated long lumens (n = 5) were placed in the same H₂O₂ container on weeks 0, 1, 2, 3 and 4. The container was shaken for 30 seconds and left aside for 2, 3 or 6 hours. Remaining bacteria was then quantified using the Maki et al. method to assess CFU formation [22]. To further validate the effectiveness of used H₂O₂ over time, the same long-term experiment was also done in which catheters were incubated with high load urobacteria.

Data quality control

To ensure adequate quality control, aseptic techniques were used throughout the experiment to minimize risk of contamination. In addition, a negative control plate was incubated in each stack of petri dishes placed in the incubator to verify there was no cross-contamination between samples. These plates were not exposed to bacteria and served as a baseline to confirm sterility of the experimental set up. Replicates (n = 5) were used to account for variability between samples.

Data analysis

The analysis involved a binary classification to determine whether bacterial colonies were present or absent on the disinfected surfaces after the cleaning procedures. If CFUs were observed on the nutrient agar plates following incubation, it indicated that the cleaning method was ineffective.

Results

Soap and water cleaning efficacy

Bacteria colonies remained on the long lumen samples contaminated with healthy urine concentration of E. coli and S. aureus after being washed via the commonly practiced method of rinsing with or soaking in warm water and soap for various times (Figure 2). The more physically involved method of holding the catheter head under continuous running water (Figure 2a) yielded 2–3 CFUs per sample. Washing the lumen surface in a container of warm water and soap for 3, 8 and even 24 hours (Figure 2b–d) showed the formation of a bacterial lawn, where individual colonies become overconfluent and indistinguishable from one another. Overall, the current most recommended practices of washing did not disinfect the contaminated lumen catheters.

Figure 2.

Bacteria colonies remain after washing contaminated long lumen samples with soap and water. Surfaces contaminated with a healthy urine concentration of E. coli and S. aureus (a) flushed with continuous running water and soap or soaked in water and soap for (b) 3 hours, (c) 8 hours or (d) 24 hours produced bacteria colonies.

Hydrogen peroxide cleaning efficacy

As discussed previously, disinfecting urine-contaminated surfaces can be complicated by the presence of UTIs which greatly increase the concentration of pathogens. Therefore, it was important to test the extremes to determine if surfaces could be cleaned even when contaminated with a high urobacterial load. In addition, human urine contains many organic and inorganic compounds, along with a slightly acidic pH, which can influence bacterial adhesion to the catheter and, consequently, its cleaning process [19]. Assessing healthy urine was therefore essential, as the nutrient broth used for culturing bacterial biofilms on the catheter typically has a pH between 7 and 8, whereas urine pH ranges from 4.5 to 7 [25]. As such, smaller plastic samples were contaminated with a high concentration of E. coli, S. aureus or human urine. Bacterial colonies persisted after soaking the contaminated pieces in water and soap for one hour (Figure 3a–c) and remained present even when the soaking time was extended to three hours (data not shown). Similarly treating the surfaces with H₂O₂ for 20 minutes and 30 minutes did not remove all the colonies (data not shown). On the other hand, after one hour of treatment, no colonies were observed (Figure 3d–f).

Figure 3.

Comparison of cleaning methods on flat plastic pieces contaminated with high concentrations of E. coli, S. aureus, and healthy human urine. (a–c) Soaking in water and soap for one hour failed to remove bacterial contamination. (d–f) Catheter pieces soaked in 3% H₂O₂ for 1 hour showed no bacterial colony formation regardless of the contamination source.

Reuse of hydrogen peroxide stock solution

The long-term efficacy of reusing the same stock solution of H₂O₂ to disinfect long lumen samples was evaluated. Because H₂O₂ decomposes when exposed to light and oxygen, it was important to investigate both its cleaning capacity and reactivity to minimize the need for rigorous quality control of the stock solution and its storage conditions. The long-term study was designed to replicate how a user would clean multiple urine-contaminated surfaces a day using the same H₂O₂.

To simulate repeated contamination of the H₂O₂ stock solution, the jar was initially inoculated with a high concentration of bacteria. The H₂O₂ solution was then exposed to light and air for a total of eight hours per week to simulate the exaggerated conditions of when a user opens the container to insert a contaminated object equivalent to approximately 10 minutes of time to open and close the jar seven times a day. Shaking the jar for 30 seconds was done for the H₂O₂ to reach the centre of the lumen surface, which would later be cut in half and swabbed on the growth plate to ensure the hardest to reach places were disinfected. Under these conditions H₂O₂ effectively removed pathogens from surfaces contaminated with a healthy urine concentration co-culture of E. coli and S. aureus (Figure 4a–e) and surfaces contaminated with high urobacterial load. (Figure 4f–j). [19]. As shown in Figure 4, reused H₂O₂ eliminated bacteria colony formation for up to 3 weeks.

Figure 4.

Long-term bacteria removal efficacy of reusing the same H₂O₂ stock solution. Each week, lumen tubing, freshly contaminated with (a–e) healthy urine concentration or (f–j) a high urobacterial load of E. coli and S. aureus were soaked in the same 3% H₂O₂ stock solution for 3 hours. Black circles highlight the appearance of the first CFU detected at (d) week 3 for the healthy urine concentration and at (j) week 4 for the high urobacterial load indicating the efficacy of reusing the same solution for up to 3 weeks.

Discussion

The result of this study indicates that urine bacterial contamination persisted after washing with a common protocol of applying warm water and soap while H₂O₂ was effective at eliminating bacteria from separate or co-cultures of E. coli and S. aureus. The efficacy of H₂O₂ can potentially be explained by its oxidizing properties. The reactive oxygen species disrupts the biological processes of bacteria leading to death of the microbe [26], while water and soap (i.e. surfactant) works by dislocating the pathogens [17]. This explains why using continuous running water and soap worked better than soaking in soap and water for 24 hours. However, neither method completely removed the bacterial colonies. In contrast, H₂O₂ treatment resulted in the absence of CFU under all tested conditions. From the presented long-term study, it is suggested that the reuse of a 250 ml container of H₂O₂ for up to three weeks disinfected urine-contaminated surfaces. In addition, it was observed that after the three-week period, the solution starts becoming cloudy and produces fewer bubbles. These changes may serve as visual cues to know when it is time to replace the solution.

In addition, this protocol offers a minimally involved cleaning method with fewer steps and no scrubbing that can be conducted for less laborious use and by users with limited hand dexterity, such as users of urine interfacing medical devices. Unlike the traditional soap and water method which involves scrubbing and holding a small tube under the running tap, this approach eliminates the need for fine motor control or sustained grip strength. H₂O₂ is also a common household agent and is generally highly accessible and low cost.

Limitations

This study provides results of using H₂O₂ to remove bacteria from urine-contaminated surfaces. Follow up studies are needed before it can be used for any clinical recommendation. For example, it is important to test if the discussed protocol can also disinfect other biological contaminants such as blood, epithelial cells or other human secretion. In addition, hematuria, a condition where erythrocytes leech into urine, can occur after certain urinary tract infections, kidney stones and cancer [27]. Thus, to confirm if H₂O₂ effectively disinfects even the most extreme cases of urine-contamination, a study could be done on UTI hematuria urine.

Conclusion

The H₂O₂ disinfection method was found effective and easy-to-use procedure to remove bacteria colonies from urine-contaminated materials. In addition, H₂O₂ effectively disinfected hard-to-clean surfaces such as the long lumen of a catheter contaminated with E. coli, S. aureus, as well as human urine and may offer an accessible, reusable and sustainable disinfectant for urine-contaminated plastic surfaces.

CRediT authorship contribution statement

Brianna Tsuyuki: Data curation, Formal analysis, Investigation, Methodology, Project administration, Validation, Roles/Writing – original draft. Dena Shahriari: Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Validation, Roles/Writing – original draft, Writing – review & editing.

Ethics

Not required.

Funding

This work was partially funded by the Government of Canada New Frontiers in Research Fund - Transformation (NFRFT-2020-00238). D.S. acknowledges funding from the Michael Smith Health Research British Columbia Scholar Award.

Conflict of interest statement

The authors claim no relevant conflict of interest.