Tap Water has No Influence on Microbial Colonization of Skin Wounds in Rats

  Abstract: The aim of this study was to investigate the influence of tap water on microbial colonization of skin wounds in rats. Methods. A circular wound 2.4 cm in diameter was created on the back of 40 Wistar rats, which were randomly assigned to the control (n = 20) or study (n = 20) group. The wounds were cleansed daily using a sterile 0.9% sodium chloride solution (control group) or tap water (study group) for 6 days, following a standardized protocol. Sequential samples were collected for microbiological analysis every other day. Results. Microbial growth occurred in 49% of the samples from the control group and in 47% of the samples from the study group (P = 0.39). There was no significant difference in microbial growth between groups for the different culture media: thioglycolate (P = 0.20), mannitol salt agar (P = 0.53), blood agar (P = 0.61), eosin methylene blue agar (P = 0.51), and Sabouraud agar (P = 0.34). The following microorganisms were identified: Bacillus subtilis, Staphylococcus sp, Aureobasidium sp, Penicillium sp, and Cladosporium sp. Conclusion. The use of tap water for cleansing skin wounds in rats had no effect on microbial colonization compared with the use of a sterile saline solution.

Introduction

  Tap water has previously been investigated as an alternative for cleansing wounds.^1-4 It is readily available, convenient, requires no preparation, and is cost effective, which benefits low-income patients and health care providers.^1,4,5   Studies comparing results obtained using tap water for cleansing wounds with those using a sterile 0.9% sodium chloride solution found no significant differences in wound infection rates.^1,6-10 However, a recent systematic review⁴ concluded that most studies in the literature have some methodological flaws, such as lack of randomized group assignment, standard eligibility criteria, and standard wound assessment criteria, as well as differences in the pressure, temperature, and volume of the irrigation fluids.^1,4,11   Therefore, the objective of this study was to investigate the influence of using tap water for cleansing skin wounds in rats on microbial colonization, ensuring that issues identified in other studies were properly addressed.

Material and Methods

  This study was approved by the Research Ethics Committee of the Universidade Federal de São Paulo (UNIFESP), Brazil. All animals received humane care in accordance with the principles of the Brazilian College on Animal Experimentation and the National Research Council’s criteria for humane care as outlined in the “Guide for the Care and Use of Laboratory Animals, 7th Edition” prepared by the Institute of Laboratory Animal Resources and published by the National Academies Press.   Forty adult male EPM1-Wistar rats (Rattus norvegicus albinus) were used in this study. The mean weight of the animals was 352 g (standard deviation [SD], 0.05 g]) in the control group and 344 g (SD, 0.05 g) in the study group. The rats were housed in individual cages in a temperature-controlled environment (21° C), on a 12:12 hour light-dark cycle, and fed standard rat chow and water ad libitum. After a 10-day adaptation period, the rats were randomly allocated into 2 groups of 20 animals each: the control group (wounds cleansed with a sterile 0.9% sodium chloride solution), and the study group (wounds cleansed with tap water).   The assignment of the animals to different groups, the distribution of cleansing fluids into groups, and the sequence in which the animals were cleansed every day were randomly determined using the BioEstat version 5.0 software (Instituto Mamirauá, Manaus, AM, Brazil). The researcher who cleansed the wounds and the statistician who analyzed the experimental data were blinded to group assignment.   Before the surgical procedure, the rats were anesthetized intramuscularly with ketamine hydrochloride (10 g/100 ml at a dose of 1 mg/100 g body weight [bw]; Syntec, Cotia, São Paulo, Brazil) and xylazine hydrochloride (2 g/100 ml at a dose of 2 mg/100 g bw; União Química, Pouso Alegre, Minas Gerais, Brazil). Next, the dorsal hair of the rats was removed and the skin was swabbed with 10% povidone-iodine solution (Rioquimica, São José do Rio Preto, São Paulo, Brazil). A circular wound 2.4 cm in diameter was then created on the back of each animal. The center of the wound was positioned on the mid-dorsal line, 1.0 cm from a transverse line defined by the lower border of the scapulas, in the caudal direction. The dorsal skin was resected together with the panniculus carnosus; the incision was deepened until the superficial muscular fascia.   Following, the wounds were cleansed either with sterile 0.9% sodium chloride solution (Linhamax®, Segmenta, Ribeirão Preto, Brazil) or tap water. All animals received oral acetaminophen (0.1 mg/g bw; Janssen-Cilag, São José dos Campos, São Paulo, Brazil) for postoperative analgesia. The wounds were cleansed once a day for 6 consecutive days with the animals under the same anesthetic conditions.   Bacteriological analysis of the tap water was performed at the beginning and end of the experiment in 100-ml samples, and the results were negative for Escherichia coli and thermotolerant coliform bacteria. Using an aseptic technique, a bottle of 0.9% sodium chloride solution was emptied and filled with the same amount of tap water so the researcher was blinded to the contents of the bottle of cleansing solution. Both cleansing solutions were warmed to 37° C in an oven (EES0B, Olidef CZ, Ribeirão Preto, São Paulo Brazil) before use.   The wounds were cleansed using an irrigation system consisting of a macrodrip infusion set (Equipo®, Jiangsu Xuyi Kangning, Jiangsu, China) with a 150-ml graduated burette connected to a bottle of irrigation fluid. The bottle of irrigation fluid was fixed to a support at a height of 2.1 m from the floor, the burette was at a height of 100 cm from the rat, and the end of the connection tubing was at a height of 15 cm from the wound. The rat was placed in the prone position on the operating board (Figure 1). For the cleansing of the wound, the clamp at the end of the tubing was removed, allowing the fluid in the burette (150 ml) to flow directed at the center of the wound. This volume of liquid was sufficient in removing loosely adherent debris, as determined in a previous pilot study. The wound was irrigated for a period of 2 minutes. The pressure of the fluid supplied was measured using a pressure gauge (Universal Pressure Meter Fluke DPM2Plus, Biotek, São Gonçalo, Rio de Janeiro, Brazil). The fluid pressure at the end of the tubing was 2.8 mm Hg (5.4 psi) at the beginning of the irrigation, and 2.4 mm Hg (4.6 psi) at the end. After irrigation, the skin surrounding the wound was dried using sterile gauze and the wound was covered with a transparent sterile adhesive film (Opsite Flexigrid, Smith & Nephew, Gilberdyke, United Kingdom).   Following the cleansing of the wound on postoperative days 2, 4, and 6, a wound swab was taken for culture; a swab was placed perpendicular to the center of the wound and then transferred to a test tube containing 1.0 ml of sterile 0.9% sodium chloride solution. The suspension was seeded in thioglycollate medium using aseptic technique. The inoculum suspension was diluted to 10-3, and 0.1 ml of the solution was added to 4 Petri dishes, each containing 1 of the following culture media: Sabouraud agar, mannitol salt agar, eosin methylene blue (EMB) agar, or blood agar. The culture dishes were incubated at 37° C for 72 hours.   Microbial colony growth was assessed by counting the number of colony-forming units (CFUs). For bacterial identification, smears were made from colonies, stained by Gram’s method, and examined with an optical microscope (Olympus, Tokyo, Japan) at a magnification of x1000. Culture dishes containing Sabouraud agar medium were screened for yeast-like fungi, then incubated at room temperature for 1 week, and examined for the presence of filamentous fungi. For fungal identification, colony smears were stained with lactophenol cotton blue and examined by light microscopy at a magnification of x1000. Culture dishes containing mannitol salt agar medium and presenting colonies surrounded by a yellow halo were tested for Staphylococcus aureus using the coagulase test. At the end of the experiment, the rats were euthanized by anesthetic overdose.   Statistical analysis was performed using the IBM Statistical Package for the Social Sciences (SPSS) version 18.0 (IBM, New York, United States). Fisher’s exact test was used to evaluate qualitative differences in microbial growth between groups and the Mann-Whitney U test was used to assess quantitative differences in the number of colonies between groups. All statistical tests were performed at a significance level of 0.05 (P < 0.05).

Results

  Sixty samples were collected from each group, yielding a total of 300 microbial cultures per group. Microbial growth occurred in 146 (49%) cultures in the control group and in 114 (47%) cultures in the study group (P = 0.39). The total number of CFUs was 70,030 in the control group and 64,405 in the study group (P = 0.52), with no significant differences between groups. Moreover, there was no significant difference in the number of positive cultures between groups for all 5 culture media (Table 1). Also, no significant difference was found in the number of CFUs between groups for the mannitol salt agar, blood agar, and EMB agar media (Table 2). The types of microorganisms identified in each culture medium were similar in both groups (Table 3).

Discussion

  Sterile 0.9% sodium chloride solution has been the gold standard for wound cleansing because it does not damage tissue, cause allergies, interfere with the healing process, or change the microbial flora of the skin. For these reasons, it has been used as a control irrigation fluid in studies evaluating the use of tap water as a cleansing fluid, as in this study.^1,5-10,12   Tap water can be an alternative to sterile saline solution because it is readily available and inexpensive for health professionals and patients. In order to be used as a cleansing solution, tap water must be considered potable and free of contaminants. According to the Brazilian drinking water standards (Regulation no. 518, March 25, 2004), drinking water must not contain detectable total coliforms, E. coli, or thermotolerant coliform bacteria per 100 ml of water; the turbidity must be less than 5 nephelometric turbidity units (NTU); and the residual chlorine level must be greater than 0.2 mg/l at the end of the lines. The tap water used in the study group was always collected from the same tap located at the hospital’s outpatient dressing clinic. The characteristics of the water were analyzed before and after the cleansing of the wound, and it was verified they were within standard limits.   Skin wounds in rats can be used as a model of microbial colonization.¹³ The swab method was used to collect biological specimens from wounds for evaluation of microbial colonization. A high rate of concordance (87.1%) between surface swab cultures and biopsy cultures after 7 days of growth was reported elsewhere.¹⁴ The swab method was chosen for this study because it is less invasive and easy to use. No studies were found in the literature indicating the optimal dilution of the inoculum suspension for counting microbial colonies grown from swabs taken from skin wounds in rats cleansed with tap water or sterile 0.9% sodium chloride solution. Thus, a pilot study was carried out to determine this value, and it was found that the optimal dilution of inoculum suspension was 10-3.   It was observed that blood agar plates yielded better growth of microorganisms followed by mannitol salt agar, Sabouraud agar, and EMB agar plates. In both groups, the same microorganisms (Bacillus subtilis and Staphylococcus sp) were identified in culture plates containing thioglycolate and in those containing blood agar. Bacteria of the genus Staphylococcus were the most frequently isolated microorganisms, followed by B. subtilis. To the authors’ knowledge, there are no studies in the literature assessing microbial growth in wounds in rats as performed in this study. In a study on humans, the most frequently isolated microorganism in chronic wounds belonged to the genus Staphylococcus.¹⁵ In the present study, the growth of Staphylococcus sp in mannitol salt agar was similar in both groups. Over the period of the study, there was an increase of 35% to 65% in coagulase-negative staphylococci counts versus 0% to 5% for coagulase-positive staphylococci counts. Coagulase-positive S. aureus is generally considered a pathogen, whereas coagulase-negative staphylococci are considered to be saprophytes and part of the microbiota of the normal skin; but this is still controversial.¹⁶   The growth of filamentous fungi was similar in both groups. No studies were found in the literature describing the growth of fungi in skin wounds in rats. But, Almeida et al¹⁷ reported on the presence of mycotoxin-producing fungi in laboratory rat chow; Cladosporium, Aspergillus, Penicillium, and Mucor were the most frequently isolated genera of fungi. Except for the genus Mucor, the other 3 genera of fungi were also identified in the present study.   Two previous studies compared the use of tap water with sterile 0.9% sodium chloride solution in the cleansing of bacteria-inoculated wounds to determine whether tap water could reduce the bacterial load, and found that the cleansing effectiveness of both irrigation fluids was similar.^5,12 It is important to note that the objectives of these studies were different from that of the current one, which was to evaluate the effects of using tap water for wound cleansing on the growth of microorganisms.^5,12 Moscati et al⁵ reported that wounds irrigated with sterile 0.9% sodium chloride solution had a reduction of 54.7% in bacterial counts, while wounds irrigated with tap water had a reduction of 80.6% (P < 0.02). Wound irrigation with tap water was made directly from the tap, whereas a syringe was used to irrigate the wound with sterile 0.9% saline solution. Therefore, the pressure and volume of the irrigation fluids were different, and the sample size was small.⁵ In a study with goats, the pressure, volume, and temperature of the irrigation fluids were the same.¹² The authors reported that irrigation with either tap water or saline solution reduced bacterial counts by 71% compared with preirrigation levels.¹² In the present study, the volume, temperature, and irrigation pressure were also the same for both the study and control groups because of the experimental design.   The author’s results are in agreement with the findings of other clinical studies comparing the infection rate of wounds irrigated with tap water with that of wounds irrigated with sterile 0.9% saline solution. No differences in wound infection rates were reported in these studies between wounds irrigated with tap water and those irrigated with saline solution.^1,6,12,18   Skin wounds affect individuals of all ages and social groups.^19,20 The use of tap water for wound cleansing has advantages over the saline solution because it is readily available and inexpensive, even for low-income populations.^1,4,5   The present study offers important perspectives from the point of view of health management. Further studies on the use of tap water for wound cleansing are necessary to determine its effects on microbial colonization of deep tissues, wound healing, and on the cell cytoplasm. Also, studies comparing its effects on the microbiota of the periwound skin and wound bed are warranted. In this study, the authors compared the microbial colonization of wounds cleaned with tap water at fluid pressures ranging from 2.8 mm Hg (5.4 psi) to 2.4 mm Hg (4.6 psi) with that of wounds cleaned with 0.9% sodium chloride solution at the same fluid pressures. Additional studies are needed to investigate the efficacy of wound irrigation at different pressures in removing microorganisms.   It is important to note that tap water for wound debridement must be potable and free of contaminants, and that not all tap water may be suitable for wound irrigation. Health professionals have to carefully verify the suitability of their own water supplies. In addition, sources of tap water, such as faucets and water tanks, must be periodically tested by a certified laboratory and the characteristics of the water must be within standard limits.

Conclusion

  The use of tap water for cleansing skin wounds in rats did not affect the microbial colonization of the wounds compared with the use of sterile 0.9% sodium chloride solution.

References

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