Introduction

Nosocomial environments can act as a reservoir for opportunistic pathogens so that healthcare-associated infections (HAIs) are commonly associated with inadequate cleaning and disinfection of environments, and can be acquired not only by patients who are more susceptible, but also by hospital staff, although less frequently by visitors.(¹,²)

HAIs represent a serious public health concern. (³,⁴) According to the World Health Organization (WHO), approximately 7% of patients in hospitals in high-income countries and 15% in low- and middle-income countries acquire some form of HAI during their hospital stay. Moreover, approximately 10% of HAI cases result in death due to complications of the infection.(⁵)

Managing these infections is further complicated by the high level of antimicrobial resistance. According to estimates from the Centers for Disease Control and Prevention (CDC), more than 2.8 million antibiotic-resistant infections and at least 35,000 deaths will occur in hospitals in the United States each year.(⁶) Antimicrobial resistance could lead to the deaths of 10 million people annually by 2050 at a total cost of $100 trillion without inter-vention.(⁷)

Controlling the spread of pathogens in hospital environments is essential to minimize the risk of HAI transmission. Methods for validating decontamination include visual inspection, microbiological analysis, and, more recently, the use of fluorescent markers, such as the adenosine triphosphate (ATP) bioluminescence method,(⁸) which provides objective assessment and immediate feedback on the presence of biological contamination.(⁹)

Some chemicals commonly used by healthcare providers for cleaning and disinfecting surfaces include 0.1% sodium hypochlorite, quaternary ammonium chloride, and hydrogen peroxide (HP). These chemicals are difficult to break down and leave harmful toxic residues that threaten human health.(¹⁰)

The use of ozonated water is an alternative and effective method for disinfecting surfaces.(¹¹) Ozone (O3), with its instability and high oxidizing reactivity, has aroused the interest of researchers. Furthermore, O3 is considered a natural broad-spectrum germicide capable of inactivating viruses, bacteria, protozoa and fungi due to its oxidative capacity, causing damage to their cell membranes.(¹²)

Another characteristic of O3 is its ability to be dissolved in water, maintaining its microbicidal action.(¹³) Thus, this study assessed the effectiveness of ozonated water in disinfecting surfaces in an adult Intensive Care Unit (ICU) of a medium and high complexity public hospital in the countryside of the Amazon, Brazil.

Methods

This is an exploratory, descriptive study with a quantitative approach. The research setting was a medium and high complexity public hospital, which assists a population of approximately 1.1 million Brazilian Health System (SUS - Sistema Único de Saúde) users. It was carried out in the first half of 2022 in two stages: the first using a luminometer (SystemSure Plus, Hygiena®) to indicate the presence of biological contamination; and the second using automated microbiology (Phoenix M50, BD®) to identify, qualify and define the microbial resistance pattern of the samples analyzed.

The methods used in this study were the ATP bioluminescence test and automated microbiological analysis. The choice of the bioluminescence test is in line with the hospital's established practices for detecting biological contamination on surfaces. The combination of these two methods enables a robust and multidimensional surface decontamination assessment, which is essential for effective control of HAIs. Each method brings its own unique advantages, and when used together, they provide a comprehensive view that guides infection control actions, promoting a safer hospital environment for patients and healthcare professionals.

The bioluminescence test uses ATP detection to indicate the presence of biological contamination, a method that does not distinguish among types of microorganisms, but signals the presence of active biological matter.(²) In this study, the UltraSnap swab and the luminometer (SystemSure Plus Hygiena®) were used, which recommends a scale of ≤100 RLU to consider a surface adequately cleaned and disinfected.

To collect microbiological samples, a Stuart swab was used on the surfaces that presented the highest ATP value. The samples were seeded in petri dishes and transferred to the Phoenix system tubes used for bacterial identification (ID) and combo for the antimicrobial susceptibility test (AST), then transferred to the corresponding panel (gram-negative and/or gram-positive) in order to guarantee viability, purity and define the microbial resistance pattern of the analyzed samples.

To select the sample, three distinct areas were chosen for each surface of the 15 healthcare units assessed (air mattress, bed rails and bedside table). Each area was subdivided into four subgroups (CG, HPG, HDEO3G and O3G) and subjected to 12 ATP tests per surface, totaling 36 tests per healthcare unit. The sample acquisition area was delimited at 100 cm² for each test.

Marking the areas on the bed rails presented challenges due to their irregular shape. We used a tape measure to precisely measure and mark the test areas, specifically in high-touch locations that are more likely to retain contamination. The test areas were marked with a circumference of 12 cm and a length of 8.33 cm, ensuring standardization of measurements.

Inclusion criteria for the samples involved beds that had been occupied for more than seven days, before concurrent cleaning and before patient bathing. Selection was based on bioluminescence results equal to or greater than 100 Relative Light Units (RLU), indicating the need for disinfection.

To determine the cost of decontaminating selected surfaces (air mattress, bed rails and bedside table) in an adult ICU with ozonated water, investments in an O3 generator (R$ 8,000.00) and additional infrastructure were necessary. The hospital already had essential elements, such as a reverse osmosis water filter (Permution RO0520l model, R$5,000.00), supply containers, fume hood (R$15,000.00), medical oxygen and personal protective equipment (PPE). Operating costs included electricity (R$209.00/ month), annual maintenance (R$4,000.00) and medical oxygen (R$300.00/month).

The cost of treated water per liter was R$0.50, considering the reverse osmosis system. The infrastructure included ventilation systems and O3 detection sensors, ensuring the safety of the researchers. The implementation followed CONITEC guidelines for safe and effective health practices.(¹⁴) Ozonated water was obtained using an O3 generator from OzonLife, Medical Systems®, Brazil. The oxygen flow rate of 1/8 L/min was adjusted at the inlet of the device, resulting in the output of O3 gas at a concentration of 50 mg/L.(¹¹) A fume hood was used as a safety measure to produce ozonated water (Figure 1). The fluid was transferred to water using the bubble column reactor (BCR) system(¹⁵) through a sintered stainless steel diffuser immersed in 500 ml of water treated by reverse osmosis. Ozonated water was produced at a temperature of 6.9°C, reaching a concentration of 2.7 mg/L and saturating in 4 minutes. To preserve the microbicidal properties of the disinfectant solution of ozonated water, it was kept at a temperature between 8°C and 10°C. Using two 10 ml ice cubes each, they were made with the same degree of purity as the water used. The solution was stored in a glass container wrapped in ice for a period of 30 minutes at an average room temperature of 23°C for immediate use.

For HPG, Oxivir Five 16 Concentrate (Diversey, Inc., Charlotte, NC) was used, with an initial HP concentration equivalent to 42.5 g/L. The solution used was obtained from dilution in a ratio of 1:64, which produces a final concentration of 664 mg/L of HP. This was sprayed onto the surfaces, and after two minutes was removed with the aid of disposable multipurpose cloths (Perfex®), according to the hospital's own decontamination protocol. Regarding HDEO3G, the surfaces were cleaned with enzymatic detergent (Neozime5®) at a concentration of 20mg/L, and then dried immediately with disposable multipurpose cloths (Perfex®). Immediately after drying, the selected surfaces were sprayed with ozonated water at a concentration of 2.7 mg/L and a temperature between 8°C and 10°C, perpendicularly and unidirectionally, at a distance of approximately 25 cm from each selected area of the surfaces. After two minutes, drying was performed with a clean compress, and repeated in the fourth and sixth minutes. For O3G, the same protocol as for HDEO3G was repeated, with the exception of the enzymatic detergent.

In order to assess the potential of ozonated water as a high-level disinfectant solution, the HDEO3G protocol was created, justified by its ability to provide more thorough cleaning and more effective disinfection compared to methods that use a single agent. The combination of enzymatic detergent and ozonated water provided a comprehensive and robust method to address the complexity of hospital surfaces and the diversity of microbial challenges encountered in hospital environments.

Quantitative analysis of the degree of disinfection was performed considering the difference before and after the disinfection procedure for each group from the ATP values (bioluminescence, RLU) and a qualitative analysis from automated microbiological assay (Phoenix, MD50, BD®). The obtained data set was statistically processed using Microsoft Excel® 2013 and Origin® 8.5. The Kolmogorov-Smirnov normality test was applied to the collected data. An intergroup statistical analysis compared the four groups using the one-way ANOVA test, followed by the Bonferroni post-hoc test. All statistical data analyses were performed with the Prism® 8.0 program (GraphPad Software Inc., La Jolla, CA, USA), with a significance level of 5% (p<0.05). For qualitative analyses and characterization of microorganisms, the relative frequency was used.

It should be noted that this study is an excerpt from a doctoral project in biomedical engineering approved by the Universidade do Estado do Pará (UEPA) Research Ethics Committee, under Opinion 4,743,235 and Certificate of Presentation of Ethical Consideration (CAAE - Certificado de Apresentação de Apreciação Ética) 39785220.4.0000.5168.

Results

A total of 540 bioluminescence tests were performed on 15 surfaces (air mattress, bed rail and bedside table) for each of the four groups (CG, HPG, HDEO3G and O3G). The results obtained showed a higher contamination rate for the bed rail with a mean of 736.2 ± 190.4 RLU, followed by the bedside table, with a mean of 718.6 ± 149.4 RLU, and the air mattress, which presented a lower degree of contamination, with a mean of 331.4 ± 30.1 RLU (Figure 2).

For all intervention groups, for all surfaces, after decontamination, a significant reduction (p<0.0001) was observed when compared to CG. HDEO3G compared to O3G did not present a significant difference (p>0.05) between them, indicating that the addition of the enzymatic detergent in this protocol did not interfere with the decontamination of the surfaces analyzed.

When comparing O3G with HPG, there was a significant difference (p<0.05), indicating that ozonated water at a concentration of 2.7 mg/L was more efficient than HP at a concentration of 664 mg/L.

The results presented in the graphs show a consistent trend in which HDEO3G and O3G demonstrate lower levels of ATP luminescence compared to HPG. In the three sets of graphs (a x b, c x d, e x f), HDEO3G is shown to be the most effective disinfection method, followed by O3G and HPG. The presence of CG in the graphs (a c e) highlights the effectiveness of the disinfection methods, showing significant reductions in contamination compared to no intervention. The data suggest that disinfection with HDEO3G and O3G is superior to HPG.

Qualitative analysis demonstrated that of the 55 samples obtained: 23.6% tested positive for MDR bacteria and 76.4% showed no microbi-al growth. Of the positive results, 50% were in the gram-negative group, (Enterobacter cloacae, Klebsiella Pneumoniae, Acinetobacter spp.) and 50% in the gram-positive group (Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus schleiferi). The most prevalent bacteria were Staphylococcus haemolyticus, Klebsiella pneumoniae and Acinetobacter spp (Chart 1).

Discussion

The present study assessed the effectiveness of ozonated water in surface decontamination compared to HP, traditionally adopted in global hospital practices.(¹⁷)

For the pneumatic mattress, an average of 331.4 RLUs was obtained, as well as a significant presence of pathogenic microorganisms, according to the microbiological analysis. This fact can be attributed to the longer contact time with patient and/or temperature and humidity conditions conducive to microbial growth.

Regarding contamination by microorganisms present in the mattress, we found Staphylococcus haemolyticus, Staphylococcus schleiferi, Klebsiella pneumoniae and Acinetobacter spp. With regard to Staphylococcus haemolyticus and Staphylococcus schleiferi, gram-positive staphylococci are described as nosocomial bacteria, with emphasis on MDR, causing HAIs such as endocarditis, bacteremia,(¹⁸) meningitis and infections.(¹⁹)

The presence of these pathogens does not pose a direct risk of transmission; however, the interaction between environmental factors and the susceptible host may facilitate the spread of HAIs.(⁴,²⁰)

Another factor to be considered is the transfer of these microorganisms that can occur through direct contact with contaminated surfaces or through aerosols in healthcare procedures. Klebsiella pneumoniae and Acinetobacter spp have been related to the length of stay in the ICU.(²⁰,²¹) They are facultatively anaerobic gram-negative bacteria capable of producing ß-lactamase enzymes capable of hydrolyzing ß-lactam rings of antibiotics such as penicillin, cephalosporins and carbapenems. Their main source of contamination is the gastrointestinal tract, which contaminates hospital surfaces and can be transferred by the hands of both staff and patients, forming biofilms that are difficult to remove.(²¹,²²)

However, the protocols used in this study managed to eliminate not only Klebsiella pneumoniae, which is recognized for its epidemiological importance, but also all pathogens found on the surfaces assessed, which suggests the effectiveness of ozonated water at a concentration of 2.7 mg/L as a high-level disinfectant. This effect is related to its oxidizing action, releasing free radicals that inactivate bacteria, fungi, viruses and protozoa.(²³,²⁴,²⁵)

These results support those found in the inte-grative literature review study on the use of O3 to disinfect surfaces, which revealed the action of O3 on different microorganisms, indicating an inhibition rate equal to or greater than 90%, electing it as a new proposal for the development of new decontamination protocols.(²⁶)

Acinetobacter spp. are gram-negative aerobic bacilli classified as members of the Moraxellaceae family that survive on dry surfaces for up to a month, and often remain on healthcare professionals' skin, increasing the likelihood of patient colonization and equipment contamination.(²⁷)

Experimental studies have shown the effectiveness of ozonated water as a microbicide.(²³,²⁴,²⁵) However, its use must be carefully managed to avoid exposure to O3 gas, which can lead to respiratory impairment. Handling chemical agents, such as oxidizing and highly reactive products used in disinfection, requires special attention, although effective in eliminating pathogens, and they can present risks through direct contact or inhalation.

Concerning ozonated water preparation, it is generally carried out by trained professionals, who may be part of a hospital's maintenance team or specialized technicians hired for this purpose. The safe preparation of ozonated water requires the use of a fume hood due to the toxicity of O3 in high concentrations. This requirement implies the need for a special planned infrastructure, including adequate ventilation systems to disperse O3 safely, O3 detection sensors to monitor levels in the environment.(²⁸)

Furthermore, it is essential that professionals use appropriate personal PPE, such as respiratory masks with activated carbon filters, silicone gloves, long-sleeved aprons and protective glasses, to mitigate the risks of exposure during the preparation and use of ozonated water.(¹⁴)

Staphylococcus schleiferi, found in the mattresses of the samples in this study, was shown to be sensitive to ozonated water at a concentration of 2.7 mg/L. These findings support the results of a study that assessed the effectiveness of ozonated water in disinfecting the surfaces of instrument tables, where the presence of gram-positive cocci was identified before the decontamination process, but after the use of ozonated water, no microbial growth was detected.(²⁸)

In this perspective, another study used ozonated water as a high-level disinfectant to inactivate Staphylococcus aureus in an experimental colonoscope contamination protocol with effective results.(²⁹) Similar results were observed in another study,(¹⁶) including MDR inactivation.(²⁰)

Concerning the bed rails, which obtained the highest contamination rate through bioluminescence with an average of 736.2 RLU, results of a study were found that suggest that the source of contamination may be related to both patients and healthcare professionals..(³⁰)

The microorganisms found in the samples from the bed rails (gram-positive Staphylococcus epidermidis and Staphylococcus haemolyticus) have been described as agents that cause bloodstream infections, in addition to being associated with HAIs, which can affect the individual, especially when the immune system is compromised.(³¹,³²,³³)

However, an unexpected finding of this study was the high contamination of the bedside table with an average of 718.6 RLUs, as it is a hospital accredited by the Brazilian National Accreditation Organization (ONA - Organização Nacional de Acreditação) level 3 that has strict cleaning and disinfection protocols aimed at patient safety with the aim of preventing HAIs.

Furthermore, the microorganisms isolated at this site were Staphylococcus haemolyticus, discussed previously, and gram-negative Enterobacter cloacae, which may be related to hand contamination during diaper changing, indicating poor hand hygiene after handling by professionals.

This result supports those found in a study that observed that surface contamination can persist even after decontamination processes due to the formation of biofilms. These complex structures protect the microbial communities attached to surfaces, hindering the action of antimicrobial agents.(¹⁰) This observation is corroborated in a study that shows how biofilms present on environmental surfaces in ICUs can harbor multiresistant bacteria, showing the difficulty of eradicating them and the need for more effective disinfection methods.(³⁴)

The HPG protocol, which used Oxivir Five 16 Concentrate at a concentration of 664 mg/L, proved to be effective in decontaminating the surfaces assessed. This method is considered the gold standard for high-level disinfection due to its rapid action, which occurs through protein denaturation and cell membrane rupture. However, it is relatively expensive and is not capable of eliminating spore forms at low concentrations.(³⁵)

Therefore, hospital environments require a multifaceted approach to HAI prevention. This includes not only the continuous improvement of decontamination techniques, but also a renewed emphasis on occupational protection to ensure the safety of all users of the healthcare environment.

The research confirmed that HDEO3G demonstrated equivalent efficacy to O3G in disinfecting the surfaces investigated. This finding is extremely important as it indicates that both methods are effective in reducing microbial contamination. However, it is important to acknowledge some limitations of this study: the investigation did not explore the residual effect of the absence of MDR on the treated surfaces.

The multifactorial nature of recontamination, which includes factors such as frequency of surface use, procedures performed, and ambient air quality, was not assessed in depth. Furthermore, the study was conducted in a single center and did not establish a direct correlation with HAI rates. The visual inspection method was also not included in the study protocol. Therefore, although the results are promising, more research is needed to confirm the effectiveness of ozonated water in preventing HAIs in different contexts and care environments, thus ensuring the maintenance of a safe hospital environment.

Conclusion

Ozonated water has proven to be a promising alternative for decontaminating surfaces contaminated by MDR in ICUs. Its advantages include its spontaneous decomposition into oxygenated substances and its low cost. This approach not only minimizes environmental risk but also acts effectively against multidrug-resistant pathogens, standing out as a high-level disinfectant, contributing to safety and infection control in critical hospital environments.

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