Efficacy of hypochlorous acid (HOCl) fog in sanitizing surfaces against Enterococcus faecalis

Background: Fogging is an efficient method when disinfection of large areas is desired.

Methods

Two methods of ultrasonic fogging, pulsed and continuous, were compared on bacteria dried on either aluminum or polystyrene surfaces. We characterized commercial and home-made hypochlorous acid (HOCl) with respect to storage and means of production.

Results

We found that the initial chlorine concentration of the commercial solution was approximately 550 ppm, and when stored open under ambient conditions, the chlorine content decreased at a rate of 30% every 100 days. The HOCl produced using the home synthesizers had a maximum chlorine content of 257.6 ppm which decayed by 65% after 100 days. A second synthesizer produced a liquid with high chlorine content and pH, 750ppm and pH = 8.55. The anti-bacterial efficacy was probed using Enterococcus faecalis, a persistent source of infection in public and clinical spaces. Time course studies determined that E. faecalis could survive dry on surfaces for more than 12 weeks, but was easily eliminated in half the fogging time.

Conclusions

The most effective mode of application was determined to be continuous fogging where a 6.59 log reduction was established in vertical geometry. The optimal pulsed fogging protocol produced a similar reduction, but required nearly 5 times as long. The home synthesized versions yielded much lower log bacterial reductions. No significant differences in outcome were determined between polymer or metal surfaces.

Introduction

There are large variations in bacterial survival on different surfaces. Aerosolized Escherichia coli can survive on paper in an office for up to 21 hours.1 Gram-positive bacteria generally out survive Gram-negative organisms. Enterococcus faecalis, a Gram-positive bacterium is an organism which causes several systemic infections, some of which are potentially fatal. These include urinary tract, neonatal and wound infections, endocarditis and meningitis. In dentistry, this bacterium is the primary cause for root canal secondary infections after other bacteria are eliminated.23 There are many factors that allow resistance of E. faecalis against disinfectants. It has a proton pump which permits maintaining a pH below the critical value of 11.5.4 E. faecalis can also withstand long durations of survival until an adequate nutritional supply becomes available.5 Lastly, E. faecalis can exhibit widespread genetic polymorphisms, possibly allowing for resistance against antimicrobials6 and over many decades, has been recognized as a notable nosocomial pathogen. As a result, E. faecalis is an important species of bacteria to control in a clinical environment.

There are many different options of disinfectants for fogging. Hydrogen peroxide is a well-known chemical used for fogging, but it is a strong oxidizing agent and hence known to be corrosive to some materials commonly found in office furniture and equipment such as copper, carbon or stainless steel, cast iron and even some plastics, such as neoprene and other kinds of rubbers.7 Chlorine dioxide is another environmentally friendly method of fogging, but is highly unstable when in contact with sunlight and explosive when its partial pressure is over 0.1 bar, making transportation and storage nearly impossible in pure form. Thus, research is still being done to determine the best method of encapsulation for chlorine dioxide.8 On the other hand, hypochlorous acid (HOCl), has been shown to be an effective anti-bacterial with minimal toxicity to eukaryotic cells.9 Previously study has shown that HOCl administered from a fogger was able to decontaminate surfaces harboring Staphylococcus aureus and Acinetobacter.10 Hypochlorous acid (HOCl) attacks bacteria by causing the inhibition of F1 ATPase, the loss of DNA replication (via loss of association of origin of replication with inner membrane), and the failure of metabolite and protein transport across the inner membrane.1112 Here we will explore whether HOCl can also be effective against E. faecalis. The method of application which we chose to study is ultrasonic fogging, which produces a thin mist that deposits on surfaces. The fogging process produces micron sized particles of HOCl, which deposit on surfaces, emitting chlorine gas as the particles dry. This method was shown to be very convenient for disinfection of large areas, where the fog could penetrate.11, 12, 13, 14

In this project, we will describe the efficacy of HOCl fogging in vertical and horizontal orientations against E. faecalis to find the optimal sequence and duration of fogging. Effectiveness of the HOCl fog against microorganisms is dependent on the angle of contact. It has been reported by Burfoot and colleagues that microbial load reduction on horizontal surfaces is 20 times greater when compared to vertical surfaces. This is due to the horizontal surfaces being more accessible to disinfectant deposition.13, 15 Hence here we will explore the effect of the fogged particles on both geometries. We will also discuss the stability of HOCl with respect to chlorine concentration, before and after fogging, and the optimal modality for application of the fog, that is, intermittent stream or steady and continuous.

Section snippets

Bacteria preparation and fogging processes

E. faecalis (ATCC 19433) was grown on Brain Heart Infusion Agar (BHI, DifcoTM) for 24 hours at 37°C then suspended in phosphate buffered saline. The number of E. faecalis in stock solution was estimated by optical density (OD) at 600 nm using a spectrophotometer. Fifty microliter droplets containing 5 × 107 E. faecalis in PBS was smeared onto 2cm2 sterilized aluminum plates and allowed to dry in an incubator at 37°C for 1 hour. The plates where then attached in either horizontal or vertical

Characterization of HOCl solutions

The HOCl solutions were tested with respect to chlorine content and pH. In Table 1 we show the pH and chlorine concentration for three EcoLogic Solutions bottles where the measurements were obtained immediately after opening. The bottles were obtained at the same time in our laboratory, but opened at different times after storage, closed, in ambient conditions. From the Figure 2A we can see that degradation were significantly slower in the sealed bottles. We also tested the chlorine content and

Discussion

Hypochlorous acid is a well-known compound that has been shown to have minimal toxicity to eukaryotic cells, while being an effective anti-bacterial agent.9 Furthermore, HOCl is straightforward to synthesize and apply using an ultrasonic fogging apparatus.18 We therefore probed the efficacy of HOCl using two distinct methods of ultrasonic fogging, pulsed and continuous. We characterized the HOCl with respect to storage and means of production, namely a commercial liquid product from EcoLogic

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Fogging for the Disinfection of Food Processing Factories and Equipment

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