Electrochemically-Activated Water Presents Bactericidal Effect Against Salmonella Heidelberg Isolated from Poultry Origin

Abstract

Salmonella spp. are among the most important pathogens in poultry farming, and Salmonella Heidelberg (SH) is one of the most frequent serotypes isolated in Brazil. SH has a zoonotic potential and stands out as a pathogen that is difficult to eliminate from the poultry chain due to its resistance to disinfectants. One alternative to traditional disinfectants is the electrochemically-activated water (ECA), a bactericidal compound produced from the electrolysis of salt and water. ECA generators produce a compound that consists of free chlorine, hypochlorous acid, and other free radicals. This alternative control method is safe for human health and reduces environmental contamination. The present study aimed at evaluating the efficacy of ECA against 30 SH isolates from poultry origin in scenarios that simulated the chiller environment (4°C, 5 and 50 parts per million [ppm], 5 and 40 min of exposure) and the cleaning and disinfection process (25°C, 200 ppm, 5 and 10 min of exposure). In the quantitative test, SH was susceptible to ECA. The mean bacterial counts decreased significantly compared to the control group, especially at 200 ppm. At this concentration, ECA inhibited the growth of almost 87% of the Salmonella strains, and the results showed a significant decrease in the mean bacterial counts for both exposure times (5 and 10 min). These findings demonstrate that ECA is effective against SH in vitro and it is a possible alternative to disinfection in the poultry industry for the control of this pathogen. However, in situ tests in the food industry are needed.

Introduction

Brazil is the second largest chicken meat producer worldwide (Associação Brasileira de Proteína Animal, 2018). Considering the importance of poultry farming in this country, chicken health is a source of constant concern. Salmonella spp. are among the microorganisms of major concern in the poultry industry. They have been isolated from poultry meat and can cause diseases in chickens and humans, being considered as one of the main causes of gastroenteritis in humans worldwide (Forsythe, 2013; Cardoso et al., 2015; World Health Organization, 2017). Salmonella Heidelberg (SH) is one of the most commonly found Salmonella serotypes responsible for outbreaks of salmonellosis in the United States and in Brazil (CDC, 2016; Brasil, 2019). The European Food Safety Authority (EFSA) has also considered SH as one of the most important serotypes in poultry farming (European Food Safety Authority, 2019a; European Food Safety Authority, 2019b). Food outbreaks are mainly associated with the consumption of raw or undercooked chicken meat or with cross-contamination (European Food Safety Authority, 2015).

Even though Brazilian poultry slaughterhouses follow rigid standards for control of microorganisms (Brasil, 2003), poultry products can be contaminated by Salmonella spp. Disinfectants are widely used in the food industry to eliminate contamination by pathogens and to avoid deterioration (Forsythe, 2013). However, resistance to disinfectants is a global concern. Previous studies have demonstrated bacterial resistance to the products that are widely used in the food industry (Sundheim et al., 1998), including those composed of quaternary ammonia (Lerma et al., 2013; Bragg et al., 2014). Considering the need for rigorous microbial control in the food industry and the increase of antimicrobial resistance to disinfectants, the search for alternative products is essential.

Electrochemically-activated water (ECA) is a technology that consists of the production of a nontoxic and biodegradable biocide compound. ECA generators produce this compound through electrolysis membranes from water, salt, and electricity. ECA has been recognized by the food industry as a promising potential decontamination compound (Hao et al., 2012). Previous studies demonstrated that ECA is effective against chicken carcasses contaminating microorganisms and several pathogens, including Escherichia coli, Salmonella Typhimurium, Salmonella Enteritidis, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis (Abadias et al., 2008; Guentzel et al., 2008; Cao et al., 2009; Xu et al., 2014; Zang et al., 2015; Wang et al., 2018).

The use of ECA consists of a cost-effective and an eco-friendly alternative to reduce bacterial contamination of food products (Huang et al., 2008; Radical Waters, 2017). The main component of ECA is the hypochlorous acid (HClO). The acid, oxidation-reduction potential (ORP), the large amount of free oxygen (>30 mg/L), the low pH, and other residual components produce a mechanism of microbial destruction by increasing the permeability of the membrane, leading to a leakage of the intracellular content, as well as reducing the dehydrogenases and nitrate reductase activities (Kiura et al., 2002; Andrade, 2008; Zeng et al., 2010). There is no evidence of bacterial resistance to ECA. Once it is not a stable solution and it can turn again into salt and water depending on the pH and the production conditions, it is possible that ECA does not cause microbial resistance, making it a potential alternative to currently used disinfectants (Thorn et al., 2012).

Based on the bactericidal action of ECA, the present study aimed at evaluating in vitro antimicrobial activity of ECA against SH strains. These results will allow determining if ECA could be a possible alternative for surface disinfection and for chiller contamination reduction in the poultry industry.

Materials and Methods

Production of ECA water

Drinking water from a local treatment plant was used for the production of ECA water. ECA was produced in an industrial ECA generator, with a production capacity of 200 L/h. Free chlorine measurements were carried out using the multiparameter chlorine meter Micro 7 Plus (Akso, São Leopoldo, Brazil) immediately after ECA production. The mean concentration detected was 250 parts per million (ppm). Then, ECA was diluted in sterile distilled water to obtain the three concentrations to be tested (5, 50, and 200 ppm). Tubes containing 9.9 mL of each concentration to be tested, with 1% bovine fetal serum (Gibco, Waltham) to simulate the presence of organic matter, were prepared. Preliminary studies, performed until 4 d after ECA production, demonstrated that the antimicrobial activity of ECA against SH remained stable throughout this period at the same conditions and concentrations of this study (data not shown).

Test concentrations, time of exposure, and temperature conditions

To simulate the chiller environment, the temperature was maintained at 4 ± 1°C, which is the maximum temperature allowed for carcasses after exiting the cooling stage (Brasil, 1998). For this simulation, two concentrations were tested: 5 ppm, which is the maximum water hyperchlorination allowed by Brazilian legislation (Brasil, 1998), and 50 ppm, which is the allowed concentration for chiller water in the United States (United States Department of Agriculture, 2017). Both concentrations were tested at two exposure times: 40 min, which corresponds to the mean time that the carcasses remain in the cooling tanks, and 5 min, which is the minimum exposure time necessary to evaluate the efficacy of the ECA. To simulate a cleaning scenario, a temperature of 25 ± 1°C, room temperature, and a concentration of 200 ppm of free chlorine were selected; this concentration is frequently used in the disinfection of equipment and surfaces in the poultry industry (Food and Drugs Administration, 2012). The exposure times tested at this concentration were 5 and 10 min, which are the closest to the real scenario in the poultry industry.

SH isolates

A total of 30 SH strains were selected for this study. The strains were isolated in 2016 from poultry sources, including drag swabs and feces, in Southern Brazil. Strains were previously isolated and biochemically characterized according to Brazilian legislation (Brasil, 1996) and were consecutively serotyped by a private laboratory belonging to the official laboratory net of the Brazilian Ministry of Agriculture, Livestock and Supply. Considering that these strains were isolated from poultry companies that are failing to eliminate SH from their flocks, they were selected for this experiment to mimic the real challenge that the companies are exposed to.

The bacterial isolates were stored frozen at −80°C in brain-heart infusion (BHI) broth (Merck, Darmstadt, Germany) supplemented with 3:1 glycerin (Synth, Diadema, Brazil). For reactivation, the isolates were seeded on xylose lysine deoxycholate (XLD) agar (Merck) and incubated at 36 ± 1°C. After 24 h, colonies morphologically consistent with those of Salmonella spp. were observed. A characteristic bacterial colony was selected and inoculated in BHI broth under the same incubation conditions previously described for the determination of the test suspension and antimicrobial assays.

Determination of test suspension

After reactivation of the SH isolates, an aliquot of the inoculated BHI broth was added to a tube containing 0.1% buffered peptone water (BPW) (Oxoid, Basingstoke, England) to obtain a turbidity consistent with the 0.5 scale of McFarland (∼10⁸ colony-forming unit [CFU]/mL). The optical density (0.08–0.1) was confirmed using a spectrophotometer (SP 22; Biospectro, Curitiba, Brazil) at 625 nm wavelength (Clinical and Laboratorial Standards Institute, 2013). Then, 1 mL of the bacterial suspension was diluted in 10 mL of 0.1% BPW to reach a concentration of ∼10⁶ CFU/mL.

BHI broth with a neutralizer

The BHI broth with neutralizer was prepared according to the protocol provided by the British Standard Institution (2006), with minor changes. Before the sterilization, 10 mL of polysorbate Tween 80 (Neon, São Paulo, Brazil), 2 g of soy lecithin (Stem, Porto Alegre, Brazil), and 2 g of sodium thiosulfate (Dynamic, Diadema, Brazil) were added for each liter of the broth.

Antimicrobial assays

Qualitative method

ECA was evaluated by the dilution test as described by the Brazilian legislation (Brasil, 1993). A measure of 0.1 mL of the SH inoculum was added to each tube containing 9.9 mL of ECA and fetal bovine serum. After the exposure time, 10 μL of the suspensions were removed and transferred in triplicate into tubes containing BHI broth with a neutralizer to inactivate the antimicrobial effect of ECA and were incubated at 36 ± 1°C for 96 h. Tubes presenting turbidity, surface film formation, or background precipitate were considered positive (Brasil, 1993). Bacterial viability was confirmed by reseeding an aliquot in plates containing XLD selective medium. Plates were incubated at 36 ± 1°C for 24 h. ECA was considered effective only when the tube remained negative after 96 h (Brasil, 1993).

Quantitative method

The quantitative suspension test was used to evaluate the bactericidal activity of ECA according to the protocol published by the European Standardization Committee (CEN) BS EN 1040:2005 (British Standards Institution, 2006). Briefly, 0.1 mL of the inoculum was added to each tube containing 9.9 mL of ECA and fetal bovine serum. After the predetermined exposure time, 1 mL of the solution was inoculated into 9 mL of BHI with a neutralizer. After 5 min, 1 mL aliquot was transferred into 9 mL of 0.85% saline solution (Neon), and serial dilutions were performed up to 10⁻⁴, followed by seeding in XLD medium using the drop plate method (Milles and Misra, 1938). Subsequently, the plates were incubated at 36 ± 1°C for 24 h. After this period, the colonies of Salmonella were counted. As controls, the 30 isolates were subjected to the same conditions of time and temperature with sterile distilled water.

Statistical analysis

The obtained data were subjected to statistical analysis using the PASW Statistic software (IBM, Hong Kong). A descriptive statistical analysis was used to determine the means of bacterial counting in each treatment and to determine the grouping of the samples according to the presence of bacterial growth. The Student's t-test was used to evaluate the count among treatment time periods. The nonparametric McNemar test was used to evaluate the comparison among the treatments for the correlated samples. A 5% level of significance was applied.

Results

In the qualitative test (Supplementary Table S1), ECA was not effective against SH strains at 5 and 50 ppm, regardless of the exposure time. At 200 ppm, ECA inhibited the growth of 15 and 16 isolates after 5 and 10 min of exposure, respectively.

Quantitative test results are shown in Tables 1, 2 and Supplementary Table S1. In the simulation of chiller environment at 4°C (Table 1), the mean bacterial count of treatments with 5 ppm did not present significant decreases (p > 0.05) compared to the control, regardless of the exposure time. At 50 ppm, bacterial count decreased significantly (p < 0.05) after 5 min (4.4 log₁₀ CFU/mL) and 40 min (3.779 log₁₀ CFU/mL) of exposure. In the simulation of a cleaning and disinfection process at 25°C (Table 2), the use of 200 ppm of ECA resulted in a significant decrease (p < 0.05) in the mean bacterial counts, 2.691 log₁₀ CFU/mL and 2.119 log₁₀ CFU/mL, for 5 and 10 min of exposure, respectively. In these conditions, only 13.3% (4/30) of the isolates showed in vitro growth.

Table 1.

Bacterial Count (log₁₀ CFU/mL) at 4°C of Thirty Salmonella Heidelberg Strains After Treatment with 5 and 50 ppm of Electro-Chemically Activated Water, According to the Exposure Time

Treatment	Contact time
Treatment	5 min	40 min
Control	4.691 ± 0.346^a	4.574 ± 0.313^a
5 ppm	4.475 ± 0.214^a	4.49 ± 0.216^a
50 ppm	4.4 ± 0.14^b	3.779 ± 0.574^b

Different letters in the same column represent a significant difference (p < 0.05).

CFU, colony-forming unit; ppm, parts per million.

Table 2.

Bacterial Count (CFU/mL) of Thirty Salmonella Heidelberg Isolates Treated with 200 ppm of Electro-Chemically Activated Water Compared to the Control Group According to the Contact Time at 25°C

Treatment	Contact time
Treatment	5 min	10 min
Control	5 × 10⁴ ± 33051.47^a	6.9 × 10⁴ ± 56308.83^a
200 ppm	4.9 × 10² ± 1366.60^b	1.3 × 10² ± 445.37^b

Different letters on the same line represent a significant difference (p < 0.05).

CFU, colony-forming unit; ppm, parts per million.

Discussion

The study of alternative compounds for pathogen reduction is urgent, especially for those microorganisms that are difficult to eliminate from the environment, like SH in Brazilian poultry companies. It has been shown that ECA, a nontoxic and biodegradable biocide compound, can be used in poultry products such as avian carcasses, chicken meat cuts, and giblets (Huang et al., 2008; Loretz et al., 2010). In addition, ECA has the advantage of not having a corrosive effect on metal surfaces (Abadias et al., 2008).

Previous studies have shown that ECA is more effective to reduce pathogens than chlorine (Ghebremichael et al., 2011), and several possible uses of this compound are described in the literature. For instance, Wang et al. (2018) tested a spray cabinet using ECA to reduce microorganisms on chicken carcasses during the processing in slaughterhouses and their results also show that it can be a substitute for traditional sodium hypochlorite. In the United States, ECA is allowed to be used in chillers at 50 ppm (United States Department of Agriculture, 2017), which could encourage its use in processing plants worldwide.

In this context, we aimed at evaluating in vitro antimicrobial activity of ECA at different concentrations and times of exposure. At 5 ppm, the bacterial count did not differ from the control, regardless of the time of exposure. The absence of a significant reduction in the microbial count at 5 ppm might be due to the low concentration of free chlorine and hypochlorous acid. Besides, the presence of organic matter may result in saturation of the product present in lower concentrations (Thorn et al., 2012). Nevertheless, ECA remains effective against SH in the presence of organic matter when higher concentrations are used. At 50 ppm, the microbial count is lower than 1 log₁₀ unit for both exposure times. This means that 50 ppm of ECA did not show a strong anti-Salmonella effect, and probably, it is not suitable for in vivo tests.

Better results were observed when a concentration of 200 ppm was tested. The highest concentration of ECA resulted in an ∼2 log₁₀ reduction in the microbial count. At this concentration, 86.7% of the isolates did not show growth, which reinforces the finding that ECA could be a viable alternative for the control of SH. The use of 200 ppm of ECA has been successfully tested by Chuang et al. (2013). They also found that this compound was effective against E. coli and Bacillus subtilis. Guentzel et al. (2008) tested the efficacy of ECA against E. coli, Salmonella Typhimurium, Staphylococcus aureus, L. monocytogenes, and Enterococcus faecalis at concentrations varying from 20 to 120 ppm with 10 min of exposure. The results showed a significant decrease in the microbial count for all species with a near neutral pH (6.3–6.5). Similarly, in the present study ECA was effective in the control of SH in nonacidic pH.

Al-Holy and Rasco (2015) inoculated E. coli O157:H7, Salmonella Typhimurium, and L. monocytogenes in various food matrices. Even though a decrease in the bacterial count was observed, the treatment with 38 ppm of ECA was not able to eliminate contamination by microorganisms, regardless of the time of exposure. According to the authors, the large amounts of organic matter present in food products may have influenced the results (Ayebah et al., 2005). Similarly, we did not observe a total elimination of SH. However, our results demonstrate a significant bacterial reduction at 50 and 200 ppm concentrations by the SH quantitative test, even in the presence of organic matter simulated by the presence of bovine fetal serum.

Akbas and Olmez (2007) found that the antimicrobial activity of ECA against L. monocytogenes and E. coli occurred in the first minutes of exposure, concluding that long periods of contact are not required for the action of ECA. However, this was not observed in the present study. Even if we could observe significant decrease in bacterial count after 5 min of exposure at 50 and 200 ppm, our data indicated that a longer period of exposure maximizes the antimicrobial activity of ECA.

It is widely accepted that regular and thorough cleaning of poultry transport crates is essential to avoid cross-contamination between batches and inhibit the spread of pathogens. Considering that the transport of the birds to the processing plant is an extremely stressful process, there is an increase in fecal excretion with the shedding of pathogens such as Salmonella (Berchieri et al., 2009). Zang et al. (2015) showed that ECA reduced Salmonella Enteritidis count in poultry crates at a near neutral pH and at a concentration of 50 ppm. However, based on our results with SH serotype, we suggest a concentration of 200 ppm of ECA to be tested in vivo. Future approaches on the bactericidal action of ECA in food products and as a surface disinfectant are needed. These results also encourage the development of new researches for the evaluation of viability of the use of ECA in the food industry.

Conclusions

Even though ECA did not completely eliminate SH, ECA was efficient at reducing significantly the bacterial contamination in poultry food products at 200 ppm in the tested conditions. The bactericidal action against SH isolates from poultry sources showed that ECA could be a possible alternative to other chlorine releasing products, but further analysis is needed.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

The authors gratefully acknowledge American Nutrients (Teutônia, Brazil) for the support and partnership to carry out the present study.

Supplementary Material

Supplementary Table S1

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Supplementary Material

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