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ISSN : 1229-1153(Print)
ISSN : 2465-9223(Online)
Journal of Food Hygiene and Safety Vol.31 No.6 pp.471-475
DOI : https://doi.org/10.13103/JFHS.2016.31.6.471

Bactericidal Efficacy of a Powder Disinfectant Containing Phosphate Compounds Against Salmonella Typhimurium

Eun-Kee Park1 , Chun-Nam Cha2 , Chang-Yeul Yoo3, Suk Kim, , Hu-Jang Lee*
Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Chinju 52828, Korea
1Department of Medical Humanities and Social Medicine, College of Medicine, Kosin University, Busan 49267, Korea
2Engineering Research Institute, Department of Industrial Systems Engineering, Gyeongsang National University, Chinju 52828, Korea
3Department of Computer Information, Gyeongnam Provincial Namhae College, Namhae 52422, Korea
Correspondence to: Hu-Jang Lee, College of Veterinary Medicine, Gyeongsang National University, 501 Jinju dae-ro, Chinju 52828, Korea 82-55-772-2352, 82-55-772-2308hujang@gnu.ac.kr
September 9, 2016 September 17, 2016 September 26, 2016

Abstract

Salmonella Typhimurium (S. Typhimurium) is one of the common food pathogens which may cause gastroenteritidis in human and animals. The microorganism also causes the economic loss in animal farming and food industry. In this study, the disinfection efficacy of a powder disinfectant containing phosphate compounds as a main ingredient, was evaluated against S. Typhimurium. A bactericidal efficacy test by broth dilution method was used to determine the lowest effective dilution of the disinfectant following exposure to test bacteria for 30 min at 4°C. The disinfectant and test bacteria were diluted with hard water (HW) or organic matter suspension (OM) according to treatment condition. On HW and OM conditions, the bactericidal activity of the disinfectant against S. Typhimurium was 4- and 2-fold dilutions, respectively. As the disinfectant composed to phosphate compounds possesses bactericidal efficacy against pathogenic bacteria such as S. Typhimurium, the powder disinfectant can be used to control the spread of bacterial diseases.


초록


    Sung-Won Co., Ltd. (Seoul, Korea)

    The bacterium Salmonella Typhimurium (S. Typhimurium) extensively causes self-limiting enteritis, fatal infection in animals, food-borne infection, and typhoid fever in humans1-3). The bacterium is a Gram-negative, motile, non-sporing, noncapsulated bacillus that can be contracted through contaminated water, milk, food or fruits and vegetables or via convalescent or chronic carriers4,5).

    Salmonella infections of food animals play an important role in public health and particularly in food safety, as food products of animal origin are considered to be the major source of human Salmonella infections6). S. Typhimurium is one of the most frequently isolated serotypes from pig farms, slaughtered swine, and associated with human foodborne diseases7,8). Salmonellosis in livestock animals and human cause enormous economic loss in the world9). In the United States and Korea, the total cost for the illness, including medical costs and loss of productivity, has been estimated at $11.4 and 1.01 won billion per annum, respectively10,11).

    In worldwide, antibiotic-resistant Salmonella spp. are increasing due to abuse and overuse of antibiotics. Therefore, the effective cleaning and disinfection regimes are essential in the prevention of infections and outbreaks12,13). The cleaning and disinfectant regimes depend on the proper use of biocides, and there is the concern that the resulting increased use of biocides in farming, food production, and hospital settings, and the home could contribute to the selection of antibiotic-resistant strains as some mechanisms of biocide resistance also confer antibiotic resistance14). Biocides are often composed of a mixture of ingredients that act upon a wide range of cellular mechanisms and targets, which makes it difficult for bacteria to become resistant to biocides9).

    The use of disinfectant is very effective for successful control of diseases from bacteria, fungi and parasites in farm animals15). Many disinfectants containing chemical components have been used for decontamination of farmed animal and food borne diseases16). However, there is not the efficacy test for the environmentally friendly disinfectant containing phosphate compounds against S. Typhimurium. Phosphates are frequently used as additives in order to improve technological and functional qualities of foodstuffs in the production of food17).

    Therefore, this study was carried out to examine bactericidal efficacy of a powder disinfectant against S. Typhimurium.

    Materials and Methods

    Bacteria and culture

    S. Typhimurium (G-B-14-21-62) was obtained from the Korean Veterinary Culture Collection (KVCC, Seoul, Korea). The strains were maintained as frozen glycerol stock. The bacteria were cultured in Luria-Bertani (LB) broth containing 1.5% agar and incubated at 37°C.

    Disinfectant

    The tested disinfectant, Stalosan-F® powder, is composed of phosphate compounds (85%), copper sulfate (2.5%), ferrous sulfate (2.1%), active chlorine (0.25%), and aluminumsilicate (10.15%). The disinfectant was provided by Sung- Won Co., Ltd. (Seoul, Korea). The powder disinfectant was stored in room temperature and prepared for dilution on the day of evaluation. Determination of the antimicrobial efficacy of the disinfectant was based on Animal and Plant Quarantine Agency (Anyang, Korea), Regulation No. 2013-34.

    Diluents and treatment condition

    Testing was based on bactericidal effects of disinfectant diluents in two treatment conditions (standard hard water (HW) condition and organic matter (OM) condition) and pathogen control (disinfectant negative control) in Table 1. HW, an ingredient of HW treatment condition, was made by adding anhydrous CaCl2 0.305 g and MgCl2·6H2O 0.139 g into one liter distilled water. Organic suspension, an ingredient of OM treatment condition, is a solution of 5% (w/v) yeast extract in HW. The test organism was prepared by titration of the cultural broth into at least 108 CFU/mL viable bacteria with the same kind of diluents of treatment condition.

    Experimental procedures

    For the efficacy test against S. Typhimurium, the disinfectant was completely dissolved in the same volume of HW and OM, respectively, and then each solution was diluted 1:1.5, 1:2, 1:2.5, 1:3 and 1:3.5 with HW and OM, correspondingly. To verify the lowest effective dilution of the disinfectant, five serial dilutions of the disinfectant were prepared and placed at 4°C prior to test reaction. 2.5 ml of each disinfectant dilution was mixed with the same amount of test organism followed by contact time of 30 min at 4°C. During this period, the mixture was shaken at 10 min interval. At the end of 30 min contact period, one mL of the mixture was neutralized with 9 mL of Nutrient broth containing 5% inactivated horse serum (BD Korea Co., Ltd., Incheon, Korea) at 37°C. 0.1 mL of the neutralized reaction mixture was subcultured into 10 mL of recovery cultural broth at 37°C for 48 h in incubator. For the test of bacteria control, 2.5 ml of hard water was mixed with the same amount of test organism followed by contact time of 30 min at 4°C, and then all procedure were undertaken in parallel for the disinfection test.

    The valid dilution of the disinfectant was determined that the greatest dilution showing no growth in more than four replicates of five tubes per treatment was confirmed. The final dilution time was statistically determined by a median value among three valid dilution of the triplicate test, but each value of which should be within 20% experimental error. In each bacteria control, the number of bacterial growth in the five replicates was counted.

    Results

    Table 2 shows the final valid dilution of the disinfectant containing phosphate compounds. When the bactericidal effect on HW condition was evaluated, the antibacterial activity of the disinfectant showed on 4-fold dilutions against S. Typhimurium. With the investigation of the bactericidal effect of the disinfectant on OM condition, S. Typhimurium was inactivated on 2-fold dilutions. As the organic material interferes with efficacy by either inactivating the disinfectant or blocking it from surface contact, the bactericidal activity of the disinfectant on the OM condition was lowered against animal pathogenic bacteria compared with HW condition.

    In the bacterial control, the number of S. Typhimurium was more than 2.0 × 105 colony forming units (CFU)/mL, which was satisfied with Animal and Plant Quarantine Agency, Regulation No. 2013-34.

    Discussion

    The powder disinfectant composed to phosphate compounds, copper sulfate, ferrous sulfate, and active chlorine, was a potential antibacterial disinfectant. The antibacterial effect on phosphate works according to the principle of chelation of mainly divalent metal ions (Ca2+, Mg2+), which can cause inactive to some essential physiological processes of bacterial growth18). The oligodynamic effect was discovered in 1893 as a toxic effect of metal ions such as copper, iron, zinc, silver and gold on bacteria and viruses, even in relatively low concentration19). In addition, active chlorine compounds are well-known antimicrobial agents in human medicine and food industry20).

    The antibacterial mechanism of polyphosphates is caused by binding of polyphosphates to the cell wall of earlyexponential phase bacterial cells, resulting in bactericidal and bacteriolytic effects21). Recent studies indicate that different metals, including copper and iron cause discrete and distinct types of injuries to microbial cells as a result of oxidative stress, protein dysfunction or membrane damage22,23). Chlorine is used in water treatment to disinfect drinking water, swimming pools, ornamental ponds and aquaria, sewage and wastewater, and other types of water reservoirs 24). When hypochlorite solutions containing chlorine are added in small amounts to pool water or industrial water systems, the chlorine atoms hydrolyze from the rest of the molecule forming hypochlorous acid, which acts as a general biocide25).

    In the present study, the antibacterial activity of the powder disinfectant showed on 4- and 2-fold dilutions against S. Typhimurium, respectively. In a previous study26), antibacterial activity was tested against S. Tyhimurium using sanitizers which are based on peracetic acid-hydrogen peroxide, sodium dichloroisocyanurate, and didecyldimethylammonium chloride (DDAC), results revealed that compared with washing parsley and cucumbers with water, treatments with all three sanitizers were not effective, resulting in a maximum reduction of only 0.7 log CFU of S. Typhimurium. In another previous study27), inhibitory effects of the hydrosols of thyme, black cumin, sage, rosemary and bay leaf were investigated against S. Typhimurium inoculated to apple and carrots, and thyme hydrosol showed the highest antibacterial effect, resulting in a maximum reduction of 1.76 log CFU of S. Typhimurium compared with control. In a study for the bactericidal efficacy of an organochlorine disinfectant, the number of S. Typhimurium was reduced from 1.4 × 108 CFU/mL to less than 102CFU/mL after exposure of the disinfectant (HOCl 100 ppm) for 10 min.

    With the consideration of dosage, concentration and contact period, the powder disinfectant is more effective than peracetic acid-hydrogen peroxide, sodium dichloroisocyanurate, DDAC and thyme hydrosol. However, the bactericidal activity of the disinfectant is lower than that of an organochlorine disinfectant. As the efficacy of the disinfectant against S. Typhimurium was investigated in vitro, a controlled field trial is required to determine whether use of the disinfectant will be able to reduce new pathogenic bacteria infection in animal farm and food-industry area.

    Conclusions

    In animal farm and food industry, salmonellosis was a very important disease because of high mortality for farmed animals, zoonoses and economic loss. In the study of the bactericide efficacy test of the powder disinfectant containing phosphate compounds, the results suggest that the disinfectant has a potential bactericidal activity against S. Typhimurium. So, the disinfectant can be used to control the spread of zoonotic bacteria such as S. Typhimurium.

    국문요약

    살모넬라증은 가축에 심각한 피해를 유발하는 질병으로, 축산업과 식품산업에 많은 경제적 손실을 초래하고 있다. 본 연구에서는, 복합 인산염을 주성분으로 하는 분말 소 독제의 Salmonella Typhimuriums에 대한 살균효과 시험을 수행하였다.

    배지희석법을 이용한 살균효력시험은 4°C에서 30분 동 안 시험 세균을 희석 소독제에 노출시켜 소독제의 가장 효과적인 낮은 희석배수를 결정하는 시험이다. 본 분말 소 독제와 시험 세균을 처리조건에 따라 경수와 유기물로 희 석하여 반응을 시켰다. 유기물 조건에서, Salmonella Typhimurium에 대한 소독제의 살균력은 경수조건에서의 살균력과 비교하여 낮게 나타났는데, 이는 유기물들에 의 한 소독제의 살균 유효성분에 대한 저해작용에 따른 것으 로 사료된다.

    분말 소독제는 Salmonella Typhimurium과 같은 병원체 에 대해 살균효과를 가지므로, 살모넬라증과 같은 세균성 질병의 확산을 제어하는데 효과적으로 이용될 수 있을 것 으로 사료된다.

    Acknowledgement

    This work was financially supported by Sung-Won Co., Ltd. (Seoul, Korea).

    Figure

    Table

    Experimental design for the determination of the bactericidal efficacy of the powder disinfectant containing phosphate compounds

    1)HW: standard hard water, OM: organic matter.
    2)+: presence, -: absence.

    Final valid dilution of the powder disinfectant containing phosphate compounds

    1)HW: standard hard water, OM: organic matter, BC: bacterial control, DF: dilution factor.
    2)No growth in more than four replicates of five tubes per treatment.
    3)Growth in more than two replicates of five tubes per treatment.
    4)All growth in each replicate.

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