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EVALUATION OF THE EFFECTIVENESS OF VARIOUS DISINFECTANTS, INCLUDING TRADITIONAL AND MODIFIED NANO TYPES, AGAINST BRUCELLA MELITENSIS

Shabaan Saber Khalafallah

https://orcid.org/0000-0002-8593-9433

Animal Poultry and Environmental Hygiene, Faculty of Veterinary Medicine, Benha University, Egypt

E-mail Address: aymansobhy2010@yahoo.com

Corresponding author

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Hoda Mohamed Zaki

https://orcid.org/0000-0001-9752-1627

Brucella Department, Animal Health Research Institute, Cairo, Egypt

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Ayman Sobhy Seada

https://orcid.org/0000-0002-1694-823X

Bacteriology Unite, Animal Health Research Institute, Tanta Branch, Egypt

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https://doi.org/10.1142/S1682648523500075Cited by:0 (Source: Crossref)

Abstract

The zoonotic illness brucellosis may spread between humans and animals in a variety of ways. The prevention and management of brucellosis depend heavily on the cleanliness of the areas around animals and the effective eradication of infection from animal housing. In our investigation, we tested a variety of disinfectants against Brucella melitensis to see how well they worked and whether they would work in various environmental settings. In addition to three different types of nano-disinfectants (Dettol with Silver-NPs, Glutaraldehyde with Silver-NPs, and Calcium oxide-NPs), our study included various conventional forms of disinfectants and antiseptics (Virkon $Ⓡ$ S, Cidex, Sodium hypochlorite, Betadine, and Dettol). For estimating the effectiveness of various types of applied disinfectants, reduction rate was employed. The findings indicated that the concentration and length of exposure time of the disinfectants employed, particularly Vircon S, had an impact on their ability to kill bacteria. However, the presence of filthy circumstances and low temperatures considerably reduced the effectiveness of disinfectants, particularly Dettol. On the other hand, nano-disinfectants, particularly glutaraldehyde containing silver nanoparticles, showed better effects than conventional ones. Our research indicated that disinfectants used in everyday life had an impact on Brucella melitensis. However, the presence of filthy environments and low temperatures reduced the bactericidal effectiveness. The impact of nano-disinfectants on Brucella was better.

Keywords:

INTRODUCTION

A Gram-negative Brucella spp. is extensively transmitted to various hosts through a variety of mechanisms.¹ Infection by brucellosis causes orichitis in males of both animals and humans, as well as late-stage premature delivery in pregnant animals.² The Brucella bacterium is eliminated in milk, after parturition or premature delivery, from the uterus or vagina, in foetal membranes, and in the urine of infected animals. Additionally, Brucella may survive in the environment for quite a while depending on environmental factors involving low temperatures, changes in pH, and humidity.³Brucella may thrive in a variety of environments, including dust, drinking water, manure, and slurry. Depending on the appropriate conditions, dirt, meat, dairy products, and aborted foetuses may also retain the bacterium for extended periods of time.⁴ Direct or indirect contact with diseased animals or a polluted environment can result in brucellosis infection.⁵Brucella despite the fact that it can survive in an unclean environment, it is known to be vulnerable to heat treatment, sanitation, and direct sunlight.⁶ Disinfection is a crucial component of both the brucellosis control programme and other initiatives, thus the type of disinfectant to use should be chosen after careful consideration.⁷ Every disinfectant in use has benefits, drawbacks, and a recommended application technique. For instance, gluteraldehyde is a potent disinfectant that is used to clean metals and other heat-sensitive materials, but it is extremely damaging to human skin. Chlorine is an intermediate level disinfectant that is used to clean surfaces in the environment as well as biological objects, machinery, and medical supplies. It is inexpensive and quick acting, but it irritates skin and corrodes metals.⁸ Numerous studies have shown that Brucella species are susceptible to the majority of available disinfectants, including halogens, ethanol, phenol, and formaldehyde, but each type must be assessed to determine the best method of application.¹ New types of nano-based disinfectants could be used to control a variety of infectious bacteria because they can be used to reduce the amount of bacteria in the environment and are effective against resistant organisms like Martha, E. coli, and salmonella spp.^9,10 Additionally, silver nanoparticles had antibacterial effects against a wide range of bacteria and increased the efficacy of other antibacterial agents when combined with it. Silver is known as a powerful antibiotic and has a wide range of industrial applications in healthcare and external medicine.¹¹ The effectiveness of silver nanoparticles against bacteria seemed to be good. Its killing effect may have happened as a result of silver’s bacteriostatic properties. Despite the fact that silver is extremely effective at killing pathogenic bacteria, the toxic product that forms inside bacterial cells that may cause some irritation at the application site.¹² Using of Silver nanoparticles (Ag-NPs) is a significant advancement in nanomedicine for the fight against multi-resistant bacteria. It is advised to add Ag-NPs to anti-bacterial agents to increase their effectiveness because in a laboratory setting, the antibacterial activities of kanamycin, erythromycin, chloramphenicol, and ampicillin were increased in the presence of Ag-NPs against tested bacterial strains.¹³ For the control of pathogens, inorganic nano-metal oxides such as CaO, ZnO, and MgO nanoparticles can be used as anti-microorganism agents. It has an oxidative effect on microbial cell walls. Additionally, it has good environmental stability and penetration power.¹⁴

Our study’s objective is to assess the effectiveness of various disinfectant types against Brucella spp. and to compare conventional and nano-disinfectants in order to determine which is most appropriate for use in brucellosis control programs.

MATERIALS AND METHOD

Preparation of Bacterial suspension of Brucella melitensis¹⁵

The strain Brucella melitensis biovar 3 is endemic in Egypt. The Brucella department of the Animal Health Research Institute in Cairo, Egypt, isolated and typed it from killed serologically positive animals. Prior to use, it was cultured and reactivated for three days. A 37°C incubation period followed its plating onto tryptone soya agar (TSA, Oxoid). Physiological saline was used to dilute a bacterial suspension with an optical density (OD) of $600 = 1.0$ , or roughly 2– $4 \times 1 0^{9}$ (colony forming unit) cfu/mL, and kept until the test.

Disinfectants Suspension Preparation¹⁶

Five types of traditional disinfectants include Potassium peroxy monosulfate (Virkon $Ⓡ$ S), Glutaraldehyde (Cidex), Sodium hypochlorite (Bleach), Povidone iodine (Betadine), and Chloroxylenol (Dettol). Three types of nano disinfectants include Chloroxylenol with silver-NPs, Glutaraldehyde with silver-NPs, and Calcium oxide NPs. All disinfectants were freshly prepared according to the manufacturer’s instructions prior to test, as shown in Table 1.

**Table 1. List of Used Disinfectants and their Recommended Concentrations and Applications.**
Commercial Name	Active Ingredient	Recommended Concentration	Application
Virkon $Ⓡ$ S	Potassium peroxy monosulfate and sodium chloride	1%	Animal house and equipment
Cidex	Glutaraldehyde	2.4%	Equipment
Bleach	Sodium hypochlorite	2g/L	Biological material smooth surfaces
Betadine	Povidone iodine	1%	Skin and mucous membranes
Dettol	Chloroxylenol (phenol)	1%	Skin of workers and skin of animals
Dettol with Silver-NPs	Chloroxylenol and Ag-NPs	1%/100ppm	Animal house and equipment
Glutaraldehyde with silver-NPs	(C5H8O2) andAg-NPs	2.4%/100ppm	Animal house and equipment
Calcium oxide NPs	Cao nanoparticle	100ppm	Animal house and equipment

Calculation of Different Disinfectants’ Minimum Bactericidal Concentration (MBC)¹⁷

Each disinfectant was serially diluted twice with sterile distilled water in test tubes, with 1.9mL of disinfectant in each tube. Test tubes containing the various disinfectant concentrations under investigation (10 folds of manufacture concentrations) received 100L of bacterial suspension (2– $4 \times 1 0^{9}$ cfu/mL), vortexed, and incubated for 20min. Control was sterile distilled water. Following the exposure period, 100L of the bacterial suspension from all disinfectant concentrations was applied to the TSA plates. Following a 72-h incubation period at 37°C, the growth was evaluated, and the minimal inhibitory concentration (MIC) values were noted, as shown in Table 2. Then, to look for any bacterial survival activity, 0.5mL MIC bacterial suspensions were sub-cultured in 4.5mL liquid media without chemicals at 37°C. MBC is the concentration at which all bacteria are completely destroyed. Testing was done in triplicate for each disinfectant dilution.

**Table 2. The MIC and MBC of Various Disinfectants at 37°C.**
Disinfectants	Vircon S	Cidex	Bleach	Betadine	Dettol	Dettol & Silver-NPs	Glutaraldehyde & Silver-NPs	Calcium Oxide NPs
MIC at 37°C	0.0750%	0.125%	0.01%	0.63%	0.250%	0.065%	0.030%	50ppm
MBC	0.088%	0.125%	0.1%	1%	0.9%	0.07%	0.030%	65ppm

The Bactericidal Impact of Disinfectant in Various Environmental Settings¹⁸

We employed the MBC of each disinfectant with saline, soil, and fecal matter to assess the effectiveness of disinfectants under various environmental circumstances. Following that, 20% suspensions of soil and fetal material that had been taken from the animal housing and autoclaved were made and kept until examination. Each test tube had 100L of bacterial suspension added to it after 1.9mL of MBC from each disinfectant had been applied. Then, 2mL of sterile fecal suspension, saline, and dirt were added to each tube. As a negative control, sterile distilled water was employed. Following that, all tubes were held at room temperature (25°C) for varying exposure times: 1, 5, and 10min. For each test tube, a 10-fold dilution was applied before the contents were plated onto TSA media to determine the number of live bacteria.

Chemical Disinfectants’ Bactericidal Effects When Used at Low Temperatures¹⁹

A suspension of bacteria with MBC from each disinfectant, together with saline, soil, and fecal matter, was made as previously described and held on ice (temperature of suspension less than 5°C) for 1, 5, and 15min. The suspension was then incubated at 37°C for 72h, and the reduction rate was assessed.

RESULTS

When compared to saline, all conventional disinfectants had good reduction rates, and their efficacy grew over time. Dettol and Cidex had the lowest reduction rates when organic matters were present (Dettol; 75% and 73%), but its reduction rate fell when these organic materials were present (soil and feces). Cidex; 70% and 69%, respectively, with soil and fecal materials as shown in Fig. 1.

Fig. 1 The effectiveness of traditional disinfectants under various situations and at various periods.

The effectiveness of chloroxylenol and glutaraldehyde following the combination of silver nanoparticles was 96%, 78%, and 77% with saline, soil, and feces, respectively, demonstrating the advancement of nano-disinfectants over traditional varieties. While the impact of calcium-NPs on saline, soil, and feces was 90%, 70%, and 75%, respectively, as shown in Fig. 2.

Fig. 2 Reduction rate of nano-disinfectants in different conditions and different times.

All traditional disinfectants had a lower reduction rate at low temperatures and a slightly lower reduction rate when saltwater was present. While Vircon S (70%, 50%, and 49% with saline, soil, and feces, respectively) and Cidex (53%, 46%, and 43% with saline, soil, and feces, respectively) had the lowest reduction rates while other types had acceptable reduction rates, it substantially dropped with the presence of organic materials, as shown in Fig. 3.

Fig. 3 Reduction percent of colony count at low temperature.

In particular, glutaraldehyde with silver-NPs (90%, 78%, and 88% with saline, soil, and feces, respectively) and Dettol with silver-NPs (84%, 80%, and 80% with saline, soil, and feces, respectively) showed the maximum reduction rates at low temperatures even when organic matter was present, as shown in Fig. 4.

Fig. 4 The reduction rate of nano-disinfectants at low temperature.

DISCUSSION

Brucellosis is a serious zoonotic disease that affects almost all animals and people. It also results in significant losses for the human and animal industries. Since Brucella is present in the secretions of affected animals and has contaminated the area, it is important to practice proper hygiene, including stringent disinfection, to lower the incidence of the disease. In Egypt, particularly in the Nile delta, brucellosis is still endemic and affects many kinds of domestic animals. Additionally, Brucella organisms were isolated from Nile catfish.^20,21

The Efficacy of Traditional Disinfectants Against Brucella melitensis

In our investigation, when administered with saline, all conventional disinfectants exhibited a good decrease rate. Dettol and Cidex had the lowest reduction rates when used in the presence of organic matters, even for extended time periods, as shown in Fig. 1. This was true even when the organic matters (soil and feces) were present. Our findings concur with those of Park and Chen¹⁶ who claimed that povidine-iodine can be employed in brucellosis control programs since it has a positive effect on Brucella microorganisms. Even in the presence of organic materials, alkaline disinfectants such as potassium monopersulphate/sodium dichloroisocyanurate, sodium dichloroisocyanurate, and quaternary ammonium compound exhibit remarkable activity against Brucella spp. According to an analysis of commonly used antiseptics and disinfectants in the veterinary profession against Brucella species, all commonly researched disinfectants had good efficiency, but some kinds required longer contact times or higher concentrations, particularly with organic materials.²² All disinfectants’ reduction rates fell in low temperatures, fell marginally with saline, and fell drastically in the presence of organic materials, especially when employing vircon S and cidex, which had the lowest reduction rates compared to other disinfectants’ higher reduction rates. These findings are consistent with those of McDonnell and Russell,²³ who noted that the bactericidal effect of disinfectants typically rises with contact duration and temperature. Liquid disinfectants, on the other hand, exhibited lower activity or were completely rendered inactive in unclean or cold settings due to a decrease in their response or because organic particles prevented the disinfectant from reaching and coming into touch with the bacterial cell. Our findings are in agreement with Wang et al.,¹⁵ who reported on the use of MBCs of each type to examine the activity of six different types of disinfectants, including quaternary ammonium compounds (QAC), aldehydes, halogens, phenol, and alkaline compounds. According to their findings, all previously tested compounds were effective against Brucella species, particularly when their concentration and the surrounding temperature rose. However, when exposed to organic materials or low temperatures, all of the compounds’ activity dropped with the exception of sodium hypochlorite and sodium hydroxide, which were less affected. When it’s unclean or cold outside, sodium hypochlorite and sodium hydroxide are preferable. Actually, because of their reduced cost and low toxicity, the two disinfectants are frequently used.

The Result of Nano Disinfectants Against Brucella mellitensis

The results of testing several nano-disinfectants to determine their effectiveness against Brucella melitensis were as follows: calcium-NPs had a lesser effect, especially in the presence of organic matter, as shown in Fig. 2, whereas the effects of Dettol and glutaraldehyde were enhanced when mixed with silver-NPs.

Even in the presence of organic materials, nano disinfectants had a good reduction rate at low temperatures, particularly Glutaraldehyde and Dettol with silver-NPs, which had the highest reduction rates, as shown in Fig. 4. That is in agreement with Hossain et al.,¹¹ who claimed that some nano elements can be utilized as disinfectants since they have antibacterial properties and a minimal risk of negative side effects from the disinfection byproducts created by typical disinfection processes.

Our findings are in line with those of Shin et al.²⁴ who claimed that silver nanoparticles may effectively kill a variety of bacteria and act as a disinfectant. Numerous nanomaterials, including carbon nanotubes, Ag, Au, CaO, ZnO, TiO2, chitosan, cationic peptides, and others, have been employed to treat infectious disorders because they exhibit antibacterial properties.²⁵

Nano-disinfectants are very effective against bacteria because they alter their shape, metabolism, and cellular membrane integrity. Due to their high surface/volume ratio, big interior volume, and other distinctive chemical and physical features, nanostructures’ antibacterial activity may be an intriguing topic for research in the near future.²⁶ Additionally, the glutaraldehyde 2.4% disinfection solution that contained 512mg/L of silver nanoparticles completely eliminated all E. coli, S. aureus, and Candida albicans from fabric strips. Furthermore, after being kept at 54°C for 14 days, the stock solution’s germicidal efficiency did not alter considerably, but the inclusion of an organic ingredient reduced it.²⁷

Both calcium oxide nanoparticles and calcium hydroxide-NPs are effective antibacterial agents because they stop bacteria from growing on surfaces where they have been applied.²⁸ According to Stoimenov et al.,²⁹ mono oxide ions such as calcium oxide and magnesium oxide are particularly effective against a variety of Gram positive and Gram negative bacteria as well as spores and remain effective for a long time and in a variety of environmental circumstances.

However, the earlier findings of certain researchers contradict our opinion because nanoparticles cannot be properly employed for disinfection due to some drawbacks like toxicity and potential carcinogenicity to humans and animals. A new generation of germs that are more resistant to disinfectants may also result from it.^12,30

CONCLUSION

Every form of disinfectant that was utilized was successful in killing Brucella melitensis. The effectiveness of disinfectants was influenced by contact duration, concentration, and temperature increases, while it was reduced by the presence of organic materials and at low temperatures. The nano type of disinfectants demonstrated good activity against Brucella melitensis and its efficacy declines to a lower extent with the presence of organic matter and low temperature; therefore, further testing is required to determine its performance and whether it is safe for use in dairy farms.

ORCID

Shabaan Saber Khalafallah https://orcid.org/0000-0002-8593-9433

Hoda Mohamed Zaki https://orcid.org/0000-0001-9752-1627

Ayman Sobhy Seada https://orcid.org/0000-0002-1694-823X

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Received 16 June 2023

Accepted 26 September 2023

Published: 30 November 2023

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This is an Open Access article published by World Scientific Publishing Company. It is distributed under the terms of the Creative Commons Attribution 4.0 (CC BY) License which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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