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22veterinary sciences
Soltys M. P.1, Gunchak V. M.1, Rudyk2G. V., GutyjB. V.,
Brezvin2O. M., Vasiv R. 0.1
1Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies, Lviv, Ukraine 2State Scientific-Research Control Institute of Veterinary Medicinal Products and Feed Additives, Lviv, Ukraine DOI: 10.24412/2520-2480-2020-3082-11-16 TO ASSESS THE BIOCIDAL ACTION OF THE DRUG BASED ON SODIUM HYPOCHLORITE.
Abstract
The article presents data on the antiseptic and disinfectant effect of the drug «Vitosept». The peculiarity of this biocidal product is that the active substance in it is high-purity sodium hypochlorite, obtained in a specially designed diaphragm-free flow electrolyzer using isotonic sodium chlorite solution prepared in water purified by a separate technology.
In the course of the conducted researches it was found out that the investigated remedy has the expressed bactericidal, fungicidal and sporocidal properties. Its high bactericidal activity against E. coli, S. aureus, P. aeruginosa has been established. There was a decrease in the number of microorganisms by 6.5-7.5 lg in the first 30 minutes of contact both in the presence of organic matter (5% bovine albumin - B.S.A.) and in its absence.
Resistance of A. niger to «Vitosept», in the presence of high organic load (5% of bovine albumin-B.S.A.) allows to recommend a differentiated approach to the mode of disinfection in mycosis foci; to wipe twice with an interval of 15 minutes, and when immersed in 60 minutes to replace « Vitosept» and keep the items in solution for 30 minutes. It was confirmed that when disinfecting objects contaminated with fungi, it is necessary to use higher concentrations of the studied drug and increase the duration of contact, in particular, the concentration should be maintained at 600 mg /1 for more than three hours. The use of lower concentrations is not appropriate, because it requires a long period ofaction and does not guarantee the desired result.
Increasing the temperature of« Vitosept» to 50 ° C and keeping it at this level for 30 minutes dramatically increases the sporicidal activity of the drug. In this case, the time required for sterilization is reduced several times. However, given the technical difficulties associated with heating, the use of the temperature factor can be considered as one ofthe possible options.
Key words: biocides, sodium hypochlorite, bactericidal, fungicidal, sporocidal action.
Introduction
Recently, there has been considerable interest in the problem of bacterial resistance to antiseptics and disinfectants, combined under the term "biocides" [1-4].
Providing the population with ecologically safe livestock products is an urgent problem. Toxic substances, in pure form or in various combinations, accumulate in livestock products, which is dangerous for human consumption. Based on the above, an important aspect of improving the technology of veterinary preventive measures is the introduction into veterinary practice of new tools and methods of prevention and treatment of animals. In recent years, drugs based on sodium hypochlorite solution (sodium hypochlorite) have been increasingly used in medicine and veterinary medicine, as all higher multicellular organisms, including humans, synthesize hypochlorous acid and highly active oxygen-containing compounds in special cellular structures to neutralize foreign microorganisms and toxic substances [5, 6].
The pharmacodynamics of oxygen-containing drugs is reduced to the oxidation of bacterial protein structures and is associated with the ability to bind to amino groups of proteins and hydrogen in tissues and disrupt their properties. Hydrochloric and hydrochloric acids in aqueous solutions dissociate with the formation of cations and ions, and the strength of their antiseptic, astringent, anti-inflammatory and antitoxic effects depends on the amount of cations formed. Reacting with
living tissues, they cause dehydration of the protein with the formation of albuminates and due to changes in pH and dehydration of bacterial cells have a strong antimicrobial effect [7]. Sodium hypochlorite as a low molecular weight compound is a generator of active oxygen, which freely penetrates cell membranes and, accordingly, oxidizes toxins that are not only in the blood but also in tissues [8, 9].
The advantage of sodium hypochlorite over other antiseptics is the ease of obtaining, relative cheapness, no contraindications, complications, side effects and resistance of microorganisms; wide range of antimicrobial action, etc. [10, 11]. The described characteristics of drugs with the inclusion of sodium hypochlorite in their composition are determined, as a rule, by different methods of electrochemical production of this active substance [12-14].
The question of finding and developing new antiseptics and disinfectants remains relevant, because absolutely ideal drugs with a pronounced bactericidal effect simply do not exist, and the requirements for such biocides are constantly growing.
We used the drug «Vitosept» for the study, which is based on a solution of high-purity sodium hypo-chlorite, which is obtained in a specially designed diaphragm-free flow electrolyzer, in the process of direct electrochemical reaction, bypassing the formation of molecular chlorine. Isotonic sodium chloride solution
(0.9% NaCl) prepared in water purified by a special technology was used as the starting electrolyte. At the same time, it was important to find out the bactericidal, sporocidal and fungicidal properties of the drug «Vi-tosept», which was the purpose of our further research.
Materials and methods.
In the process of microbiological studies as test cultures used strains of microorganisms: to study the bactericidal activity - E. coli (ATCC 25922),
S. aureus (ATCC 6583), P. aeruginosa (ATCC 9027), to determine the fungicidal activity - C. albicans (ATCC 885-653) and A. niger (ATCC 16404); sporocidal effect was investigated in the model of B. subtilis (ATCC 6633). These microorganisms were used in a final concentration of 105-107 column-forming units per ml (CFU / ml) (571% / ml). A. niger was grown at t 36 ° C for five days, and B. subtilis - at 30 ° C for nine days, until the formation of about 80% of spores. The latter were washed three times in physiological sodium chloride solution by centrifugation.
The bacteriostatic concentration of the drug «Vi-tosept» was determined by serial dilutions in a liquid nutrient medium. After the introduction of the test culture in the serial dilutions of the test tool was incubated in a thermostat at a temperature of 37 ° ± 0.5 ° C for 1824 hours. The results of the studies were calculated on the absence of visible growth of microorganisms in test tubes.
To assess the antimicrobial activity of the drug «Vitosept» in vitro used the method of diffusion into agar. For this purpose, 0.1 ml of test culture at a concentration of 1 billion cells per 1 ml of saline was inoculated onto the dried surface of meat-peptone agar poured into Petri dishes. The cups were again dried in a thermostat for 20 minutes Stainless metal metal cylinders were placed on the surface of the contaminated medium, after which 0.2 ml of the test solution was introduced into the cylinder. The plates were incubated at 37 ° C for 24 hours. At the end of the incubation, the zones of growth inhibition of test cultures in mm were evaluated. All experiments with test cultures were performed in 5 replicates and then calculated the average (M), error (m) and error probability (P). Other test cultures were also used to determine the antagonistic activity of the test drugs, in particular Klebsiella ozaenae, Streptococcus pyogenes, Enterococcus faecalis, Staph-ylococcus aureus, Staphylococcus epidermidis, Esherichia coli, Proteus vulgaris, Pseudicophyphyas a Caucasus.
The disinfecting properties of «Vitosept» were investigated by suspension method by introducing microorganisms into appropriate concentrations of the drug with and without organic load.
Bovine albumin(B.S.A.) at a concentration of 5% was used as organic contamination of objects; 0.3%; 0.03%. For the purpose of disinfection, «Vitosept» was used at room temperature (18 ° C) at a concentration of 650 mg / l. Samples to study the effect of disinfection were taken after 30, 60, 120, 180 and 240 minutes. contact.
The sterilization process was studied in the model of B. subtilis in four stages:
1) disinfection of the infected object;
2) mechanical cleaning in disinfectant solution from organic pollution; 3) washing in saline;
4) sterilization with «Vitosept».
Sterilization was investigated using rubber products based on rubber, glass, stainless steel, linoleum. The test microorganism was applied to the objects at a concentration of 106 CFU / ml (6 1g) with 0.3% B.A. After drying of the microbial culture, the experimental objects were filled with disinfectant solution for 30 minutes and the control ones with physiological NaCl solution. After 30 min, the items were washed from the organic load in disinfectant and saline. The presence of B. subtilis was determined in the last wash water. Then the test items were filled with a fresh solution of «Vi-tosept». After 30 min, the contact determined the presence of B. subtilis in the disinfectant solution. Finally, the items were removed, washed with sterile tap water and immersed in thioglycol medium and Saburo medium under sterile conditions.
The main volume of research concerned the determination of the disinfectant effect of «Vitosept» by the method of immersion. The bactericidal effect on gramnegative microorganisms was studied in models of E. coli and P. aeruginosa. The initial concentration of cultures was 1x107 - 3x107 x (7-7.48 lg) and 6.2 x 106 -7.7 x 106 x (6.79 -6.89 lg) CFU / ml, respectively. The initial concentration of S. aureus was 4.2 x106 - 9.9 x 106 CFU / ml (6.62 - 7.0 lg) .
Research results.
Characteristics of bacteriostatic activity of microorganisms under the conditions of application of the drug «Vitosept» are given in Table 1, 2.
Table 1.
Bacteriostatic concentration of the drug «Vitosept» in relation to the studied strains of microorganisms
Concentration, mg/l Cultures of microorganisms
E. coli St. aureus P. aeruginosa B. subtilis C. albicans
525 — — — — —
250 — — — — —
130 — — — — —
125 + — + + +
65 + + + + +
Note in this and the following tables *: + - growth of microorganisms; — - no growth of microorganisms
As can be seen from the data in table. 1, bacteriostatic concentration of the drug «Vitosept» against E. coli, St. aureus, P. aeruginosa, B. subtilis, C. albicans was 525; 250 and 130 mg / l. According to the analysis of the microbial spectrum, it can be stated that it is effective on both gram-positive and gram-negative microorganisms. In addition, its fungiostatic action at a concentration of 130 mg / l is important against C. albicans.
At a concentration of 125 mg / l, the drug «Vitosept» showed a clear antiseptic effect against Staphylococcus aureus 209. After studying the biochemical properties of Staphylococcus aureus 209, it was found that under the action of the study drug cultures lose or reduce the activity of pathogenic enzymes, in particular lecithinase, hemolytic and DNA activity were inhibited.
Table 2.
Bactericidal action of the drug «Vitosept» depending on the duration of contact, lg
Test-culture Duration of con 30 min tact, min / Concentrati 60 min on of «Vitosept», m 120 min g / l Culture control
650 320 650 320 650 320
E. coli — — — — — — 7,0
S. aureus — — — — — — 6,62
P. aeruginosa — — — — — — 6,89
С. albicans — — — — — — 6,32
and 320 mg / l after 30 min of contact with microorganisms showed 100% bactericidal action (> 7 lg).
The results of the bactericidal action of the drug «Vitosept» under conditions of organic load 5% B.S.A. are presented in Table 3.
Table 3.
Bactericidal action of the drug «Vitosept» depending on the duration of contact and protein load (5% __B.S.A.), lg_
Test-culture Duration of contact, min / Concentration of «Vitosept», mg / l
30 min 650 60 min 650 120 min 650 Culture control
E. coli — — — 7,48
S. aureus — — — 7,0
P. aeruginosa — — — 6,79
С. albicans — — — 6,59
Regarding the effectiveness of the antiseptic effect of «Vitosept» depending on other factors, in particular the duration of exposure (Table 2), we confirmed that the drug at a concentration of sodium hypochlorite 650
pend on the presence in the study environment of significant organic contamination - 5% B.S.A. Fungi of the genus Candida were not detected after 30 minutes of contact with the drug.
The study of the fungicidal activity of the drug «Vitosept» was also conducted on a culture of A. niger, as this fungus has a higher resistance to phys-icochemical factors, including disinfectants. In the table. Figure 4 shows the results obtained with a protein load at the level of 5% E.A, which differed significantly from the results of the drug «Vitosept» on S. albicans.
Table 4.
Fungicidal effect of the drug «Vitosept» on the test culture of the fungus A. niger, depending on the duration _of contact, concentration and protein load (5% B.S.A.), lg_
Concentration, mg / l Contact duration, min
30 60 120 Culture control
650 3,7 3,48 2,95 6,57
320 4,48 4,53 4,2
160 5,11 4,99 4,8
It was found that the bactericidal effect of «Vitosept» against E. coli, S. aureus, P. aeruginosa and C. albicans was not reduced with the introduction of protein load. The obtained result with respect to 100% death of microorganisms indicates that even with such a sufficiently high organic pollution, the residual oxygen was in a concentration that was sufficient for complete disinfection of the object from these microorganisms.
It was found that the study drug had a high fun-gicidal activity against C. albicans, which did not de-
Under the action of the drug «Vitosept» at a concentration of 650 mg / l after 3 hours of contact, the number of A. niger decreased by almost 3.5 lg (4.1 x 103), at a concentration of 320 mg / l - by 2.37 lg (2, 3 x 102) and a concentration of 160 mg / l - 1.77 lg (5.9 x102).
Therefore, in our opinion, when disinfecting objects contaminated with fungi, it is necessary to use higher concentrations of the studied drug and increase the duration of contact. In our case, it should be a concentration of 600 mg / l for a period of more than three hours. The use of lower concentrations is not appropriate, because it requires a
long period of action and does not guarantee the «Vitosept» 1% ethanol, because according to the achievement of the desired result. data, the latter enhances the disinfectant effect of
In connection with obtaining these results, we hypochlorites. used a technological technique - adding to the drug
Table 5.
Fungicidal effect of the drug «Vitosept» on the test culture of the fungus A. niger depending on the duration of
contact, concentration and protein load
Test-culture Concentration of «Vitosept», mg / l Concentration of B.S.A., % Duration of contact, min Culture control
30 60 120
A. niger 600 + 1% ethanol 5,0 3,4 3,43 3,0 5,4
0,3 — — —
The obtained research results are given in table. 5 did not confirm the existing hypothesis. The disinfecting effect of «Vitosept» remained at the same level. For a more in-depth study of the effect
of the study drug on fungus, which include A. niger, the organic load of B.S.A. up to 0.3%, as required by European and other international standards. The results obtained are given in Table 6.
Table 6.
Fungicidal effect of the drug «Vitosept» on the test culture of the fungus A. niger depending on the duration of
Test-culture Concentration of «Vi- Concentration of Duration of contact, min Culture
tosept», mg / l B.S.A., % 30 60 120 control
600 0,3 — — —
A.niger 320 0,3 — — — 6,42
600 0 — — —
320 0 — — —
As the results show, reducing the protein load to 0.3% gave a 100% disinfection effect (> 6.42 lg). The same table shows the results on the effect of the drug «Vitosept» on A. niger in the absence of organic contamination. The effect of fungicidal action is 100% (> 6.42 lg).
Therefore, based on the results of the studies, it can be concluded that the drug «Vitosept» at a concentration of 600 and 320 mg / l shows a fairly high
fungicidal activity against fungi, both in the absence of organic contamination and in its presence (0.3% BSA .), which is most common in the practice of veterinary medicine.
The study of the sporocidal effect of the drug «Vitosept» was performed on a model of spores of B. subtilis. The results of the research are presented in Table. 7.
Table 7.
Sporocidal effect of the drug «Vitosept» on the culture of B. subtilis depending on the duration of contact,
As the results show, reducing the protein load to 0.3% gave a 100% disinfection effect (> 6.42 lg). The same table shows the results on the effect of the drug «Vi-tosept» on A. niger in the absence of organic contamination. The effect of fungicidal action is 100% (> 6.42 lg).
Therefore, based on the results of the studies, it can be concluded that the drug «Vitosept» at a concentration of 600 and 320 mg / l shows a fairly high
fungicidal activity against fungi, both in the absence of organic contamination and in its presence (0.3% BSA .), which is most common in the practice of veterinary medicine.
The study of the sporocidal effect of the drug «Vitosept» was performed on a model of spores of B. subtilis. The results of the research are presented in Table. 7.
Table 7.
Sporocidal effect of the drug «Vitosept» on the culture of B. subtilis depending on the duration of contact,
Test-culture Concentration of «Vitosept», mg / l Concentration of B.S.A., % Duration o 30 ' contact, m 60 in 120 240 Culture control
B. subtilis 650 0,3 3,08 3,01 2,82 2,02 5,89
320 0,3 3,85 3,45 3,07 2,38
650 0 2,6 2,08 1,18 0,18
320 0 2,93 2,95 1,66 0,55
The obtained research results indicate that the drug «Vitosept» has a sporicidal effect. In particular, at a concentration of 650 mg / l, the number of spores decreased by 5.71 lg, and 320 mg / l - by 5.24 lg.
It is known that increasing the temperature of disinfectant solutions leads to the activation of oxi-dative processes, as well as to a decrease in interfacial tension. Therefore, one of the methods of activating the action of disinfectants is to increase their temperature. Table 8 shows the results obtained by heating «Vitosept» to 50 ° C.
Table 8.
Sporocidal effect of the drug «Vitosept», under conditions of heating to 50 ° C, on B. subtilis depending on the
Concentration of Concentration of «Vi- Duration of contact, min Culture control
B.S.A., % tosept», mg / l 30 60 120 180 240
0 650 3,94 — — — —
320 1,95 — — — —
0,03% 650 3,15 3,45 3,30 3,04 3,1 6,3
320 3,42 3,3 3,05 2,9 2,81
0,3% 650 3,26 3,2 2,96 2,75 2,62
320 3,13 3,0 2,6 2,4 2,3
As the results show (Table 8), in the absence of organic contamination, the effect of the heated drug «Vitosept» for one hour was sufficient to achieve a 100% disinfection effect. The increase in temperature of the studied solution of sodium hypochlorite increased the sporocidal activity of the drug «Vitosept» much less. It should be noted that the lack of the expected effect, to some extent, could be provoked by a sufficiently rapid decrease in temperature
Table 9.
Sporocidal effect of the drug «Vitosept» on spores of culture of B. subtilis, under conditions of heating through-
due to the small volume of the solution of the study drug and organic load.
The obtained result was the reason for the application of a more severe temperature regime - heating of the drug «Vitosept» to 50 ° C during the entire period of contact - 4 hours. The results of the research are presented in Table. 9.
Test-culture Concentration of «Vitosept», mg / l Concentration of B.S.A., % Duration of contact, min Culture control
30 60 120
B. subtilis 650 0,3 — — — 5,38
320 0,3 — — —
650 0 — — —
320 0 — — —
Therefore, under conditions of heating the drug «Vitosept» throughout the experiment to 50 ° C, disinfection from spores of B. subtilis at the level of 100% was detected during the first sampling - after 30 minutes. No further changes were observed.
One option to study sporocidal activity was to reduce the microbial load to 2.8 lg (6.4 x 102 CFU / ml). The results of these studies are presented in Table 10.
Table 10.
The effect of sporocidal action of the drug «Vitosept», under conditions of slight contamination with
Concentration of Concentration of «Vitosept», Duration of contact, min Culture control
B.S.A., % mg / l 30 60 120 180 240
0,3% 650 320 650 — — — — — 2,81
0,03%
320 — — — — —
They confirm the ability of «Vitosept», in the presence of protein load, to disinfect the object from spore-forming microorganisms at the level of 2.8 lg.
The results of the study of the dynamics of the concentration of sodium hypochlorite, under the conditions of the introduction of organic load - 0.3% and 0.03% bovine albumin, are presented in table 11.
Table 11.
Dynamics of sodium hypochlorite in the drug «Vitosept», under conditions of different concentrations of
Concentration of B.S.A., % Duration of contact/Concentration of sodium hypochlorite, mg / l
Output In the process +B. А. 10 min. 30 min. 20 hours
0,3% 475 340 264 216 173
0,03% 475 394 351 326 212
The process of sodium hypochlorite binding was centration of bovine albumin of 0.3%, the level of so-found to be prolonged over time. In particular, at a con- dium hypochlorite after 30 minutes decreased to 216
mg/l, which is twice lower than the initial concentration. At 0.03% B.S.A. concentration, the amount of sodium hypochlorite decreased 1.5 times (to 326 mg/l), despite the difference in albumin concentrations of 10 times. Comparative analysis of the obtained results indicated the absence of a linear relationship between the amount of organic load and oxidation. It can be assumed that the peculiarity of this process to some extent influenced the above results of sporocidal activity of «Vitosept» against B. subtilis, when there was no direct relationship between sodium hypochlorite concentration, organic load and the amount of sporocidal activity.
Confirmation of the developed modes of efficiency of disinfection of surfaces we carried out on linoleum, tile and faience. E. coli, S. aureus, P. aeruginosa, C. albicans, A. niger were applied to an area of 100 cm2 at a concentration of 2 x 106 - 8 x 106 CFU/ml (6.3-6.9 lg) with a load of bovine albumin. 5%.
The calculated concentration was expressed as 10 CFU / cm area (~ 41g). After the surfaces dried, they were wiped with a gauze napkin soaked in «Vitosept». The consumption of the drug was ~ 5 ml per 100 cm2, and the contact period was 30 and 60 minutes. After this period, the surface was applied a neutralizer - 0.5% solution of sodium hyposulfite. After 5 minutes, samples were taken by wiping the surfaces with sterile cotton swabs. Tampons were immersed in appropriate nutrient media. The results were recorded by visual observation (turbidity of the medium) and seeding on dense nutrient media. From 90 cases of observation in 5 cases (5.5%) A.niger and other spore-forming species of microorganisms were isolated at a contact period of 30 minutes. This gives grounds to recommend in the foci of mycoses to carry out double wiping of surfaces with the drug «Vitosept» at a concentration of 0.05% with an interval of 15 minutes.
Conclusions
1. The drug «Vitosept» has an effective bacterio-static effect against E. coli, P. aeruginosa, B. subtilis, C. albicans at a concentration of sodium hypochlorite 130 mg / liter. For S.aureus, a similar effect is achieved at a dose of 125 mg / l.
2. The biocidal effect of effective disinfection of objects contaminated with fungi (A. niger) is manifested at a concentration of the drug at 600 mg / l and increasing the duration of contact to 3 hours or more.
3. The sporocidal effect of «Vitosept» is manifested at concentrations of 320 and 600 mg / l, but the drug must be heated to 50 ° C and the drug has a contact time of at least 4 hours.
Prospects for further research. Establishing the possibility of using the drug «Vitosept» as a means for sterilization of objects and products for artificial insemination, obstetrics and gynecology and surgery.
Acknowledgments
This scientific work was financially supported by the Ministry of Education and Science of Ukraine (0120U101999)
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