EFFECT OF DISINFECTANTS BASED ON POTASSIUM PERSULFATE, HYDROGEN PEROXIDE, GLUTARALDEHYDE AND QUATERNARY AMMONIUM COMPOUNDS ON THE GENETIC MATERIAL OF THE PATHOGEN BACTERIA SPECIFIC TO MEAT PROCESSING INDUSTRY ВЛИЯНИЕ ДЕЗИНФИЦИРУЮЩИХ СРЕДСТВ НА ОСНОВЕ ПЕРСУЛЬФАТА КАЛИЯ, ПЕРЕКИСИ ВОДОРОДА, ГЛУТАРАЛЬДЕ

Afonyushkin V.N.,1,2 Tabanyukhov K.A.,1,4 Cherepushkina V.S.,1 Khomenko Yu.S.,1 Tatarchuk O.P.3 1The Institute of the Experimental veterinary of the Siberia and the Far East, Krasnoobsk, Novosibirsk region, Russia 2The Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia 3KRKA PHARMA, Moscow, Russia 4Novosibirsk State Agrarian University, Novosibirsk, Russia EFFECT OF DISINFECTANTS BASED ON POTASSIUM PERSULFATE, HYDROGEN PEROXIDE, GLUTARALDEHYDE AND QUATERNARY AMMONIUM COMPOUNDS ON THE GENETIC MATERIAL OF THE PATHOGEN BACTERIA SPECIFIC TO MEAT PROCESSING INDUSTRY

Based on the study of the data on physico-chemical and biological properties of the disinfectants that are more often used in the agro-industrial enterprises and in the meat industry, it was suggested that the overwhelming majority of disinfectants did not significantly affect the genetic information nor prevent the horizontal gene transfer.The aim of this work was an experimental verification of this hypothesis and search for prospective disinfectants that allow prevention of the horizontal transfer of undesirable genetic information upon disinfection of the industrial equipment in enterprises of the meat industry.

Materials and methods
The research work was conducted in the Sector of Molecular Biology of FGBNU "The Institute of the Experimental Veterinary of the Siberia and the Far East" and the Laboratory of Pharmacogenomics of FGBNU "The Institute of Chemical Biology and Fundamental Medicine" of the Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS) (Novosibirsk).
The following disinfectants were used for the study: Viroshield on the basis of glutaraldehyde and quaternary ammonium compounds (QAC), HPPI on the basis of hydrogen peroxide and Ecocid on the basis of potassium persulfate, which is produced by the LLC KRKA PHARMA.
The following reference strains of the sanitary indicator microorganisms were used: Clostridium perfringens ATCC 13124, Salmonella enterica var typhimurium TA100 (collection of ICBFM SB RAS), E. coli XL Blue with the pBluescript plasmid carrying the ampicillin resistance gene.Isolation of the bacterial DNA was carried out by the standard silica adsorption method; isolation of the plasmid DNA by phenol-chloroform method (Maniatis and Fritch, 1984).
To assess the influence of the disinfectants under investigation on the genetic information of the bacterial pathogens, the disinfectants were added in the concentration up to 0.5% (w/vol.for Ecocid; vol./vol.for Viroshield and HPPI) to the samples of Cl. perfringens DNA (4.9х10⁶GE/ml).The physiological solution was added to the intact control in the same volume.After incubation for 10 min., the reaction mixture was diluted with TE buffer ten times, and then used for performing PCR.The primers and fluorescent probe for detecting the genetic material (chromosomal DNA) of Clostridium perfringens had the following structure: -CPF1 5'-ACATGTTCAGCTGACCGATACT-3' , -CPF2 5'-CACGTGCTCTACCGACTGA-3' , -Taqman probe FAM-CATCGGCTTCTAAAGGCTTAACCGTC-BHQ To detect the concentration of the genetic material (chromosomal DNA) of Salmonella enterica, real-time дезинфицирующие средства, не только уменьшающие микробную обсемененность обрабатываемых поверхностей, но и снижающие вероятность горизонтального переноса нежелательной генетической информации путем разрушения или инактивации ДНК.
Purification of the reaction mixtures from the residues of the disinfectants was carried out using gel-filtration on the micro columns with Bio-Gel P-6 (BioRad).The competent cells of E.coli were transformed with the plasmid using the method of heat shock: 10 µl of the solution of the plasmid DNA (positive control) or the reaction mixture after treatment with the disinfectants were added to 100 µl of the bacterial culture, incubated at 0 °С for 40 min., then spread on the agar medium with ampicillin (agar Luria-Bertani) heated to 37 °С (Maniatis and Fritch, 1984).
All experiments were carried out in four replications.

Results of the experiments
The degree of destruction of the isolated of Cl. perfringens DNA under the influence of the tested disinfectants was assessed according to the change in the threshold cycle C(t), which occurred later upon decreasing a quantity of DNA suitable for amplification (Fig. 1-2, Table 1).
As result of the experiment, it was found that all disinfectants, to one extent or another, change the beginning of the threshold cycle; that is, are able to significantly (28-49 times) reduce the concentration of DNA suitable for PCR.In this regard, the highest activity had Ecocid, which induced the change in the threshold cycle C(t) on average by 2.7, which was the best result among the studied disinfectants.It is necessary to note that in this experiment, it was impossible to achieve the full destruction of DNA as the experimental conditions envisage detection of a comparatively short DNA region with a length of only 200 b.p.On the other hand, the real amount of chromosomal DNA destruction after treatment with Ecicid would be proportional to the genome size; that is, 2000-4000 times more.To clarify the capacity of Ecocid to damage DNA molecules located in the cells of Salmonella enterica, the samples of the isolated DNA of Salmonella enterica and the suspension of viable bacterial cells, which were taken in the same concentration as for DNA isolation, were treated with the disinfectant for 30 min.After purification by gel filtration, the reaction mixture was used for realtime PCR (Fig. 3).
Real-time quantitative PCR is quite a convenient system for assessing effectiveness of disinfectants in regard to a genetic material allowing investigation of a mechanism of this action.As can be seen from the results of the PCR, the samples of the isolated Salmonella enterica DNA were completely inactivated after treatment with Ecocid.The degree of inactivation of the genetic material within the Salmonella cells upon treatment with Ecocid, was assessed by the change in the threshold cycle C(t) (Table 2).
The obtained data on the concentration of the Salmonella enterica DNA after treatment of live bacteria with disinfectant Ecocid suggest the statistically significant (P=0.0096)decrease in the quantity of DNA suitable for amplification (more than 280 times, that is by 98.53%).Thus, the Ecocid action on the Salmonella culture was accompanied not only by death of bacteria but also by DNA destruction.
As the system of DNA reparation in live Salmonella was active, the degree of the destruction of the genetic material within the bacterial cells was less than that upon the action of Ecocid on the isolated DNA.Nevertheless, use of DNA as a matrix for PCR is possible only in the absence of two-stranded disruptions inside a target.In this experiment the amplicon size was 150 b.p.; thus, to полного разрушения ДНК в этом эксперименте достигнуть не возможно, так как условиями опыта предусмотрена детекция сравнительно короткого участка ДНК длиной всего 200 п.н.С другой стороны, реальное количество повреждений в хромосомной ДНК после обработки дезсредством Экоцид будет пропорционально размеру генома -то есть в 2000-4000 раз больше.
Полученные данные о концентрациях ДНК Salmonella enterica после обработки живых бактерий дезсредством Экоцид свидетельствуют о статистически достоверном (P=0,0096) уменьшении количества reduce the concentration of Salmonella DNA detectable by PCR, on average one damage of DNA molecule per each 150 nucleotides is necessary.Taking into account that the Salmonella genome size in about 4.8 mln b.p, it is perfectly evident that after treatment of live bacterial cells with disinfectant Ecocid, the probability of preserving intact chromosomal DNA tends to zero.
Besides the chromosomal DNA, bacteria have nonchromosomal carriers of the genetic information -plasmids.They present two-stranded, more frequently circular DNA molecules and usually contain genes that increase resistance of bacteria to unfavorable external factors, for example, antibiotics, UV radiation and several disinfectants, as well as genes of toxin production and other determinants of virulence.Plasmids are mobile genetic elements, they are replicated autonomously and independently of the chromosomal DNA and a bacterial cell can contain up to several hundreds of plasmid copies.Bacteria can exchange plasmids in conjugation, or acquiring so called competence can absorb plasmids from an environment.Being a circular DNA molecule, plasmids are distinguished by their high resistance in an environment; thus, the highest risk of transfer of undesirable genetic information is associated precisely with plasmids.
To assess the impact of disinfectants on the plasmid DNA, the methods of the genetic engineering were used; namely, transformation of the competent E. coli cells by the pBluescript plasmid.The pBluescript plasmid contains the ampicillin resistance gene and the gene of β-galactosidase in the Lac-operon; therefore, the transformed bacteria on the medium with ampicillin in presence of the chromogenic substrate form blue color colonies (Fig 4).
The intensity of plasmid DNA destruction by the tested disinfectants was determined by the number of the stained E.coli XL Blue colonies after transformation of bacteria both by the isolated plasmids treated with the disinfectants, and by the plasmids within the bacterial cells during the disinfectant exposure.The results of the bacteria transformation by the pBluescript plasmid are presented in Fig. 5 and in Table 3. пригодной к амплификации ДНК в более чем 280 раз, то есть на 98,53%.Таким образом, действие дезсредства Экоцид на культуру сальмонелл сопровождалось не только гибелью бактерий, но и разрушением ДНК.
As can be seen from the table, the tested disinfectants destroy to a variable degree the pBluscript plasmid with the ampicillin resistance gene; thereby, preventing to a variable degree the growth of the transformed bacteria on the medium with this antibiotic.Disinfectant Veroshield on the basis of glutaraldehyde and quaternary ammonium compounds (QAC) showed extremely low effectiveness regarding the plasmid DNA: high numbers of colonies of the transformed bacteria were recorded in the sample.Disinfectant HPPI on the basis of hydrogen peroxide inactivated the plasmids to a greater degree in this experiment; thus, the individual colonies of the transformed bacteria were found.Most probably, insufficient effectiveness of this disinfectant is associated with the special characteristics of action of catalases -the enzymes, which protect bacterial cells from hydrogen peroxide.Catalases are less fitted to inactivation of inorganic peroxides, such as potassium persulfate, which is a constituent of the disinfectant Ecocid.In the samples processed with the disinfectant Ecocid, no colonies of the transformed bacteria were found; therefore, the disinfectant Ecocid demonstrated 100% effectiveness in destruction of the bacterial plasmid DNA.
Unfortunately, the criteria of an impact of disinfectants on the genetic material of the bacterial and viral pathogens specific for the meat industry have not been regulated by law up to the present day.Nevertheless, it is expedient to use disinfectants that allow prevention of the horizontal transfer of undesired genetic information to ensure high level of biological safety of meat products upon disinfection of industrial equipment in enterprises of the meat industry.

Conclusion
The tested disinfectants on the basis of potassium persulfate, hydrogen peroxide, glutaraldehyde and quaternary ammonium compounds (QAC) can reduce the concentration of the chromosomal DNA of Cl. perfringens 28-49 times.
The disinfectant Ecocid destroys the isolated genetic material of Salmonella enterica and with less intensity the chromosomal DNA located within a bacterial cell during treatment.
The disinfectant Ecocid effectively destroys plasmid DNA, both isolated and located within a bacterial cell during treatment.
Use of the disinfectant Ecocid for sanitary treatment of equipment on enterprises of the meat industry reduces the risk of the horizontal gene transfer, thereby, reducing the distribution of the undesirable genetic information such as bacterial antimicrobial resistance genes.