Сomparative study of antibacterial activity of peroxydisuccinic acid, hydrogen peroxide and their mixture

Abstract

Introduction. It is known that reactive oxygen species (ROS) generated in vivo by cell aerobic metabolism cause multiple damage in different cell organelles and kill not only obligate anaerobes and microaerophilles, but also aerobes. ROS generated by phagocytes and representatives of normal microflora are an important component of macroorganism defense from most pathogens, which is explained by their ability to damage different biological structures. ROS have high reactivity and let us use them in vitro as effective biocides. Hydrogen peroxide is widely used in many industries, in particular, in medicine and veterinary as antiseptic and disinfectant agent due to its safety for environment and broad spectrum of antimicrobial activity including spore-forming bacteria.However, in the recent years certain decrease of background sensitivity of microorganisms to hydrogen peroxide and occurrence of resistant strains of pathogenic microorganismsto this agent has been noted.

The aim of this work is to carry out a comparative study of antimicrobial activity of hydrogen peroxide, peroxydisuccinic acid (PDSA), monoperoxysuccinic acid (MPSA), and mixture of PDSA and hydrogen peroxide (Н₂О₂). Materialsandmethods. The substances of peroxydisuccinic acid (PDSA) and monoperoxysuccinic acid (MPSA) were prepared by well known methods. Thefollowingtest-strainswereusedtoassessantimicrobialactivityoftheagents: Staphylococcusaureus АТСС 25923, Escherichiacoli АТСС 25922, Pseudomonasaeruginosa АТСС 27853, Pseudomonasaeruginosa АТСС 9027, Basillusсereus АТСС 10702, Basillusсereus АТСС 96, Basillussubtilis АТСС 6633, ProteusvulgarisATCC 4636, Candidaalbicans АТСС 885/653, andCandidaalbicans АТСС 10231.All disinfectant agents were diluted in distilled water at 40 ºС and stirred. The microbial burden was 2∙109 CFU/ml of the medium, and for kinetic studies 105 CFU/ml of the medium, it was standardizing according to McFarland standard. Microorganisms’ resistance to disinfectant agents was determined by seeding of the diluted cell suspension on agar medium after their incubation with MPSA, PDSA, Н₂О₂, and also after MPSA generation in the reaction between PDSA and Н₂О₂, and seeding of the cell suspension with exclusion of these factors was taken as control. 18-24-hour microorganism culture was taken into work. Mueller-Hinton agar and MPA were used for bacteria. Sabouraud agar was used for Candida albicans. Agar well diffusion method was applied. Determination of antimicrobial activity of the agents were carried out on two layers of solid medium in Petri dishes. In kinetic study of microorganism’s death rate due to incubation period with peroxide solution tested, the bactericidal action was stopped with 5 % sodium thiosulfate sterile solution. All studies were carried out in 3 replicates. In statistical analysis methods, significance level P was equal to 0.95. Student’s test was used for statistical processing of the data obtained. Results and discussion. It has been found that treatment of S.aureus АТСС 25923, E.coli АТСС 25922test-strains for 60 min with 3% (and even 6% Н₂О₂forE.coli)does not cause total death of microorganisms. The fact of resistance of spore-forming cultures B. cereus, P.aeruginosa and C.albicans to hydrogen peroxide should be noted, in 60 min of their incubation with 6% Н₂О₂, the number of viable cells decreased from 5.0 to 1.58 lg CFU/ml, as well as to 3.00 (to 1.84 for 120-min exposition) and 1.65 lg CFU/ml, respectively. However, pretreatment of test-stains by 5-fold diluted mixture of 3% Н₂О₂ and 1% PDSA for 60 min caused total death of S.aureusandE.сoli, and decreased the number of viable cells of B. cereusspore-forming cells and relatively resistant to hydrogen peroxide cultures of P.aeruginosa и C.albicans from 5.00 to 1.34 and 0.95 lg CFU/ml, respectively, and in 120 min to their total deathConclusions. The study have determined that antimicrobial effect of reactive oxygen species (ROS) produced by interaction of hydrogen peroxide and peroxydisuccinic acid (PDSA) mixture in the synergistic system is many times higher and faster achievable than hydrogen peroxide or peroxydisuccinic acid only. We have determined the principles of optimum conditions for ROS generation by choice of respective concentrations of peroxydisuccinic acid and hydrogen peroxide. ROS demonstrate high biocidal activity and short lifetime, which allows us to consider the combination of hydrogen peroxide and peroxydisuccinic acid as a promising means for development of a highly efficient disinfectant with a broad spectrum of antimicrobial activity and without a tendency to accumulate in the environment.