COMPARATIVE STUDY OF ANTIBACTERIAL ACTIVITY OF PEROXYDISUCCINIC ACID, HYDROGEN PEROXIDE AND THEIR MIXTURE

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 sporeforming
bacteria. However, in the recent years certain
decrease of background sensitivity of microorganisms to
hydrogen peroxide and occurrence of resistant strains of
pathogenic microorganisms to 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). Materials and methods. The substances of
peroxydisuccinic acid (PDSA) and monoperoxysuccinic
acid (MPSA) were prepared by well known methods. The
following test-strains were used to assess antimicrobial
activity of the agents: Staphylococcus aureus АТСС
25923, Escherichia coli АТСС 25922, Pseudomonas
aeruginosa АТСС 27853, Pseudomonas aeruginosa
АТСС 9027, Basillus сereus АТСС 10702, Basillus
сereus АТСС 96, Basillus subtilis АТСС 6633, Proteus
vulgaris ATCC 4636, Candida albicans АТСС 885/653,
and Candida albicans 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 25922 test-strains for 60 min with
3% (and even 6% Н2О2 for E. coli) does not cause total
death of microorganisms. The fact of resistance of sporeforming
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. aureus and E. сoli, and
decreased the number of viable cells of B. cereus sporeforming
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 death Conclusions. 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.