RESISTANCE OF S. AUREUS ATCC 25923, E. COLI 055K59 No. 3912/41 AND P. AERUGINOSA 27/99 TO THE WASH-DISINFECTANT «MILKODEZ»
Authors: Verkholyuk Mykola , Peleno Ruslan , Turko Iaromyr 
Published: February 2020

The aim of the work – the article presents the results of determining of the resistance of S. aureus ATCC 25923, E. coli 055K59 No. 3912/4 and P. aeruginosa 27/99 test cultures in planktonic form and in biofilm to our developed «Milkodez» acid detergent.

Materials and methods. Microbial biofilms were grown on MPB in 5 cm disposable plastic Petri dishes. To determine the effect of disinfectants on microbial biofilms, 3 Petri dishes with biofilms of each of the test cultures were used. One of the Petri dishes served as control and she had for 15 minutes made 5 cm3 of saline NaCl solution, in the second – 5 cm3 of hot water (t=70±5 °C), and in the third – 5 cm3 of acidic detergent «Milkodez». Microbial biofilms were fixed for 10 min. 96º with ethyl alcohol for 10 min. were stained with a 0.1 % solution of crystalline violet, and the remnants of the unabsorbed paint were removed with phosphate buffer. The biofilm dye was extracted with 96º of ethyl alcohol, which was photocolometrically investigated at 570 nm to establish the density of the formed biofilms. The density of the formed microbial biofilms was considered low in optical density of the extract up to 0.5 units, average – from 0.5 to 1.0 units; and high – over 1.0 units

The resistance of planktonic forms of test cultures of microorganisms to disinfectants was determined in sterile tubes, which made 10 cm3 (t=70±5 °C) of 0.5 % of their working solutions and 0.1 cm3 (1 billion microbial bodies) of the standard test – cultures. The culture was maintained for 15 min. and made ten – fold plantings on IPA in Petri dishes.

Incubation of mesophilic microorganisms was carried out in a thermostat at a temperature of 30 °C, and psychrophilic – 20 °C. After 48 hours the calculation of the growing colonies were carried out. The results were expressed in colony forming units (CFU).

Results. Due to the impact on microbial biofilms formed by the test cultures of S. aureus ATCC 25923, E. coli 055K59 No. 3912/41 and P. aeruginosa 27/99 for 15 min. 0.5 % solution of acid detergent «Milkodez» the optical density of the solutions was respectively 0.64, 0.72, 0.45 units. The results obtained indicate that the melkodez caused a decrease in the biofilm–forming ability of S. aureus ATCC 25923 3.2 times, in E. coli 055K59 No. 3912/41 – 1,7 times and in P. aeruginosa 27/99 – 2.8 times, compared to control. However, the density of one – day microbial biofilms formed by S. aureus ATCC 25923 and E. coli 055K59 No. 3912/41 was medium, and P. aeruginosa 27/99 was low. It has been proven that the «Milkodez» acid detergent developed is more effective than the prototype «Hypracid», since it caused the death of 100 % of planktonic test cultures and the number of S. aureus ATCC 25923, E. coli 055K59 No. 3912/41 and P. aeruginosa 27/99 formed in the biofilm that survived after its application was 2.7, 3.2 and 1.4 times lower, respectively.

Conclusions. It was found that the test cultures were able to form high – density biofilms, since the optical density of the extract in the control was in the range from 1.28 to 2.05 units, which is greater than 1.0 units. Acid wash detergent «Milkodez» for 15 minutes of exposure causes the formation of S. aureus ATCC 25923, E. coli 055K59 No. 3912/41 and P. aeruginosa 27/99 biofilms of low and medium density and reduces their biofilm capacity by 3.2, 1.7 and 2.8 times, respectively. Its use provides the death of 100 % of the planktonic forms of the test cultures under study and reduces their number in the biofilm by 2.7, 3.2 and 1,4 times more, respectively, compared to «Hypracid» detergent.