RU2621306C1 - Method of estimation of antibacterial action of antiseptics on microbial films - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: evaluating the effect of the antiseptic drug on the microbial biofilm formed by a mixed culture of bacteria, represented by pathogens of infectious complications. To do this, polymicrobial microbial biofilms formed in vitro are subjected to a 60-minute action of the antiseptic, after which two factors are evaluated and compared with the control (without the influence of an antiseptic): the bioavailability of the biofilm (in U OP) and the viability (by the number of CFUs) composition of bacteria. If the UE decreased by 70% and CFU was not detected, the antiseptic was considered highly effective; If the U OP decreased from 30% to 70% and the number of CFU decreased by 4 orders of magnitude or more, the antiseptic was considered to be medium-effective; with a decrease in U OP less than 30% and a decrease in the number of CFUs by less than 4 orders of magnitude, the antiseptic is assessed as being ineffective.

EFFECT: increasing the accuracy and objectivity of the method by evaluating the antibacterial action of the antiseptic on polymicrobial biofilms.

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Description

The invention relates to medicine, namely combustiology, purulent surgery, clinical microbiology, and can be used to evaluate the antibacterial effect of an antiseptic on microbial biofilms formed by several bacteria. A known suspension method for studying the antimicrobial activity of antiseptics (Gudkova E.I., Krasilnikov A.P., Adarchenko, A.A. Test methods for the antimicrobial activity of antiseptics for preventive purposes: guidelines No. 11-13-197, approved by the Ministry of Health of the Republic of Belarus 16.01.97 , Minsk, 1997. 8 pp.), In which the effectiveness of antiseptics is assessed for only monovid planktonic cultures of bacteria. Pure bacterial cultures isolated using standard techniques are used to test antiseptic resistance.

The disadvantages of the prototype: the determination of the antibacterial activity of the antiseptic according to the prototype is carried out in relation to only monovirus culture (one strain) of bacteria, while in the material of surgical, including burn patients, polymicrobial associations of bacteria are more often found.

Effect: increasing the accuracy and objectivity of the method by evaluating the antibacterial effect of an antiseptic on polymicrobial biofilms.

The essence of the method is that they evaluate the effect of an antiseptic drug on a biofilm formed by a mixed culture of bacteria represented by pathogens of infectious complications. For this, polymicrobial biofilms formed in vitro are subjected to a short-term exposure to an antiseptic, after which two indicators are evaluated and compared with a control (without the effect of an antiseptic): the biofilm mass and the viability of its constituent bacteria.

The method is as follows according to O'Toole GF and KolterR. (1998): diurnal cultures of two bacterial strains, for example Pseudomonas aeruginosa and Staphylicoccus aureus, were standardized to 2.0 according to McFarland and diluted 1: 100 in Luria Bertani broth (LB broth). 0.1 ml broth cultures of both types of bacteria are introduced into the wells of a polystyrene 96-well flat-bottomed tablet (Medpolymer, Russia) and closed plates are kept statically in an thermostat at 37 ° C for 20 hours to form biofilms. After planktonic culture was removed and the biofilm was washed 3 times with phosphate-buffered medium (PBS), 200 μl of antiseptic was added to the experimental part of the wells, 0.89% NaCl was added to the control wells, the mixture was incubated for 1 hour and the PBS was washed 3 times. Then 200 μl of a 0.1% aqueous solution of gentian violet is added and incubated for 30 minutes. Biofilm biofilms are evaluated by the level of ethanol extraction of the dye, which is measured on a Benchmark Plus microplate reader (Bio-Rad, USA) at a wavelength of 580 nm in optical density units (U, OD ₅₈₀ ). To assess cell viability in biofilms, the latter are washed 3 times, 100 μl of PBS are added to the wells and sonicated 5 times for 1 min at 37 kHz by placing the plates in an Elma Ultrasonic 30S ultrasonic bath (Elma, Germany). Cell viability is assessed by the number of colony forming units (CFU) after plating from successive decimal dilutions of bacterial suspensions on selective media. Next, averaged indicators of the optical density of the eluate from mixed biofilms treated with an antiseptic are compared with the same in the control. If the indicator characterizing the biofilm bulkness (in units of OD) after exposure to an antiseptic has decreased by 70%) from the control, an antiseptic is highly effective, a decrease of 30-70% is average, less than 30% is low. In addition, assess the number of viable cells (according to the number of CFU) in the biofilm treated with an antiseptic, and compare it with the control. If viable cells are not found, then an antiseptic is highly effective, if the number of cells has decreased by 4 orders of magnitude or more - average efficiency, less than 4 orders of magnitude - low efficiency.

Case Studies

Example 1: The effect of Prontosan ^® on a binary biofilm was evaluated using clinical strains of Pseudomonas aeruginosa and Staphylicoccus aureus isolated from a wound discharge treated in a burn center using the proposed method. For this, a 20-hour mixed biofilm was formed in the wells of the polystyrene plate, after which plankton cells and culture medium were removed, and biofilms were washed 3 times. The formed biofilms were kept with Prontosan ^® for 1 h and the mass of the biofilm and the viability of the cells in its composition were evaluated. Result: Ed OD decreased by 70% and CFU were not detected, the antiseptic is highly effective (see table. 1).

Example 2: The effect of a 0.05% chlorhexidine solution on a binary biofilm was evaluated using clinical strains of Pseudomonas aeruginosa and Staphylicoccus aureus isolated from a wound discharge treated in a burn center, the procedure was performed in a similar way. Result: Unit OD decreased by 60% and the number of CFU decreased by 4 orders of magnitude, the antiseptic is medium effective (see table. 1).

Example 3: The effect of a 0.02% solution of furatsilin on a binary biofilm was evaluated using clinical strains of Pseudomonas aeruginosa and Staphylicoccus aureus isolated from a wound discharge treated in a burn center, the procedure was performed in a similar way. Result: Unit OD decreased by 20% and the number of CFU decreased less than 4 orders of magnitude, the antiseptic was ineffective (see Table 1).

The positive effect of the proposed method is as follows: the method allows to evaluate the effectiveness of the use of an antiseptic to suppress the viability of bacterial cells in mixed biofilms, and also ensures the objectivity of the results.

Claims (1)

A method for evaluating the antibacterial effect of antiseptics on microbial biofilms by exposing the antiseptic to microbial biofilms for no more than 60 minutes, washing the samples, assessing cell viability in the composition of the biofilm by the number of colony forming units (CFU) and comparing with the control, characterized in that they use polymicrobial biofilms, formed by several bacteria, measure the biomass of the polymicrobial biofilm in units of optical density (OD), calculate the viability of the cells in its composition, compare with control, and if Unit OD decreased by 70% and CFU were not detected, antiseptics are considered highly effective, if Unit OD decreased from 30 to 70% and the number of CFU decreased by 4 orders of magnitude or more, the antiseptic is considered medium effective, with a decrease in Unit OD less than 30% and a decrease in the number of CFU by less than 4 orders of magnitude, the antiseptic is rated as low effective.