Method for forming a static biofilm on various surfaces to evaluate the effectiveness of disinfectants

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Introduction

Microbial biofilms are the main form of existence of microorganisms in nature [1–3] and represent an important strategy for the survival of microorganisms and colonization of environmental objects [4, 5]. Biofilms are described as microbial communities attached to the surface of a material, formed by bacterial cells embedded in their own extracellular matrix consisting of several types of biopolymers, including extracellular polysaccharides, extracellular DNA, proteins, and lipids [5–8].

The presence of microorganisms in biofilms contributes to their resistance to disinfectants, which is due to the difficulty or inability of disinfectant active pharmaceutical ingredients (APIs) to penetrate the matrix; their binding and inactivation by matrix polymers; the slow rate of bacterial division in biofilms; the presence of metabolically inactive bacterial cells in biofilm that are not particularly susceptible to disinfectants [8–13].

Most biofilms, with the exception of laboratory models, consist of several types of bacteria; there is no single biofilm architecture [14]. However, biofilms have certain common properties, including the presence of gradients of nutrients and metabolic products, relatively high cell density, deposition of extracellular polymeric substances, and pronounced resistance to antimicrobial drugs [15–17]. Studies of the physiology and microecology of biofilms are based on experiments using in vitro models [18].

The best conditions for biofilm growth are a constant supply of nutrients to the environment, for example, in medical urinary catheters, drainage tubes, and oxygen tubes [19]. With the flow of fluid, biofilms develop more effectively and spread more quickly in the environment. Such biofilms, growing on surfaces with a continuous flow of the environment, are called dynamic [19, 20], and dynamic cultivation methods are used to create them in laboratory conditions. Dry biofilms are found on surfaces in rooms, equipment, and devices and static cultivation methods are used to model them [21–23].

Guideline R 4.2.3676-20 “Methods for laboratory research and testing of disinfectants to assess their effectiveness and safety”¹ presents methods for evaluating the effectiveness of disinfectants using only planktonic microorganisms, but the regimes developed in accordance with these methods are not sufficiently effective against microorganisms in a biofilm state.

The method we propose for studying the effectiveness of disinfectants against microorganisms in biofilms on abiotic surfaces will enable the development of disinfection regimes for use in medical organizations, food industry enterprises, etc., with the aim of ensuring the destruction of infectious disease pathogens on abiotic objects in a biofilm state.

The aim of the study is to develop and validate a method for evaluating the effectiveness of disinfectants against microorganisms in a biofilm state on abiotic surfaces.

Materials and methods

The study used the main chemical APIs included in the composition of the disinfectants:

• from the group of oxygen-active compounds — hydrogen peroxide (HP) in concentrations of 3.0; 6.0; 8.0%;
• from the group of chlorine-active compounds — sodium salt of dichloroisocyanuric acid (SDIC) in concentrations of 0.03, 0.06, and 0.10% by active chlorine;
• from the group of quaternary ammonium compounds — alkyl dimethyl benzylammonium chloride (ADBAC) in concentrations of 0.5 and 1.0%.

Working solutions of APIs were prepared immediately before the studies on sterile distilled water at room temperature (20.0 ± 2.0) °C.

Reference strains from the American Type Culture Collection were used as test microorganisms: Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 6538-P.

Biofilms of bacteria were cultivated using a static method on sterile test surfaces made of various materials (plastic, metal, glass, linoleum, tile) measuring 5 × 5 cm, placed at the bottom of Petri dishes. A suspension of daily bacterial cultures prepared using sterile GRM broth at a concentration of 1 × 10⁶ cells per 1 cm³ in an amount of 10 cm³ was applied to the test surface. The surfaces were then placed in a thermostat and incubated at 37°C for 24 hours. After that, the test surfaces covered with biofilm were removed from the Petri dish and washed three times with sterile distilled water to remove planktonic cells. After washing, the test surfaces were placed on the bottom of a sterile Petri dish and dried with the Petri dish slightly open (until completely dry) at 20–22°C and a relative humidity of 40–50%, after which they were treated with a disinfectant solution by wiping. The control test surfaces were treated in the same way as the experimental ones, using sterile tap water instead of disinfectant solution. After the end of the exposure, the dishes with the test surfaces were filled with 10 cm³ of neutralizer solution, then after 10 minutes the test surfaces were removed and rinsed with sterile distilled water, after which they were transferred to sterile Petri dishes.

The effectiveness of disinfectant exposure on formed biofilms was assessed using a quantitative method. For this purpose, 10 cm³ of sterile saline solution was added to Petri dishes with test surfaces and treated with ultrasound for 1 min at 37 kHz in an Elma Ultrasonic 30S ultrasonic bath (Elma). Then, 0.1 cm³ was seeded onto a dense nutrient medium (GRM agar).

Furthermore, to assess the condition of biofilm on the surface of objects before and after DS exposure, a fluorescent staining method was used on an Olympus BX53M fluorescent microscope with a 100× lens. The LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen) was used to stain viable and non-viable cells, and fluorescence was excited by a blue LED with an emission range of 420–480 nm. The recorded emission was in the range of 500–550 nm for viable cells and 620–650 nm for non-viable cells. Ruby dye (FilmTracer SYPRO Ruby Biofilm Matrix Stain solution, Invitrogen) was used to stain the biofilm matrix at excitation wavelengths of 420–480 nm. The recorded radiation was in the range of 610 nm.

The method was validated by calculating the validation parameters: “precision,” “accuracy,” “limit of quantification,” and “stability,” using data from comparative studies [24, 25]. To determine the validation parameter “precision,” the study was conducted in three replicates on different days by different performers, and the coefficient of variation (CV) was calculated. To assess the compliance of the results with the established values, the acceptability criterion “percentage of viable microorganism cells recovered” was used. The “limit of quantification” was established using suspensions of test microorganisms, which were inoculated into the test object. The culture control was the actual number of cells in the working suspension of test microorganisms, for which the CV values were calculated in experiments performed by different operators.

To obtain reliable results, the study was conducted in three replicates and the average CFU count was calculated. The data were processed using the MS Excel statistical software package for Windows. A value of p < 0.05 was considered statistically significant.

Results

The studied cultures of microorganisms P. aeruginosa ATCC 15442 and S. aureus ATCC 6538-P after 24 hours of static cultivation on test surfaces (metal, plastic, linoleum, glass, smooth tile) formed biofilms with varying densities depending on the type of microorganism and the structure of the test surfaces. The densest biofilms formed on rough surfaces compared to smooth ones. The average number of viable cells in the biofilm ranged from (2.5 ± 0.7) × 10³ to (5.7 ± 2.5) × 10⁷ CFU/cm² depending on the type of test surface.

The results of evaluating the effectiveness of DS solutions against S. aureus ATCC 6538-P and P. aeruginosa ATCC 15442 in biofilms cultivated by static method on abiotic surfaces are presented in Table 1 and Table 2.

 

Table 1. Efficacy of disinfectants against S. aureus ATCC 6538-P in biofilms, cultured statically on abiotic surfaces, at 60 min exposure (wiping method)

Disinfection surfaces	Control, CFU/cm2	Concentration of disinfectant solution by API, %	Mean value, CFU/cm2 (М ± σ)	Disinfection effectiveness, % (М ± σ)
Metal	(4.5 ± 2.5) × 106	0.03 SDIC	(3.5 ± 1.2) × 105	92.23 ± 1.05
		0.06 SDIC	(1.9 ± 0.7) × 103	99.56 ± 0.36
		0.1 SDIC	0	100.0
		3.0 HP	(5.5 ± 1.4) × 105	87.44 ± 1.25
		6.0 HP	(1.5 ± 0.6) × 103	99.99 ± 0.01
		8.0 HP	0	100.0
		0.5 ADBAC	(5.6 ± 2.2) × 105	87.56 ± 1.05
		1.0 ADBAC	35 ± 9.5	99.99
Plastic	(1.7 ± 1.1) × 106	0.03 SDIC	(3.7 ± 1.7) × 104	97.82 ± 0.25
		0.06 SDIC	(1.8 ± 0.5) × 103	99.94 ± 0.04
		0.1 SDIC	0	100.0
		3.0 HP	(4.7 ± 2.5) × 105	72.35 ± 1.33
		6.0 HP	(2.4 ± 1.5) × 103	99.85 ± 0.15
		8.0 HP	0	100.0
		0.5 ADBAC	(5.7 ± 2.5) × 104	88.23 ± 1.02
		1.0 ADBAC	0	100.0
Linoleum	(3.7 ± 2.1) × 107	0.03 SDIC	(5.7 ± 2.3) × 106	84.59 ± 1.62
		0.06 SDIC	(2.1 ± 0.7) × 103	99.98 ± 0.01
		0.1 SDIC	0	100.0
		3.0 HP	(5.1 ± 1.5) × 106	86.21 ± 1.32
		6.0 HP	(1.7 ± 0.4) × 105	99.54 ± 0.36
		8.0 HP	0	100.0
		0.5 ADBAC	(4.6 ± 1.1) × 105	98.76 ± 0.45
		1.0 ADBAC	9 ± 5	99.99
Glass	(2.5 ± 1.5) × 105	0.03 SDIC	(1.5 ± 0.8) × 104	90.80 ± 2.55
		0.06 SDIC	(3.7 ± 1.2) × 103	98.52 ± 2.23
		0.1 SDIC	0	100.0
		3.0 HP	(1.3 ± 0.4) × 104	94.82 ± 1.05
		6.0 HP	(2.2 ± 0.3) × 103	99.12 ± 0.43
		8.0 HP	0	100.0
		0.5 ADBAC	(1.9 ± 0.4) × 103	99.24 ± 0.34
		1.0 ADBAC	0	100.0
Smooth tile	(1.5 ± 0.6) × 104	0.03 SDIC	(5.6 ± 1.3) × 102	99.64 ± 0.35
		0.06 SDIC	(2.1 ± 0.3) × 102	99.98 ± 0.01
		0.1 SDIC	0	100.0
		3.0 HP	(2.5 ± 1.7) × 102	93.33 ± 1.25
		6.0 HP	24 ± 9	99.99
		8.0 HP	0	100.0
		0.5 ADBAC	(1.7 ± 0.8) × 103	88.66 ± 0.66
		1.0 ADBAC	0	100.0

 

Table 2. Efficacy of disinfectants against P. aeruginosa ATCC 15442 in biofilms, cultured statically on abiotic surfaces, at 60 min exposure (wiping method)

Disinfection surfaces	Control, CFU/cm2	Concentration of disinfectant solution by API, %	Mean value, CFU/cm2 (М ± σ)	Disinfection effectiveness, % (М ± σ)
Metal	(5.5 ± 1.4) × 106	0.03 SDIC	(3.7 ± 1.2) × 103	98.45 ± 1.34
		0.06 SDIC	(3.1 ± 0.2) × 103	99.96 ± 0.04
		0.1 SDIC	0	100.0
		3.0 HP	(3.5 ± 1.3) × 104	99.36 ± 0.37
		6.0 HP	(1.3 ± 0.4) × 102	99.99 ± 0.01
		8.0 HP	0	100.0
		0.5 ADBAC	(3.5 ± 1.6) × 103	99.82 ± 0.22
		1.0 ADBAC	7 ± 5.5	99.99
Plastic	(3.7 ± 1.5) × 106	0.03 SDIC	(1.8 ± 0.7) × 104	99.13 ± 0.27
		0.06 SDIC	(1.4 ± 0.5) × 103	99.98 ± 0.01
		0.1 SDIC	0	100.0
		3.0 HP	(1.4 ± 0.2) × 105	96.13 ± 0.67
		6.0 HP	(1.2 ± 0.6) × 103	99.98 ± 0.05
		8.0 HP	0	100.0
		0.5 ADBAC	(2.2 ± 1.2) × 103	99.54 ± 1.25
		1.0 ADBAC	11 ± 3.5	99.99
Linoleum	(3.8 ± 2.5) × 107	0.03 SDIC	(4.8 ± 2.2) × 105	91.57 ± 1.55
		0.06 SDIC	(1.9 ± 0.4) × 103	99.73 ± 0.05
		0.1 SDIC	0	100.0
		3.0 HP	(3.7 ± 1.9) × 105	99.35 ± 0.46
		6.0 HP	(4.4 ± 1.7) × 102	99.98 ± 0.01
		8.0 HP	0	100.0
		0.5 ADBAC	(2.1 ± 0.5) × 103	99.95 ± 0.23
		1.0 ADBAC	11 ± 8	99.99
Glass	(2.7 ± 0.6) × 105	0.03 SDIC	(2.5 ± 0.3) × 104	90.74 ± 0.16
		0.06 SDIC	(1.8 ± 0.6) × 103	99.33 ± 0.07
		0.1 SDIC	0	100.0
		3.0 HP	(1.4 ± 0.3) × 103	96.70 ± 0.05
		6.0 HP	(1.5 ± 0.4) × 102	99.94 ± 0.04
		8.0 HP	0	100.0
		0.5 ADBAC	(3.5 ± 0.3) × 103	87.04 ± 0.48
		1.0 ADBAC	15.4 ± 6.6	99.99
Smooth tile	(2.5 ± 0.7) × 103	0.03 SDIC	(6.4 ± 0.8) × 102	97.92 ± 0.25
		0.06 SDIC	(1.1 ± 0.4) × 102	99.56 ± 0.26
		0.1 SDIC	0	100.0
		3.0 HP	(1.4 ± 0.7) × 102	94.21 ± 0.34
		6.0 HP	21 ± 4	99.99
		8.0 HP	0	100.0
		0.5 ADBAC	(1.9 ± 0.7) × 102	94.24 ± 0.45
		1.0 ADBAC	0	100.0

 

The criterion for the effectiveness of disinfectants in surface disinfection is the destruction of at least 99.99% of microorganisms on all types of surfaces in accordance with Appendix 6 “Effectiveness of disinfectants taken into account when organizing disinfection measures” to SanPiN 3.3686-21².

It has been established that the effectiveness of disinfection depends on the structure of the surfaces and the type of contamination.

When disinfecting various types of surfaces contaminated with S. aureus and P. aeruginosa bacteria in a biofilm state, with DHCK solutions at concentrations of 0.03 and 0.06% active chlorine, recommended by the instructions for use for disinfecting surfaces in bactericidal and virucidal modes by wiping with an exposure time of 60 minutes, the disinfection of surfaces was insufficient. Disinfection of 99.99–100.00% of all types of surfaces was achieved only with solutions containing 0.1% active chlorine.

Working solutions of biofilm in concentrations of 3.0 and 6.0% were also insufficiently effective in disinfecting surfaces contaminated with S. aureus and P. aeruginosa bacteria in a biofilm state. To achieve the criterion of effective disinfection of surfaces contaminated with both types of microorganisms in biofilms, it was necessary to increase the concentration of biofilm to 8.0%.

The effectiveness of surface disinfection after exposure to a 0.5% ADBAC solution was not achieved. ADBAC solutions at a concentration of 1.0% provided 99.99–100.00% disinfection of various types of surfaces.

The presence of biofilms on surfaces and the antimicrobial effect of ADBAC were confirmed by microscopic methods (Figs. 1, 2). The results of fluorescent staining were evaluated based on the intensity of the fluorescent signal of microorganisms. Using the LIVE/DEAD BacLight method, two types of cells were observed: cells with green fluorescence — viable cells with intact (intact) cell membranes; cells with red fluorescence — non-viable cells with damaged cell membranes.

 

Fig. 1. BP S. aureus ATCC 6538-P, stained LIVE/DEAD, before (a, c, e) and after (b, d, f) treatment with 0.5% ADBAC on glass surface (a, b), smooth tile (c, d), stained metal (e, f).

 

Fig. 2. P. aeruginosa ATCC 15442 before (a, c, e) and after (b, d, f) treatment with 0.5% ADBAC on glass surfaces (a, b), smooth tiles (c, d), and painted metal (e, f).

 

S. aureus and P. aeruginosa showed the most pronounced biofilm formation on the glass surface. S. aureus also formed dense biofilms on the tile and metal surfaces. At the same time, P. aeruginosa formed weakly expressed biofilms on tiles, and on metal surfaces, P. aeruginosa biofilms were in the initial stage of formation. It is likely that this microorganism requires a longer incubation period to achieve higher density on metal surfaces.

According to microscopy data, S. aureus demonstrated the highest resistance to ADBAC. Despite a decrease in biofilm density and the death of a significant portion of cells, the cellular biomass of S. aureus retained its integrity after treatment. Biofilms of P. aeruginosa on all types of surfaces showed high sensitivity to DS. As a result, there are no signs of structured biofilm in the images, only debris (remnants) of dead cells and isolated viable bacteria are observed.

Additional staining of the biofilm matrix with ruby red confirmed the presence of structured biofilm in the microorganisms under study (Figs. 3, 4).

 

Fig. 3. Biofilm of S. aureus ATCC 6538-P, stained with ruby dye, before (a) and after (b) treatment with 0.5% ADBAC on the glass surface.

 

Fig. 4. Biofilm of P. aeruginosa ATCC 15442, stained with ruby dye, before (a) and after (b) treatment with 0.5% ADBAC on a glass surface.

 

After treating the surfaces with a 0.5% ADBAC solution by wiping for 60 minutes, destruction of the biofilm structures was observed in both strains, but the most pronounced changes were recorded in P. aeruginosa (Fig. 4). S. aureus retained a significant portion of its biomass (Fig. 3), indicating increased resistance to the disinfectant due to dense biofilm formation.

When assessing precision (repeatability), the CV was 5.5–13.4%, which met the requirements of the methodology.

The percentage of viable microbial cells recovered in biofilms after ultrasonic exposure ranged from 75 to 96%, with values within the confidence intervals corresponding to the average values of the research results, confirming the correctness of the chosen methodology.

When assessing the limit of quantitative determination, the CV was 6.4–11.5%.

The stability of the method to controlled changes was confirmed: when using DS from different chemical groups and different types of surfaces. The Fisher criterion was 3.3–8.4%, which corresponded to the maximum permissible values (F_max ≤ 19.0).

Thus, the data obtained during the validation of the method for evaluating the effectiveness of disinfectants against microorganisms in biofilms on abiotic surfaces prove the possibility of its use in laboratories for the development of effective disinfection regimes against biofilm microorganisms.

Discussion

Studies on the effectiveness of disinfectants on planktonic forms of microorganisms do not take into account the high resistance of microorganisms in biofilms. However, the formation of biofilms, for example, on medical devices or surfaces in medical organizations, is a natural state of bacterial life, and disinfection regimes must ensure the death of all types of pathogens under any conditions [26–28].

The developed method differs from the generally accepted one in that surfaces are contaminated with a liquid culture of test microorganisms and cultivated in a thermostat at 37°C for 24 hours. Under these conditions, a static biofilm of moderate density forms on the surface. After incubation, the biofilm on the surfaces is dried, and only then are they treated with a disinfectant solution. The presented cultivation method allows creating conditions that are closest to the practical conditions of biofilm formation on abiotic surfaces [30].

Biofilms of different densities are formed on surfaces made of different materials, since the surface structure determines the key properties of biofilm: porosity, density, charge, sorption and ion exchange properties, hydrophobicity and mechanical stability.

The presence of biofilms on the test surfaces was confirmed using the fluorescent microscopy method with the application of specific dyes. The results of microscopic studies confirmed the formation of microbial communities on all types of surfaces examined. The presence of an extracellular polymer matrix, characteristic of mature biofilms, was reliably established using specific staining with crystal violet dye, which is consistent with data obtained in other studies [31].

Based on the study of the effectiveness of disinfectants from the main groups (sodium dichloroisocyanurate, hydrogen peroxide, ADBAC) against microorganisms in the biofilm state, it has been shown that disinfection regimens using disinfectants at minimally effective concentrations from the main groups of disinfectants, including chlorine-active and oxygen-active compounds, and cationic surfactants, against bacteria in planktonic form [29] are not sufficiently effective for disinfecting various surfaces contaminated with bacteria in the biofilm state. To achieve the required level of surface disinfection, a 99.99% concentration of the API was necessary, which had to be increased 2–3 times: SDIC solutions from 0.03 to 0.1% by active chlorine, surfactants from 3.0 to 8.0%, and the concentration of ADBAC from 0.5 to 1.0%.

Validation data confirms the possibility of using the new method for developing surface disinfection regimens.

Further research is advisable to continue in the direction of selecting the most significant types of surfaces and microorganisms to create the densest biofilm.

Conclusion

The newly developed method for assessing the disinfecting activity of disinfectants against microorganisms present in static biofilms on surfaces will allow for the recommendation of adequate disinfection regimens for practical application, effective against pathogenic and conditionally pathogenic microorganisms. The newly developed method is reflected in MG No. 3.5.1.4130-25 "Assessment of the Effectiveness of Disinfectants Against Microorganisms in Microbial Associations (Biofilms)", approved by Rospotrebnadzor.

 

¹ Guideline R 4.2.3676-20 "Methods of laboratory research and testing of disinfectants to assess their effectiveness and safety" (approved by the Head of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Chief State Sanitary Doctor of the Russian Federation A.Yu. Popova on 12.18.2020).

² Sanitary rules and regulations 3.3686-21 "Sanitary and epidemiological requirements for the prevention of infectious diseases", approved by the Resolution of the Chief State Sanitary Doctor of the Russian Federation dated January 28, 2021 No. 4 (registered by the Ministry of Justice of Russia on February 15, 2021, registration No. 62500).

About the authors

Lyudmila S. Fedorova

Scientific Research Institute for Systems Biology and Medicine

Author for correspondence.
Email: fedorova-ls@yandex.ru
ORCID iD: 0000-0002-3345-2631

D. Sci. (Med.), Professor, Head, Laboratory of overcoming microbial resistance

Russian Federation, Moscow

Anastasia V. Ilyakova

F.F. Erisman Federal Scientific Center of Hygiene

Email: ilyakova.av@fncg.ru
ORCID iD: 0000-0002-1867-3495

researcher, Department of disinfection and sterilization, Institute of disinfectology, Institute of Disinfectology

Russian Federation, Moscow

Alina S. Ivkina

Scientific Research Institute for Systems Biology and Medicine

Email: alinaIvkina21@mail.ru
ORCID iD: 0009-0005-7886-6159

junior researcher, Laboratory of overcoming microbial resistance

Russian Federation, Moscow

Stepan P. Dudik

Scientific Research Institute for Systems Biology and Medicine

Email: stepan_maestro@mail.ru
ORCID iD: 0000-0002-3157-5902

junior researcher, Micro- and nanofluidics laboratory

Russian Federation, Moscow

Elena N. Ilina

Scientific Research Institute for Systems Biology and Medicine

Email: ilinaen@gmail.com
ORCID iD: 0000-0003-0130-5079

D. Sci. (Biol.), Professor, Corresponding Member of the Russian Academy of Sciences, Deputy director for research

Russian Federation, Moscow

References

Supplementary files