RU2737417C1 - Method for controlling bacterial biofilms - Google Patents

Abstract

FIELD: medicine; veterinary.

SUBSTANCE: invention relates to a method for controlling bacterial biofilms and can be used in medicine and veterinary science. Method of controlling bacterial biofilms is to provide a substrate of plastic, transparent for radiation of subsequently used laser; metal layer of silver or copper of submicron thickness is applied on one side of said substrate; applying said substrate by applying a metal layer on said bacterial biofilm; scanning said metal layer through said substrate by radiation pulses of said laser with pulse energy of 0.2 mJ, thereby providing an application transfer of substance of said metal layer in the form of nanoparticles on said bacterial biofilm.

EFFECT: invention provides simplified use and prevention of direct effect of laser radiation on body tissues at high efficiency.

3 cl, 1 tbl, 2 dwg

Description

The technical field to which the invention relates

This invention relates to a method for controlling bacterial biofilms and can be used in medicine and veterinary medicine.

State of the art

G.A. et al. Biofilm formation as microbial development // Ann Rev Microbiol 2000. P. 49-79; Hunt S.M. et al. Hypothesis for the role of nutrient starvation in biofilm detachment // Appl Environ Microbiol. 2004. P. 7418-25; Романова Ю.М. и др. «Биопленки патогенных бактерий и их роль в хронизации инфекционного процесса. Поиск средств борьбы с биопленками» // ВРАМН. 2011. №10. С. 31-39). Сложная инфраструктура и иерархия бактерий в бактериальной биопленке, формирование ею специальных средств жизнеобеспечения и защиты в виде матрикса приводят к тому, что биопленки становятся практически неуязвимыми для антибиотиков.The most widely used drugs in the fight against microorganisms are antibiotics. However, their activity is decreasing every year. Pathogens capable of forming bacterial communities or biofilms are especially resistant to the action of antibacterial drugs. It was found that organized communities of pathogenic bacteria with a complex structure can form on almost any surface and are the cause of many problems, including medical (

GA et al. Biofilm formation as microbial development // Ann Rev Microbiol 2000. P. 49-79; Hunt SM et al. Hypothesis for the role of nutrient starvation in biofilm detachment // Appl Environ Microbiol. 2004. P. 7418-25; Romanova Yu.M. et al. “Biofilms of pathogenic bacteria and their role in the chronicity of the infectious process. Search for means of combating biofilms "// VRAMN. 2011. No. 10. S. 31-39). The complex infrastructure and hierarchy of bacteria in the bacterial biofilm, the formation of special means of life support and protection in the form of a matrix lead to the fact that biofilms become practically invulnerable to antibiotics.

The growing resistance of bacteria to existing drugs and the lack of structures that could potentially form the basis of new antibiotics have put on the agenda the search for alternative ways to combat pathogenic microorganisms.

The known method of photodynamic therapy for inactivation of bacteria and biofilms, which uses cationic purpurinimide as a photosensitizer for photodynamic inactivation of bacterial biofilms (RF patent No. 2565450, publ. 20.10.2015). The disadvantages of this method of photodynamic therapy include the low level of photostability of the composition used, which leads to a short shelf life and to significant restrictions during therapy. Another limitation when using the above composition is the low level of effectiveness of therapy of foci of bacterial lesions due to the low level of bioavailability of molecules. In addition, the solutions of chemical agents proposed for application use have significant chemical activity, which leads to their accelerated withdrawal or inactivation.

Also known are chemical and microbiological methods for inactivating biofilms with chemical agents by direct application of their solutions - a mixture of enzymes (RF application No. 2009106069, publ. 27.08.2010), bacteriophage (RF patent No. 2565824, publ. 20.10.2015; RF patent No. 2646102, publ. 03/01/2018), as well as bacterial strains (RF patent No. 2576008, publ. 02.27.2016), a complex of antimicrobial insect peptides (RF patent No. 2664708, publ. 08.21.2018; RF patent No. 2699712, publ. 09.09.2019) , one or more unsaturated aliphatic long-chain alcohols or aldehydes of a certain formula (RU 2012126070), sypros (RF application No. 2014145270, publ. 10.107111.2016), silver ions in the composition (RF patent No. 2553363, publ. 10.06.2015). The disadvantage of these methods is their relatively short-term effect (due to metabolism), the need to repeat, but avoid overdose. The use of bacteriophages in the fight against biofilms is gaining popularity every year. But to date, no reliable results have been obtained that show the effectiveness of their application. The bacteriophage is highly specific, i.e. acts only against one type of bacteria and is not effective against others. It has been experimentally proven that the use of low concentrations of phage particles or the use of phages against bacteria that exhibit resistance to phages stimulates the formation of biofilms. Isolation and / or production of genetically modified phages, the action of which will be directed to the destruction of biofilms, is a very complex, expensive and difficult task. The action of enzymes is mainly aimed at the destruction of the matrix (for example, DNase). At the same time, the bacteria remain viable and may well form the biomatrix anew and form a new focus of infection.

There is a known method for preventing the formation of biofilms on a substrate on which particles with local plasmon resonance (copper, silver, gold, semiconductors, metal oxides) with a density of 1-100 particles / μm ^{2 are} applied (RF patent No. 2650376, publ. 11.04.2018). It is assumed that when illuminating the surface, absorption and heating of nanoparticles will prevent the attachment of the microorganism to the surface, inhibition of biofilm formation, and / or destruction of the already formed biofilm. The disadvantages of this method lie in the removal of nanoparticles due to metabolism, oxidation and physicochemical processes accompanying heating in biosystems (boiling, cavitation, flotation, etc.).

There is also known a method of destroying biofilms by direct exposure to radiation of a femtosecond laser (patent of Ukraine No. 104321, 01/27/2014), which involves prolonged (10-20 minutes) high-intensity irradiation with ultraviolet laser pulses of varying power and wavelength. The disadvantages of this method include possible damage to healthy cells by intense ultraviolet laser radiation - up to DNA damage and mutations.

It is possible to detach the biofilm from the surface on which it was located under the action of laser radiation in the liquid layer (Japanese application No. 2004-275979, publ. 07.10.2004). Although the principle of operation is not disclosed, it can be assumed that in this case shock waves are generated that tear off the biofilm from the surface (Song, WD, Hong, MN, Lukyanchuk, B., & Chong, TS (2004). Laser -induced cavitation bubbles for cleaning of solid surfaces.Journal of applied physics 95 (6), 2952-2956). This method with certain assumptions (in terms of mechanical strength) is applicable to abiotic surfaces, but on the surface of tissues it can cause destruction of healthy cells and blood microvessels (Shen, N., Datta, D., Schaffer, S.V., LeDuc, P. , Ingber, DE, & Mazur, E. (2005) Ablation of cytoskeletal filaments and mitochondria in live cells using a femtosecond laser nanoscissor Mech Chem Biosyst 2 (1) 17-25).

The closest analogue is a method of destroying biofilms by laser radiation using a composition containing silver (publication of the international application WO 2014/089552, publ. 12.06.2014). The destruction of the biofilm on the surface of the wound is assumed under the action of a local shock wave generated by nanosecond laser radiation (wavelength 1064 nm) in the layer of the silver-containing composition on the surface of the biofilm and pressing the bactericidal composition deep into the wound under the action of successive laser pulses. The main disadvantage of the method is the direct laser effect on tissues, as well as the complexity of optimal focusing, which allows, under the action of a shock wave, to ensure the destruction of the biofilm and transport the composition into the interior, but at the same time to avoid the destruction of components and whole cells of a healthy tissue, as well as blood microvessels.

Disclosure of invention

The objective of the present invention is to develop such a method for combating bacterial biofilms that would overcome the disadvantages of the closest analogue, providing a technical result in the form of simplification of use and prevention of direct exposure to laser radiation on body tissues with high efficiency.

To solve this problem and achieve the specified technical result, the present invention proposes a method for combating bacterial biofilms, which consists in the fact that: provide a substrate made of plastic, transparent for the radiation of the laser used subsequently; a submicron-thick metal layer is applied to one side of the substrate; the substrate is applied with a deposited metal layer to the bacterial biofilm; scanning the metal layer through the substrate with laser pulses, resulting in the application transfer of the substance of the metal layer in the form of nanoparticles to the bacterial biofilm.

A feature of the method according to the present invention is that silver or copper can be chosen as the material for the metal layer.

Another feature of the method according to the present invention is that the metal layer can be made of at least two sublayers of different metals.

Another feature of the method according to the present invention is that a laser of the visible or near infrared range with pulse durations from units to hundreds of nanoseconds can be used.

Brief Description of Drawings

The present invention is illustrated in the accompanying drawings.

FIG. 1 shows a schematic diagram explaining the principle of application laser transfer of nanoparticles from a transparent substrate onto biofilms of pathogenic microorganisms.

FIG. 2 shows images of the microbiological results of the application of the method according to the present invention.

Detailed Description of Embodiments

The method according to the present invention is carried out as follows. A metal layer of submicron thickness is applied to one side of the plastic substrate. The plastic of the substrate transmits the radiation of the subsequently used laser, which is a laser of the visible or near infrared range with a pulse duration from units to hundreds of nanoseconds. Silver or copper is chosen as the metal, and two or more subcoats of different metals can be applied.

The support with the applied metal layer is applied to the biofilm so that the metal layer contacts the biofilm. After that, the metal layer is scanned through the substrate with laser pulses, as a result of which the application transfer of the substance from the metal layer in the form of nanoparticles to the bacterial biofilm is provided. This is shown schematically in FIG. one.

The method according to the present invention was tested in the following experiment. Overnight broth culture is diluted 100 times with nutrient medium and transferred into 24 well plates, 1 ml. Sterile slides of the same size are placed in each well of the plate. Then incubated for 24 hours in a thermostat at 37 ° C. A fairly dense layer of biofilm forms on the surface of the glasses over a 24-hour period. Then, the formed biofilm is coated on both sides with a layer of nanoparticles for a few seconds. Silver and copper can be used as the nanoparticle material. Application transfer is carried out by scanning the substrate with a layer of nanoparticles with a nanosecond IR laser (pulse duration 120 nm, wavelength 1064 nm) with a pulse energy of 0.2 mJ and a pulse repetition rate of 20 kHz when focusing with a lens with a focal length of 163 mm. After transferring the particles, the glasses are transferred into test tubes with a physiological solution of DNase and vigorously shaken on a shaker for 1 hour. Under the influence of DNase, the biofilm matrix is destroyed, but the bacterial cells remain unharmed. Then the resulting suspension is titrated by a standard microbiological method and to determine the CFU (colony-forming unit) is sown on a solid nutrient medium.

Experimental studies have shown that the use of the method of application laser transfer of silver and copper nanoparticles is a highly effective method in the fight against biofilms formed on a solid substrate. At the same time, in a control experiment with a plastic substrate without a metal layer, it was shown that this effect is not associated with the action of the laser itself.

FIG. 2 illustrates what has been said. The left and right photographs refer, respectively, to the data from the study of colloidal inactivation of biofilms Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Staphylococcus aureus (Staphylococcus aureus) using silver, copper and chemically inert gold nanoparticles, as well as a control experiment with laser exposure without nanoparticles. Light gray corresponds to red staining with Live / dead dye for dead bacteria, dark gray color corresponds to green staining with Live / dead dye of live bacteria.

The quantitative data of the studies performed are given in the attached table showing the effect of application laser transfer of nanoparticles of various materials (with control by similar laser action through a plastic substrate without a metal layer) on the formed biofilms and their CFU / ml values.

Thus, in the proposed method, a transparent plastic substrate allows the use of relatively low-intensity pulsed laser radiation for transferring nanoparticles from a metal layer, which weakly heats these nanoparticles. This method uses a laser with radiation pulses in the visible and near-IR ranges, having a nanosecond duration, well absorbed by the metal layer and extremely insignificantly transparent substrate. In this case, copper and silver particles with a high bactericidal effect are used, while chemically inert gold nanoparticles show an insignificant effect of inactivating biofilms.

All this indicates that the proposed method of combating bacterial biofilms, having a high efficiency, provides a technical result in the form of simplifying the application and preventing the direct effect of laser radiation on body tissues.

Claims (7)

1. A way to combat bacterial biofilms, which consists in the fact that: - provide a substrate of plastic, transparent to the radiation of the subsequently used laser; - applying on one side of said substrate a metal layer of silver or copper of submicron thickness; - imposing said substrate with an applied metal layer on said bacterial biofilm; - scanning said metal layer through said substrate with radiation pulses of said laser with a pulse energy of 0.2 mJ, thereby providing application transfer of the substance of said metal layer in the form of nanoparticles to said bacterial biofilm. 2. A method according to claim 1 or 2, wherein said metal layer is made of at least two sublayers of different metals. 3. The method according to claim 1, in which a laser is used in the visible or near infrared range with a pulse duration from units to hundreds of nanoseconds.

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