JP2019195281A - Biofilm suppression and/or removal agent - Google Patents

Abstract

To provide biofilm suppression and/or removal agents that can remove formed biofilms while effectively suppressing various biofilm formation.SOLUTION: Provided is a biofilm suppression and/or removal agent characterized by having at least one of PLA, PLC and PLD. It is a biofilm suppression and/or removal agent having preferably PLA, DNA-degrading enzyme (DNase I) and polysaccharide-degrading enzyme (Dispersin B). It can suppress the formation of biofilms of Staphylococcus aureus, Staphylococcus epidermidis and rot staphylococci and remove the formed biofilm.SELECTED DRAWING: Figure 4

Description

  The present invention relates to a biofilm suppression and / or removal agent that suppresses biofilm formation and / or removes the formed biofilm.

  A biofilm is a structure composed of a matrix of bacteria and the like, which are secreted by bacteria from outside the cells, and an aggregate of the cells. When bacteria adhere and grow, treatment is difficult because they are covered with a matrix, that is, a biofilm is formed to show resistance to the drug, and also escape from the defense mechanism of the living body.

  In recent years, infections related to biofilms (biofilm infections) are increasing with treatment using artificial medical materials such as central venous catheters and artificial joints. Examples of biofilm infections include catheter-related bloodstream infection (CR-BSI) and urethral indwelling catheter-related urinary tract infection (UA-UTI). Once a biofilm infection occurs, it is difficult to control the infection, and medical materials must be removed.

  Staphylococcus aureus is a causative agent for surgical site infection, bloodstream infection, respiratory tract infection, urinary tract infection, etc. in highly infectious patients in medical institutions, and it becomes refractory depending on the infected site and sometimes causes death. It may become. In particular, methicillin-resistant Staphylococcus aureus (MRSA), which is the causative agent of nosocomial infections, is said to be intractable because 48% is due to indwelling catheter and 14% is due to indwelling urinary catheter. At the same time, becoming serious becomes a problem. Thus, biofilm infections are extremely important issues in all clinical departments including surgical departments, and the development of fundamental preventive and therapeutic methods is urgently needed.

  Biofilm research so far has been conducted at the laboratory level, but what kind of bacteria the biofilm is composed of at the infection site and how it relates to the collection site and clinical findings At present, no important basic data is obtained in clinical practice, such as whether or not it has sex (Non-Patent Documents 1 and 2).

  Biofilms have different properties not only at the bacterial species but also at the bacterial strain level. Accurate evaluation of the components that make up the extracellular matrix (ECM) of individual biofilms is an understanding of the molecular mechanism of biofilm formation. It is considered to be important for the planning of a flexible biofilm infectious disease countermeasure adapted to each property (Non-Patent Documents 3, 4, 5, 6, 7).

  In recent years, biofilm formation inhibitors including enzymes have been developed. For example, biofilm formation inhibitors containing origin or basiloricin as an active ingredient have been proposed (Patent Document 1). This biofilm formation inhibitor is effective to some extent in suppressing the formation of biofilms derived from Candida, but does not effectively suppress the formation of various biofilms.

  Moreover, for example, a biofilm formation inhibitor containing hexyl resorcinol at a predetermined concentration has been proposed (Patent Document 2). This biofilm formation inhibitor is effective to some extent in suppressing the formation of biofilms derived from Pseudomonas aeruginosa, but does not effectively suppress the formation of various biofilms. In addition, these biofilm formation inhibitors exhibit only a biofilm formation inhibitory effect, and do not remove the formed biofilm.

Japanese Patent Laid-Open No. 2018-030819 JP 2017-218390 A

Mizunoe Yoshimitsu, Sugimoto Shinya, Iwase Tadayuki: Staphylococcus aureus elimination mechanism by resident bacteria. Respiratory Medicine. 26 (1): 49-52, 2014 Mizunoe Yoshimitsu, Sugimoto Shinya, Okuda Kenichi: Prospects for Biofilm Infectious Disease Control. Journal of Livestock Infectious Diseases. 5 (4): 113-120. 2016 Yoshimitsu Mizue, Akio Chiba, Tadayuki Iwase, Shinya Sugimoto: Separation and analysis of extracellular matrix of biofilm. Chemotherapy area. 31 (11): 2158-2165, 2015 Sugimoto S, Sato F, Miyakawa R, Chiba A, Onodera S, Hori S, Mizunoe Y: Broad impact of extracellular DNA on biofilm formation by clinically isolated Methicillin-resistant and -sensitive strains of Staphylococcus aureus.Sci Rep. 8 (1) : 2254, 2018 Sugimoto S, Okuda K, Miyakawa R, Sato M, Arita-Morioka K, Chiba A, Yamanaka K, Ogura T, Mizunoe Y, Sato C: Imaging of bacterial multicellular behavior in biofilms in liquid by atmospheric scanning electron microscopy.Sci Rep. 6: 25889, 2016 Chiba A, Sugimoto S, Sato F, Hori S, Mizunoe Y: A refined technique for extraction of extracellular matrices from bacterial biofilms and its applicability.Microb Biotechnol. 8 (3): 392-403, 2015. Sugimoto S, Iwamoto T, Takada K, Okuda K, Tajima A, Iwase T, Mizunoe Y: Staphylococcus epidermidis Esp degrades specific proteins associated with Staphylococcus aureus biofilm formation and host-pathogen interaction. J Bacteriol. 195 (8): 1645-55 , 2013.

  The present invention has been made in view of such a problem, and provides a biofilm suppression and / or removal agent capable of effectively suppressing the formation of various biofilms and removing the formed biofilm. For the purpose.

  The biofilm suppressing and / or removing agent according to the present invention contains at least one of PLA (Phospholipase A, phospholipase A), PLC (Phospholipase C, phospholipase C), and PLD (Phospholipase D, phospholipase D). It is characterized by that.

  According to the present invention, the formation of various biofilms can be effectively suppressed, and the formed biofilm can be removed.

It is a figure explaining the cutting site | part of the chemical bond by phospholipase. It is a figure which shows the biofilm formation inhibitory effect by various single-piece | unit enzymes (PLA, PLC, PI-PLC, PLD, Lipase) with respect to the biofilm which MRSA, MSSA, and Staphylococcus epidermidis form. It is a figure which shows the biofilm formation inhibitory effect by various single-piece | unit enzymes (PLA, PLC, PK, DNase, DispB) with respect to the biofilm which MRSA10 strain | stump | stock, MSSA7 strain | stump | stock, and S. epidermidis 4 strain | stump | stock form. It is a figure which shows the biofilm formation inhibitory effect by various cocktail enzymes (PK / DNase / DispB, PLC / DNase / DispB and PLA / DNase / DispB) with respect to the biofilm which MRSA10 strain, MSSA7 strain and S. epidermidis 4 strain form is there. It is an image figure using the confocal laser microscope (CLSM) which shows the biofilm destruction effect by PLA with respect to the biofilm which MRSA11 forms.

  Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating understanding of the principle of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.

  The biofilm suppressing and / or removing agent according to the present invention contains at least one of PLA (Phospholipase A, phospholipase A), PLC (Phospholipase C, phospholipase C), and PLD (Phospholipase D, phospholipase D). .

  Phospholipases are enzymes that hydrolyze phospholipids into fatty acids and other lipophilic substances, and are roughly classified into four types, A, B, C, and D, depending on the type of reaction catalyzed. As shown in the examples described later, PLA that does not exhibit hemolysis is preferable. FIG. 1 is a diagram illustrating a phospholipase cleavage site. As shown in FIG. 1, PLA1 cleaves SN-1 acyl group, PLA2 cleaves SN-2 acyl group to produce arachidonic acid, PLD cleaves phosphate ester bond, phosphatidic acid and alcohol Is generated. In the biofilm suppressing and / or removing agent according to this embodiment, PLA may be either PLA1 or PLA2, or a mixture thereof, but preferably, PLA is PLA1.

  In the present invention, the suppression of biofilm means not only the growth of biofilm but also the prevention of biofilm adhesion and the prevention of deposition. The removal of the biofilm formed in the present invention means the destruction of the attached biofilm and the suppression, killing, and removal of bacteria in the biofilm.

  The origin of the biofilm on which the biofilm suppression and / or removal agent according to this embodiment acts is not particularly limited, but the biofilm suppression and / or removal agent according to this embodiment is a gram belonging to Staphylococcus. In particular, it has inhibitory activity against those formed by positive cocci. The biofilm suppressing and / or removing agent according to the present embodiment is preferably S. aureus, S. epidermidis or S. saprophyticus among the genus Staphylococcus. Effectively suppresses biofilm formation. The biofilm suppressing and / or removing agent according to the present embodiment is effective for inhibiting biofilm formation from Staphylococcus aureus, MRSA, MSSA and vancomycin-resistant Staphylococcus aureus (VRSA). Works.

  The biofilm suppressing and / or removing agent according to this embodiment can further include a DNA degrading enzyme (DNase I) and a polysaccharide degrading enzyme (Dispersin B).

  DNA degrading enzyme (DNase I) is an endonuclease that non-specifically degrades DNA to produce dinucleotides, trinucleotides, and oligonucleotides having 5′-phosphate groups and 3′-hydroxyl group ends. Polysaccharide-degrading enzyme (Dispersin B) is an enzyme that cleaves the glucoside bond of various polysaccharides by hydrolysis to produce constituent monosaccharides or oligosaccharides (sugars with several monosaccharides bonded) or their derivatives. It is.

  A substance that fills cells in a biofilm is a matrix, and examples of matrix components include extracellular nucleic acids called eDNA (extracellular DNA), proteins, and polysaccharides. Proteins as matrix components include not only biofilm-related proteins and host cell adhesion factors, but also numerous cytoplasmic proteins. As polysaccharides as matrix components, in Pseudomonas aeruginosa, polysaccharides mainly composed of mannose, galactose or glucose synthesized by psl (polysaccharide synthesis locus) or pel (pellicle formation locus) gene products are the key to biofilm formation. However, in Gram-negative bacteria and Staphylococcus aureus, PNAG (poly-β (1, 6) -N-acetyl-D-glucosamine), also called PIA (polysaccharide intercellular adhesin), is known.

  The main component of the matrix differs depending on the bacterial species that forms the biofilm.For example, in MRSA, eDNA is the main component, in MSSA, protein is the main component, and in Staphylococcus epidermidis, PIA is the main component. The biofilm suppressing and / or removing agent according to the embodiment can further effectively suppress the formation of various biofilms by further comprising a DNA degrading enzyme (DNase I) and a polysaccharide degrading enzyme (Dispersin B). It becomes.

Therefore, the biofilm inhibitor according to the present embodiment has the following configuration, for example.
Biofilm suppression and / or removal agent with PLA Biofilm suppression and / or removal agent with PLC Biofilm suppression and / or removal agent with PLD Biofilm suppression and / or removal agent with PLA and PLC -Biofilm inhibition and / or removal agent with PLA and PLD-Biofilm inhibition and / or removal agent with PLC and PLD-Biofilm inhibition and / or removal agent with PLA, PLC and PLD-PLA, DNase I and Biofilm Suppression and / or Removal Agent with Dispersin B ・ PLC, Biofilm Suppression and / or Removal Agent with DNase I and Dispersin B ・ Biofilm Suppression and / or Removal Agent with PLA, DNase I and Dispersin B ・ PLA Biofilm suppression and / or removal agent with PLC, DNase I and Dispersin B ・ Biofilm suppression with PLA, PLD, DNase I and Dispersin B and Or removers · PLC, PLD, DNase I and biofilm inhibition and / or removal agents · PLA having Dispersin B, PLC, PLD, DNase I and biofilm inhibition and / or removing agent having Dispersin B

  In the biofilm suppression and / or removal agent according to this embodiment, although not particularly limited, for example, PLA 20-200 μg / mL, PLC 20-200 μg / mL, PLD 100-1,000 U / mL, DNase I 10-100 U / mL, Dispersin B 2-20 μg / mL.

  In the biofilm suppression and / or removal agent having PLA, DNase I and Dispersin B, the mixing ratio of each enzyme is not particularly limited. For example, PLA 20-200 μg / mL, DNase I 10-100 U / mL and Dispersin B 2-20 μg / mL.

  In the biofilm suppression and / or removal agent having PLC, DNase I and Dispersin B, the mixing ratio of each enzyme is not particularly limited. For example, PLC 20-200 μg / mL, DNase I 10-100 U / mL and Dispersin B 2-20 μg / mL.

  In the biofilm suppression and / or removal agent having PLD, DNase I and Dispersin B, the mixing ratio of each enzyme is not particularly limited. For example, PLD 100 to 1,000 U / mL, DNase I 10 to 100 U / mL mL and Dispersin B 2-20 μg / mL.

  The medical instrument according to this embodiment is a medical instrument processed with the biofilm suppression and / or removal agent, for example, a medical device, a medical dressing, a medical tape, or the like. A medical device treated with a biofilm suppressing and / or removing agent is a part of or the entire surface of the medical device coated with a biofilm suppressing and / or removing agent, or a medical device. The biofilm suppression and / or removal agent penetrates partly or entirely.

  The medical device is, for example, an implant. Examples of implants include, but are not limited to, for example, catheters, artificial teeth (dental implants), bolts for fixing bones in the treatment of fractures, rheumatism, pacemakers, artificial heart valves, artificial joints, voice prostheses, contact lenses, etc. Is mentioned. Examples of the catheter include a urinary catheter, an abdominal catheter, and a central venous catheter.

  In addition, the biofilm suppressing and / or removing agent according to the present embodiment includes an antiseptic effective for bacteria capable of forming a biofilm, for example, chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, an amphoteric interface, if necessary. An activator, alcohol, sodium hypochlorite, povidone iodine, glutaral and the like can be used in combination. In addition, chlorhexidine is effective in terms of a sustained effect on staphylococci.

1. Strains, enzymes and reagents The following strains were used.
-Staphylococcus aureus clinical isolates: 10 methicillin-resistant Staphylococcus aureus (MRSA) strains and 7 methicillin-sensitive Staphylococcus aureus (MSSA) isolates from patients at Jikei University Hospital: Staphylococcus epidermidis clinical isolates: at Jikei University Hospital Four strains of Staphylococcus epidermidis isolated from patients The following enzymes were used.
・ PLA: Phospholipase A ₁ from Thermomyces lanuginosus (Sigma-Aldrich, Cat #: L3295)
・ PLC: Phospholipase C from Clostridium perfringens (Sigma-Aldrich, Cat #: P4039)
PI-PLC: Phosphatidylinositol-specific phospholipase C from Bacillus cereus (Sigma-Aldrich, Cat #: P5542)
・ PLD: Phospholipase D from Streptomyces chromofuscus (Sigma-Aldrich, Cat #: P0065)
・ Lipase from Candida rugosa (Sigma-Aldrich, Cat #: L1754)
・ DNase: DNase I recombinant, RNase-free from Bovine pancreas, expressed in Pichia pastoris (Roche, Cat #: 4716728001)
DispB: Dispersin B (beta-1,6-N-acetylglucosaminidase) from Aggregatibacter actinomycetemcomitans (Kane Biotech)
・ PK: Proteinase K from Tritirachium album (Sigma-Aldrich, Cat #: P6556)
The following reagents were used.
Brain heart infusion (BHI) medium (Becton Dickinson, Cat #: 211065): Dissolved in deionized water to 37 g / L, autoclaved at 121 ° C for 15 minutes and sterilized.
-BHIG medium: BHI medium containing 1% glucose.
・ Glucose (Nacalai Tesque, Cat #: 16805-35)
Crystal violet (CV): 50 mg of CV was weighed, dissolved in 1 mL of ethanol, and diluted to 500 mL with sterilized water.
・ Phosphate buffered saline (PBS)
・ Sterile water

2. Biofilm formation inhibition test
(a) Staphylococcal clinical isolates were inoculated into 2 mL of BHI medium sterilized from a glycerol stock using a sterile toothpick, and cultured overnight at 37 ° C. (shaking speed: 150 rpm).

  (b) The overnight culture was diluted 1,000 times in a sterilized BHIG medium.

  (c) Dispense 200 μL of diluted bacterial solution into a sterilized 96-well polystyrene plate per well, and use various enzymes (200 μg / mL PLA, 200 μg / mL PLC, 200 μg / mL PI-PLC, 1,000 U / mL PLD, 200 μg / mL Lipase, 100 μg / mL PK, 100 U / mL DNase I, 20 μg / mL Dispersin B) alone or in various combinations.

  (d) The culture was stationary at 37 ° C for 24 hours.

  (e) The culture solution and the floating bacteria were sucked up using an aspirator, and the hole of the plate on which the biofilm was formed was washed with 150 μL of PBS.

  (f) The washing solution was sucked using an aspirator, 0.05% CV was added at 150 μL per well, and staining was performed at 25 ° C. for about 3 minutes.

  (g) The staining solution was sucked using an aspirator, and the plate hole on which the biofilm was formed was washed with 150 μL of PBS.

  (h) The absorbance (595 nm) of the biofilm formed on the plastic plate was measured using a plate reader Infinite F200 Pro.

  FIG. 2 is a diagram showing the biofilm formation inhibitory effect of various simple enzymes (PLA, PLC, PI-PLC, PLD, Lipase) on biofilms formed by MRSA, MSSA, and Staphylococcus epidermidis. PLD was excellent in the formation-inhibiting effect on biofilms formed by MSSA and Staphylococcus epidermidis, but showed hemolytic properties. PLA does not show hemolysis, and further has an excellent formation inhibitory effect on biofilms formed by MRSA and MSSA, but the formation inhibitory effect on biofilms formed by Staphylococcus epidermidis was almost the same as the control. The results in FIG. 2 show that the main components of the biofilm matrix formed by MRSA, MSSA, and Staphylococcus epidermidis are different, and thus there are variations in the biofilm formation inhibitory effect of various enzymes.

  FIG. 3 is a diagram showing the biofilm formation inhibitory effect of various simple enzymes (PLA, PLC, PK, DNase, DispB) on biofilms formed by MRSA10 strain, MSSA7 strain and 4 S. epidermidis strains. The results in FIG. 3 show that there are variations in the effect of inhibiting biofilm formation by various enzymes in each strain of MRSA (FIG. 3A), MSSA (FIG. 3B), and Staphylococcus epidermidis (FIG. 3C).

  FIG. 4 is a diagram showing biofilm formation inhibitory effects of various cocktail enzymes on biofilms formed by MRSA10 strain (FIG. 4A), MSSA7 strain (FIG. 4B), and 4 S. epidermidis strains (FIG. 4C). The cocktail enzyme was a mixed enzyme of PK / DNase / DispB, a mixed enzyme of PLC / DNase / DispB, and a mixed enzyme of PLA / DNase / DispB. PK / DNase / DispB was PK 100 μg / mL, DNase I 100 U / mL, and Dispersin B 20 μg / mL. PLC / DNase / DispB was PLC 200 μg / mL, DNase I 100 U / mL, and Dispersin B 20 μg / mL. PLA / DNase / DispB was PLA 200 μg / mL, DNase I 100 U / mL, and Dispersin B 20 μg / mL. The mixed enzyme of PLC / DNase / DispB was excellent in the formation inhibitory effect on the biofilm formed by MRSA10 strain, MSSA7 strain and 4 strains of S. epidermidis. The mixed enzyme of PLA / DNase / DispB was superior to the mixed enzyme of PLC / DNase / DispB in that the formation inhibitory effect on the biofilm formed by MRSA10 strain, MSSA7 strain and 4 S. epidermidis strains was superior.

3. Observation of biofilm using confocal laser microscope (CLSM)
(a) Bacteria were inoculated into 2 mL of sterilized BHI medium using a toothpick sterilized from a glycerol stock of MRSA No. 11 strain (MR11 strain) and cultured overnight at 37 ° C (shaking speed: 150) rpm).

  (b) The overnight culture was diluted 1,000 times in a sterilized BHIG medium.

  (c) 2 mL of the diluted bacterial solution was dispensed into a sterilized glass bottom culture dish (Matsunami Glass Industrial Co., Ltd., Cat #: D111300), followed by stationary culture at 37 ° C for 24 hours.

  (d) Biofilm was destroyed by adding 200 μg / mL PLA to the biofilm culture and incubating at 37 ° C. for 2 hours.

  (e) The culture solution and the floating bacteria were sucked up using an aspirator and washed twice with 2 mL of acetate buffer (pH 5.0).

  (f) 2 mL of 4% paraformaldehyde / PBS was added, and the biofilm was fixed at room temperature for 10 minutes.

  (g) The fixative was sucked using an aspirator and washed 3 times with 2 mL of acetate buffer (pH 5.0).

(h) Phospholipids (cell membrane, etc.) contained in the biofilm were stained at room temperature for 30 minutes using FilmTracer ^™ FM ^™ 1-43 Green Biofilm Cell Stain (Themo Fisher Scientific, Cat #: F10317).

  (i) The staining solution was sucked using an aspirator and washed 3 times with 2 mL of sterile water.

(j) 2 mL of sterilized water was added, and 2 drops of NucBlue ^™ Fixed Cell ReadyProbes ^™ Reagent (Themo Fisher Scientific, Cat #: R37606) was added dropwise to stain the nucleic acid.

  (k) The biofilm was observed using a confocal laser microscope LSM-880 (Carl Zeiss).

  FIG. 5 is an image diagram using a confocal laser microscope (CLSM) showing the biofilm destruction effect of PLA on the biofilm formed by MR11. The biofilm destruction effect by PLA can be visually confirmed.

  It can be used to suppress and remove biofilm formation.

Claims (10)

  A biofilm inhibitor and / or remover comprising at least one of PLA, PLC and PLD.   The biofilm suppressing and / or removing agent according to claim 1, wherein the PLA is PLA1.   The biofilm inhibiting and / or removing agent according to claim 1 or 2, further comprising a DNA degrading enzyme (DNase I) and a polysaccharide degrading enzyme (Dispersin B).   The biofilm suppression and / or removal agent according to claim 3, comprising PLA, DNA-degrading enzyme (DNase I), and polysaccharide-degrading enzyme (Dispersin B).   The biofilm suppression and / or removal agent according to claim 3, comprising a PLC, a DNA-degrading enzyme (DNase I), and a polysaccharide-degrading enzyme (Dispersin B).   The biofilm suppression and / or removal agent according to any one of claims 1 to 5, wherein the biofilm is derived from a Gram-positive cocci belonging to the genus Staphylococcus.   The biofilm suppression and / or removal agent according to claim 6, wherein the Gram-positive cocci belonging to the genus Staphylococcus is any one selected from Staphylococcus aureus, S. epidermidis and rot staphylococci.   The biofilm suppression and / or removal agent according to claim 7, wherein the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), or vancomycin-resistant Staphylococcus aureus (VRSA).   The medical instrument processed with the biofilm suppression and / or removal agent of any one of Claims 1 thru | or 8.   The medical device processed with the biofilm suppression and / or removal agent is coated with the biofilm suppression and / or removal agent on the surface of the medical device, or the medical device is biofilm suppressed and / or removed. Alternatively, the medical device according to claim 9, wherein the removing agent is permeated.