# Patent

## Title
Biofilm formation inhibitor composition

## Abstract

A drug and a composition for inhibiting biofilm formation are provided. A biofilm formation inhibitor composition containing the following component (A): (A) at least one or more selected from compounds represented by Formula (1) to Formula (4): wherein R1 to R5 each represent an alkyl group or the like; EO represents an ethyleneoxy group; p represents an integer from 0 to 5; and m+n represents a number from 0 to 30, or a salt thereof; and (B) a surfactant.

## Background
FIELD OF THE INVENTION
The present invention relates to a biofilm formation inhibitor composition, and more particularly, to a biofilm formation inhibitor composition inhibiting the formation of a biofilm which is formed of microorganisms and materials produced by microorganisms in various fields to which microorganisms are related, thus preventing the hazard caused by the biofilm.
BACKGROUND OF THE INVENTION
Biofilms, which are also called biological films or slime, refer in general to the structures formed byintracellularly producing polymeric materials such as polysaccharides, proteins and the like by microorganisms that attach themselves on the surface of a material and proliferate in an aqueoussystem. When biofilms are formed, there occurs hazard caused by microorganisms, and various industrial problems are raised. For instance, when biofilms are formed inside the pipeline of a food processing plant, these biofilms are peeled off, leading to incorporation of foreign substances into food products, as well as causing food borne disease due to the toxins derived from microorganisms. Furthermore, formation of biofilms on metal surfaces is causative of metal corrosion, thereby accelerating decrepitude of facilities.
In addition, with regard to the microbial aggregates which have already formed a biofilm, no sufficient effects are exhibited by microbial control agents such as bactericides or bacteriostatic agents in many cases, as compared with the case with regard to microorganisms which are in the state of being dispersed and floating in an aqueous system. For example, in the aspect of the medical field, there have been many reports in recent years on the cases of in-hospital infection caused by the biofilms formed by microorganisms that survive in narrow interstices or pores of medical instruments. It is well known that the biofilms formed on the teeth in human oral cavity, so-called dental plaques (tooth plaques), are causative of dental caries or gum diseases, and thus, investigation has been conducted on these problems for a long time.
Heretofore, the idea of inhibiting microbial growth by exerting a bactericidal action or bacteriostatic action on microorganisms, particularly bacteria, in order to inhibit biofilms, has been generally examined. Patent Document 1 or Patent Document 2 discloses that the number of bacterial cells is reduced by using fatty acids or aliphatic alcohols, and consequently the adhesion of bacteria onto an object material can be prevented. In particular, Patent Document 1 shows that a composition prepared from an antimicrobial oil phase and emulsifiers as an emulsion exhibits an effect of reducing the number of bacterial cells in a relatively short time, and thus discloses the idea of inhibiting adhesion of bacteria on the surface of the object material on the basis of the phenomenon that the absolute number of bacterial cells per unit volume is decreased. Furthermore, Patent Document 3 discloses a toothpaste composition and the like in which non-aqueous active ingredients such as anti-inflammatory drugs and the like are dissolved in an oily material, while Patent Document 4 discloses a method of preventing establishment of contaminative organisms (barnacles, bacterial slimes or the like) on underwater structures by contacting the contaminative organisms with specific alkylamine derivatives, but neither was effective in inhibiting the formation of biofilms. Furthermore, Patent Document 5 describes a method of inhibiting bacterial adhesion on the surfaces that are immersed in the water, using polyglycol fatty acid esters, and discloses that bacterial adhesion is effectively inhibited at a concentration far lower than the threshold level exhibiting biocidal activity of polyglycol fatty acid esters. Patent Document 6 also discloses an industrial preservative and antifungal agent containing a polyglycerine monofatty acid ester and the like.
Patent Document 1 or 2 describes an evaluation of bactericidal property (reducing the number of bacterial cells by about 4 orders of magnitude) in the case of contacting microorganisms with a bactericidal or antimicrobial composition for a relatively short time of 60 minutes or less. However, since the problem of biofilm takes place over a long time in the order of days to months, it is in fact difficult to draw a conclusion from such short-term evaluation of bactericidal property, on the control of inhibition of biofilm formation. The fatty acids or aliphatic alcohols mentioned as an antibacterial oil phase cannot be said to have a sufficient bactericidal effect on all microorganisms (bacteria), and particularly with regard to gram-negative bacteria which frequently cause problems by forming biofilms, the substances do not have the minimal inhibitory concentration (MIC), which is an index of the bactericidal effect on a long-term basis (Jon J. Kabara ed., Cosmetic and Drug Preservation; Principles and Practice, Fragrance Journal, Ltd., 1990). Moreover, according to an experiment performed by the present inventors, the composition described in Patent Document 1 or Patent Document 2 exhibits, as described therein, a short-term (up to about 3 hours) bactericidal effect on gram-negative bacteria, particularly on Pseudomonas or Serratia, but there was no indication of bactericidal property, or even of bacteriostatic effects of inhibiting microbial growth, on a long-term basis (1 day or longer), and eventually formation of biofilms was confirmed.
In addition to these, there are bactericidal drugs with a high bactericidal property, having the feature of showing an immediate effect, such as cationic surfactants, hypochlorite and the like having a high bactericidal property. However, since their bactericidal property rapidly disappears in the presence of organic substances in the system, it is difficult to maintain the effect of reducing the number of microbes over a long time as described above.
For these reasons, it was difficult to fundamentally inhibit the formation of biofilms from the viewpoint of bactericide or bacteriostasis of bacteria.

    Patent Citation 1: JP-A-2002-524257 (WO 00/15562)
    Patent Citation 2: JP-A-2004-513153 (WO 02/038181)
    Patent Citation 3: JP-A-2005-289917
    Patent Citation 4: JP-A-2004-525935 (WO 02/076928)
    Patent Citation 5: JP-A-(Hei) 11-512720 (WO 97/11912)
    Patent Citation 6: JP-A-2003-160410


DISCLOSURE OF INVENTION
The present invention provides a biofilm formation inhibitor composition including component (A):
(A) at least one or more selected from the compounds represented by Formula (1) and Formula (2):




wherein R1 and R2, which may be identical or different, each represent an alkyl group or an alkenyl group, which is a straight chain or branched chain having 8 to 14 carbon atoms; EO represents an ethyleneoxy group; p represents an integer from 0 to 5; and m and n each represent an average number of added moles, with m+n being a number from 0 to 30;
or a salt thereof, and
(B) a surfactant,
wherein the weight ratio of the component (A) and the component (B), (A)/(B), is not more than 2.
The present invention is also to provide a method of inhibiting biofilm formation by contacting the biofilm formation inhibitor composition with microorganisms.
In another aspect, the present invention is to provide use of a composition including the following component (A):
(A) at least one or more selected from the compounds represented by Formula (1) and Formula (2):




wherein R1 and R2, which may be identical or different, each represent an alkyl group or an alkenyl group, which is a straight chain or branched chain having 8 to 14 carbon atoms; EO represents an ethyleneoxy group; p represents an integer from 0 to 5; and m and n each represent an average number of added moles, with m+n being a number from 0 to 30;
or a salt thereof, and
(B) a surfactant,
wherein the weight ratio of the component (A) and the component (B), (A)/(B), is not more than 2,
as a biofilm formation inhibitor composition.
In still another aspect, the present invention is to provide a biofilm formation inhibitor composition containing the following component (C):
(C) at least one or more selected from the group consisting of the compounds represented by Formula (3) and Formula (4):




wherein R3 and R4, which may be identical or different, each represent an alkyl group or an alkenyl group, which is a straight chain or branched chain having 8 to 14 carbon atoms; and R5 represents a hydrogen atom, or a straight chain or branched chain alkyl group having 1 to 3 carbon atoms.

The present invention is also to provide a method of inhibiting biofilm formation by contacting the biofilm formation inhibitor composition with microorganisms.
In another aspect, the present invention is to provide use of the following component (C):
(C) at least one or more selected from the group consisting of the compounds represented by Formula (3) and Formula (4):




wherein R3 and R4, which may be identical or different, each represent an alkyl group or an alkenyl group, which is a straight chain or branched chain having 8 to 14 carbon atoms; and R5 represents a hydrogen atom, or a straight chain or branched chain alkyl group having 1 to 3 carbon atoms, as a biofilm formation inhibitor.


DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a biofilm formation inhibitor composition, a method of inhibiting biofilm formation, and use of the composition as a biofilm formation inhibitor.
According to the present invention, the biofilm formation can be inhibited for a prolonged time.
With regard to Formula (1) of the component (A), the alkyl group or alkenyl group represented by R1 may be a straight chain or a branched chain, and from the viewpoint of the biofilm formation inhibitory effect, it preferably has 10 to 12 carbon atoms. The number p of the ethyleneoxy group represented by EO is, from the viewpoint of the biofilm formation inhibitory effect, preferably from 0 to 4, and more preferably 0 to 3, and from the viewpoint of the dispersibility to water, p is preferably 1 to 3.
With regard to Formula (2) of the component (A), the alkyl group or alkenyl group represented by R2 may be a straight chain or a branched chain, and preferably has 10 to 12 carbon atoms. The number of the ethyleneoxy group represented by EO, that is m+n, is preferably 0 to 15, and more preferably 0 to 5. Examples of the salt of the compound represented by the Formula (2) include mineral acid salts such as hydrochloride, sulfate, phosphate and the like, and organic acid salts such as acetate, citrate and the like.
With regard to Formula (3) of the component (C), the alkyl group or alkenyl group represented by R3 may be a straight chain or a branched chain, and preferably has 10 to 12 carbon atoms.
With regard to Formula (4) of the component (C), the alkyl group or alkenyl group represented by R4 may be a straight chain or a branched chain, and preferably has 10 to 12 carbon atoms. Also for the alkyl group of R5, those having 1 or 2 carbon atoms are particularly preferred.
The component (A) or component (C) of the present invention may be favorably present in the system at a weight concentration of 1 ppm or more to manifest a long-term biofilm formation inhibitory effect, preferably 1 to 10,000 ppm from the viewpoint of economic efficiency and effect, more preferably 5 to 10,000 ppm, even more preferably 5 to 2,000 ppm, and even more preferably 10 to 1,000 ppm.
Since the compound of the component (A) of the present invention represented by Formula (1) or Formula (2) has high hydrophobicity and low solubility in water, it becomes possible to use the subject agent more effectively in an aqueous system by rendering the subject agent to exist stably in the aqueous system using the surfactant of the component (B). The compound of the component (C) represented by Formula (3) or Formula (4) exhibits an excellent biofilm formation inhibitory effect by itself, but it is also possible to use the subject agent more effectively in an aqueous system by rendering the subject agent to exist more stably in the aqueous system using the surfactant of the component (B).
Here, the phrase “exist stably in an aqueous system” means a state in which the component (A) or component (C) is emulsified, dispersed or solubilized without undergoing separation on a long-term basis, and it becomes possible to emulsify, disperse or solubilize a larger amount of the component (A) or component (C) per unit volume of the aqueous system.
The type of the surfactant of the component (B) that can be used for the biofilm formation inhibitor composition of the present invention is not particularly limited, but a surfactant which is capable of rendering the component (A) or component (C) to exist stably in an aqueous system is preferred. Particularly from the viewpoint of the capability to emulsify, disperse or solubilize, it is preferable to use anionic surfactants or nonionic surfactants among such surfactants.
Examples of the anionic surfactant include lignin sulfonic acid salts, alkylbenzenesulfonic acid salts, alkylsulfonic acid salts, polyoxyethylene (hereinafter, referred to as POE) alkylsulfonic acid salts, POE alkylphenyl ether sulfonic acid salts, POE alkylphenyl ether phosphoric acid ester salts, POE arylphenyl ether sulfonic acid salts, alkylsulfuric acid ester salts, POE alkylsulfuric acid ester salts, POE arylphenyl ether phosphoric acid ester salts, naphthalenesulfonic acid salts, naphthalenesulfonic acid formalin condensate, POE tribenzylphenyl ether sulfonic acid salts, alkylphosphoric acid salts, POE alkylphosphoric acid salts, POE tribenzylphenyl ether phosphoric acid ester salts, dialkylsulfosuccinic acid salts, fatty acid salts (soap), POE alkyl ether acetic acid salts, and the like. Among these, it is preferable to use alkylsulfuric acid ester salts, POE alkylsulfuric acid ester salts, or POE alkyl ether acetic acid salts. The number of carbon atoms in the alkyl moiety of these anionic surfactants is preferably 10 to 18, and the average number of added moles of ethylene oxide is preferably 0 to 10, and more preferably 0 to 5.
Examples of the nonionic surfactant include monovalent alcohol derivative-type nonionic surfactants such as POE alkyl ethers (with the proviso that the component (A) is excluded), POE alkylphenyl ethers, polyoxypropylene-POE (block or random) alkyl ethers, POE arylphenyl ethers, POE styrenated phenyl ethers, POE tribenzylphenyl ethers, and the like; polyvalent alcohol derivative-type nonionic surfactants such as (poly)glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, POE sorbitan fatty acid esters, alkylpolyglycosides, and the like; among these, POE alkyl ethers (with the proviso that the component (A) is excluded), (poly)glycerin fatty acid esters, alkylpolyglycosides, sorbitan fatty acid esters, and POE sorbitan fatty acid esters are preferred, with POE alkyl ethers (with the proviso that the component (A) is excluded) being particularly preferred. Inter alia, the HLB value of the POE alkyl ether is more preferably 10 or larger. The number of carbon atoms in the alkyl moiety of POE alkyl ether is preferably 12 to 18, and the average number of added moles of ethylene oxide is particularly 6 or larger.
The surfactant of the component (B) may be used individually, or in combination of two or more species so as to further enhance the capability to emulsify, disperse or solubilize the active ingredient.
In the biofilm formation inhibitor composition, the weight ratio of the component (A) or (C) to the component (B), (A)/(B) or (C)/(B), is preferably 2/1 to 1/100, more preferably 2/1 to 1/50, even more preferably 2/1 to 1/20, and even more preferably 1/1 to 1/10, in view of the long-term biofilm formation inhibitory effect. Moreover, in the case of using the component of the Formula (1) and/or Formula (2), the ratio (A)/(B) is 2 or less in view of the long-term biofilm formation inhibitory effect.
Furthermore, the biofilm formation inhibitor composition contains preferably 0.01 to 10% by weight, more preferably 0.1 to 10% by weight, and even more preferably 0.5 to 5% by weight, of the component (A) or the component (C), and contains preferably 0.1 to 30% by weight, and more preferably 1 to 20% by weight, of the component (B).
The biofilm formation inhibitor composition of the present invention can be used in combination with a bactericide or an antimicrobial agent. In general, when a biofilm is formed, there occurs a situation where it becomes difficult for the bactericide to exert an effect. However, when the biofilm formation is inhibited by the biofilm formation inhibitor composition of the present invention, it becomes possible to sufficiently elicit the efficacy of the bactericide.
The bactericide or antimicrobial agent as described above may be exemplified by a quaternary salt such as benzalkonium chloride, benzethonium chloride, didecyldimethylammonium chloride, cetylpyridinium chloride, chlorhexidine hydrochloride, and the like; chlorhexidine glyconate, polyhexamethylenebiguanidine hydrochloride, triclosan, isopropylmethylphenol, trichlorocarbanilide, ethanol, isopropyl alcohol, or the like.
The biofilm formation inhibitor composition of the present invention can also contain a thickening agent, in order to increase the viscosity of the composition and improve the adhesiveness to the object material.
Furthermore, a chelating agent may be added to the biofilm formation inhibitor composition of the present invention. Examples of the chelating agent include ethylenediamine tetraacetic acid (EDTA), succinic acid, salicylic acid, oxalic acid, malic acid, lactic acid, fumaric acid, tartaric acid, citric acid, gluconic acid, tripolyphosphoric acid, 1-hydroxyethane-1,1-diphosphonic acid, polyacrylic acid, acrylic acid/maleic acid copolymers and salts thereof.
The biofilm formation inhibitor composition of the present invention can take various forms such as liquid, paste, powder, tablet and the like, in accordance with the application. The biofilm formation inhibitor composition may be used as a single formulation containing all of the components as a mixture, but may also be in the form of several divided packages according to the application.
The biofilm formation inhibitor composition of the present invention can be effectively used as a water-diluted system. Certain amounts of water dilutions of the composition are collected, and an object material is immersed therein. In the case where the object material is present extensively, the composition may be sprayed as a mist using a sprayer, or may be sprayed as a froth using a frothing machine. The composition may also be poured over the object material, or may be applied thereon with a brush or the like. In addition to those, a towel or the like may be impregnated with a water dilution of the composition and used to wipe out the object material. As long as the conditions for contacting the composition with microorganisms are satisfied, a water dilution of the composition may be adhered or daubed on the surface where microorganism are possibly present. For the water dilution of the composition, the concentration by weight of the component (A) or component (C) at the time of use is preferably 1 to 10,000 ppm, and more preferably 5 to 10,000 ppm.
For some object materials, it is also possible to smear and spread the composition as a cream or ointment, without diluting the composition in water. In this case, the component (A) or component (C) is provided in the form of being dissolved, dispersed or emulsified in a suitable solvent, and the concentration by weight of the component (A) or component (C) at the time of use is preferably 1 to 10,000 ppm, and more preferably 5 to 10,000 ppm.
The present invention is also to provide a method of inhibiting the formation of a biofilm by contacting the biofilm formation inhibitor composition with microorganisms. Here, it is desirable to carry out the contact between the biofilm formation inhibitor composition and microorganisms continuously. The present invention also provides the use of the component (A) or component (C) as a biofilm formation inhibitor.
The biofilm formation inhibitor composition of the present invention can be used in a wide range of applications where the harm of biofilms is a problem of concern. For example, the composition can be applied to detergents for food or beverage processing plants where the risk of microbial contamination is high. Furthermore, the composition can be applied also to detergents for medical instruments that are susceptible to biofilm formation, such as, for example, endoscope, artificial dialyzer and the like. Moreover, from the viewpoint of being highly safe, the composition can also be used in cleansing agents, toothpastes, oral care products, denture care products and the like for human use.
The following examples further describe and demonstrate embodiments of the present invention. The examples are given solely for the purpose of illustration and are not to be constructed as limitations of the present invention.
EXAMPLES
(1) Example of Using the Compound Represented by Formula (1)
Example 1
Blending of a Biofilm Formation Inhibitor Composition, and Verification of Ability to Inhibit Biofilm Formation
Component (A) R1O-(EO)p—H
(A-1) C8 alcohol [KALCOL 0898, manufactured by Kao Corporation, R1=C8 alkyl, p=0]
(A-2) C10 alcohol [KALCOL 1098, manufactured by Kao Corporation, R1=C10 alkyl, p=0]
(A-3) C12 alcohol [KALCOL 2098, manufactured by Kao Corporation, R1=C12 alkyl, p=0]
(A-4) C14 alcohol [KALCOL 4098, manufactured by Kao Corporation, R1=C14 alkyl, p=0]
(A-5) Adduct of C12 alcohol and 1 mole of ethylene oxide [NIKKOL BL-1SY, manufactured by Nikko Chemicals Co., Ltd., R1=C12 alkyl, p=1]
(A-6) Adduct of C12 alcohol and 2 moles of ethylene oxide [NIKKOL BL-2SY, manufactured by Nikko Chemicals Co., Ltd., R1=C12 alkyl, p=2]
(A-7) Adduct of C12 alcohol and 3 moles of ethylene oxide [NIKKOL BL-3SY, manufactured by Nikko Chemicals Co., Ltd., R1=C12 alkyl, p=3]
(A-8) Adduct of C12 alcohol and 5 moles of ethylene oxide [NIKKOL BL-5SY, manufactured by Nikko Chemicals Co., Ltd., R1=C12 alkyl, p=5]
(A-9) C12 alcohol [trans-2-dodecen-1-ol, manufactured by Wako Pure Chemical Industries, Ltd., R=C12 alkenyl, p=0]
(A-10) C12 alcohol (secondary) [2-dodecanol, manufactured by Wako Pure Chemical Industries, Ltd., R1=C12 secondary alkyl, p=0]
(A-11) C12 branched alcohol [2-butyl-1-octanol, manufactured by Sigma-Aldrich, Inc., R1=C12 branched alkyl, p=0]
Component (A′) R1′O-(EO)p—H
(A′-1) C1 alcohol [methanol, manufactured by Wako Pure Chemical Industries, Ltd., R1′=C1 alkyl, p=0]
(A′-2) C2 alcohol [ethanol, manufactured by Wako Pure Chemical Industries, Ltd., R1=C2 alkyl, p=0]
(A′-3) C3 alcohol [L-propanol, manufactured by Wako Pure Chemical Industries, Ltd., R1′=C3 alkyl, p=0]
(A′-4) C4 alcohol [1-butanol, manufactured by Wako Pure Chemical Industries, Ltd., R1′=C4 alkyl, p=0]
(A′-5) C16 alcohol [KALCOL 6098, manufactured by Kao Corporation, R1′=C16 alkyl, p=0]
(A′-6) C18 alcohol [KALCOL 8098, manufactured by Kao Corporation, R1′=C18 alkyl, p=0]
(A′-7) Adduct of C1 alcohol and 1 mole of ethylene oxide [2-methoxyethanol, manufactured by Wako Pure Chemical Industries, Ltd., R1′=C1 alkyl, p=1]
(A′-8) Adduct of C1 alcohol and 2 moles of ethylene oxide [2-(2-methoxyethoxy)ethanol, manufactured by Wako Pure Chemical Industries, Ltd., R1′=C1 alkyl, p=2]
(A′-9) Adduct of C2 alcohol and 1 mole of ethylene oxide [2-ethoxyethanol, manufactured by Wako Pure Chemical Industries, Ltd., R1′=C2 alkyl, p=1]
(A′-10) Adduct of C2 alcohol and 2 moles of ethylene oxide [2-(2-ethoxyethoxy)ethanol, manufactured by Wako Pure Chemical Industries, Ltd., R1′=C2 alkyl, p=2]
(A′-11) Adduct of C4 alcohol and 1 mole of ethylene oxide [2-butoxyethanol, manufactured by Wako Pure Chemical Industries, Ltd., R1′=C4 alkyl, p=1]
(A′-12) Adduct of C4 alcohol and 2 moles of ethylene oxide [2-(2-butoxyethoxy)ethanol, manufactured by Wako Pure Chemical Industries, Ltd., R1′=C4 alkyl, p=2]
(A′-13) C20 branched alcohol [2-octyl-1-dodecanol, manufactured by Sigma-Aldrich, Inc., R1′=C20 branched alkyl, p=0]
(A′-14) Adduct of C12 alcohol and 8 moles of ethylene oxide [NIKKOL BL-8SY, manufactured by Nikko Chemicals, Ltd., R1′=C12alkyl, p=8]
Component (B) Surfactant [the number within the parentheses represents the average number of added moles of ethylene oxide.]
<Anionic Surfactants>
(B-1) Sodium lauryl sulfate [EMAL 0, manufactured by Kao Corporation]
(B-2) Sodium polyoxyethylene(2) lauryl ethersulfate [EMAL 20C, manufactured by Kao Corporation; active ingredient 25% by weight]
(B-3) Sodium polyoxyethylene (4.5) lauryl ether acetate [KAO AKYPO RLM45-NV, manufactured by Kao Corporation; active ingredient 24% by weight]
<Nonionic Surfactants>
(B-4) Polyoxyethylene (6) lauryl ether [EMULGEN 106, manufactured by Kao Corporation]
(B-5) Polyoxyethylene (12) lauryl ether [EMULGEN 120, manufactured by Kao Corporation]
(B-6) Lauryl glucoside [MYDOL 12, manufactured by Kao Corporation]
(B-7) Decyl glyceryl monocaprylate [SY-GLYSTER MCA750, manufactured by SAKAMOTO YAKUHIN KOGYO CO., LTD.]
(B-8) Sorbitan monolaurate [RHEODOLSP-L10, manufactured by Kao Corporation]
(B-9) Polyoxyethylene (6) sorbitan monolaurate [RHEODOL TW-L106, manufactured by Kao Corporation]
(B-10) Polyoxyethylene (20) sorbitan monolaurate [RHEODOL TW-L120, manufactured by Kao Corporation]
The active ingredients were blended on a weight basis such that the concentration of the component (A) or component (A′) was fixed at 1% by weight, while the concentration of the component (B) was selected from 1% by weight, 3% by weight, 6% by weight and 10% by weight, with the balance being ion-exchanged water. The blend was diluted using Mueller-Hinton Broth [manufactured by Nippon Becton Dickinson Co., Ltd.], to a concentration of 100 ppm of the component (A) or component (A′), and 2 mL of the dilution was taken and placed on a 24-well microplate [manufactured by ASAHI TECHNO GLASS CORPORATION].
Pseudomonas (Pseudomonas aeruginosa NBRC 13275), Serratia (Serratia marcescens NBRC 12648), and Klebsiella (Klebsiella pneumoniae ATCC 13883) were each pre-cultured using soybean-casein digest agar [SCD Agar Medium; manufactured by Nihon Pharmaceutical Co., Ltd.], at 37 degrees Celsius for 24 hours to form colonies. A very small amount of bacterial clusters from the colonies formed was inoculated into each of the above-described test solutions on the microplate, using a sterilized bamboo skewer. The inoculated solution was incubated at 37 degrees Celsius for 48 hours, then the culture solution was discarded, and the state of formation of the biofilm adhering on the microplate wall was observed by visual evaluation. The state of the biofilm formation was graded such that the state in which the biofilm covered 0 to less than 20% of the plate wall surface was graded A, the state in which the biofilm covered 20% or more to less than 40% was graded B, the state in which the biofilm covered 40% or more to less than 60% was graded C, and the state in which the biofilm covered 60% or more was graded D.
The results are presented in Table 1-1 to Table 1-5.















TABLE 1-1








Example
Example
Example
Example
Example
Example
Example
Example



product 1
product 2
product 3
product 4
product 5
product 6
product 7
product 8





Component (A)










RO-(EO)n-H










A-1
1.0
1.0
1.0







A-2



1.0
1.0
1.0
1.0
1.0


A-3










A-4










A-5










A-6










A-7










A-8










Surfactant (B)










[Anionic










Surfactant]










B-1
3.0


1.0






B-2

3.0


3.0
6.0




B-3






3.0



[Nonionic










Surfactant]










B-4







3.0


B-5


3.0







B-6










B-7










B-8










B-9










B-10










Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Water










Total
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0


State of Biofilm










Inhibition










P. aeruginosa
B
B
B
A
A
A
A
A


S. marcescens
B
B
B
A
A
A
A
A


K. neumoniae
B
B
B
A
A
A
A
A






Example
Example
Example
Example
Example
Example
Example
Example



product 9
product 10
product 11
product 12
product 13
product 14
product 15
product 16





Component (A)










RO-(EO)n-H










A-1










A-2
1.0
1.0








A-3


1.0
1.0
1.0
1.0
1.0
1.0


A-4










A-5










A-6










A-7










A-8










Surfactant (B)










[Anionic










Surfactant]










B-1


3.0
6.0






B-2










B-3










[Nonionic










Surfactant]










B-4




1.0
3.0




B-5
1.0





3.0
10.0


B-6










B-7










B-8










B-9

3.0








B-10










Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Water










Total
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0


State of Biofilm










Inhibition










P. aeruginosa
A
A
A
A
A
A
A
A


S. marcescens
A
A
A
A
A
A
A
A


K. neumoniae
A
A
A
A
A
A
A
A

























TABLE 1-2








Example
Example
Example
Example
Example
Example
Example
Example
Example



product
product
product
product
product
product
product
product
product



17
18
19
20
21
22
23
24
25





Component (A)











RO-(EO)n-H











A-1











A-2











A-3
1.0










A-4

1.0
1.0
1.0







A-5




1.0
1.0
1.0
1.0



A-6








1.0


A-7











Surfactant (B)











[Anionic











Surfactant]











B-1




3.0






B-2

3.0






3.0


B-3











[Nonionic











Surfactant]











B-4





3.0





B-5






3.0
6.0



B-6











B-7


10.0








B-8



3.0







B-9











B-10
6.0










Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Water











Total
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0


State of Biofilm











Inhibition











P. aeruginosa
A
B
B
B
A
A
A
A
A


S. marcescens
A
B
B
B
A
A
A
A
A


K. pneumoniae
A
B
B
B
A
A
A
A
A






Example
Example
Example
Example
Example
Example
Example
Example
Example



product
product
product
product
product
product
product
product
product



26
27
28
29
30
31
32
33
34





Component (A)











RO-(EO)n-H











A-1











A-2











A-3











A-4











A-5











A-6
1.0
1.0
1.0








A-7



1.0
1.0
1.0
1.0
1.0
1.0


Surfactant (B)











[Anionic











Surfactant]











B-1



6.0







B-2











B-3











[Nonionic











Surfactant]











B-4











B-5
3.0



10.0






B-6

3.0



3.0





B-7






6.0




B-8







3.0



B-9








3.0


B-10


3.0








Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Water











Total
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0


State of Biofilm











Inhibition











P. aeruginosa
A
A
A
A
A
A
A
A
A


S. marcescens
A
A
A
A
A
A
A
A
A


K. pneumoniae
A
A
A
A
A
A
A
A
A
























TABLE 1-3








Example
Example
Example
Example
Example
Example
Example
Example



product 35
product 36
product 37
product 38
product 39
product 40
product 41
product 42





Component (A)










RO-(EO)n-H










A-8
1.0
1.0
1.0







A-9



1.0
1.0
1.0




A-10






1.0
1.0


A-11










Surfactant (B)










[Anionic Surfactant]










B-1


6.0







B-2







6.0


B-3










[Nonionic Surfactant]










B-4



3.0






B-5
3.0



3.0
6.0
3.0



B-6

3.0








B-7










B-8










B-9










B-10






3.0



Ion-Exchanged Water
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Total
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0


State of Biofilm










Inhibition










P. aeruginosa
A
A
B
A
A
A
A
A


S. marcescens
B
B
B
A
A
A
A
A


K. pneumoniae
A
B
B
A
A
A
A
A

















Example
Example
Example
Example




product 43
product 44
product 45
product 46






Component (A)







A-8







A-9







A-10
1.0






A-11

1.0
1.0
1.0



Surfactant (B)







[Anionic Surfactant]







B-1

3.0





B-2







B-3
3.0






[Nonionic Surfactant]







B-4







B-5







B-6


6.0




B-7







B-8







B-9







B-10



3.0



Ion-Exchanged Water
Balance
Balance
Balance
Balance



Total
100.0
100.0
100.0
100.0



State of Biofilm







Inhibition







P. aeruginosa
A
A
A
A



S. marcescens
A
A
A
A



K. pneumoniae
A
A
A
A
























TABLE 1-4








Comparative
Comparative
Comparative
Comparative
Comparative
Comparative
Comparative
Comparative



Product 1
Product 2
Product 3
Product 4
Product 5
Product 6
Product 7
Product 8





Component










(A′)










R′O-(EO)n-H










A′-1
1.0
1.0








A′-2


1.0
1.0
1.0





A′-3





1.0
1.0
1.0


A′-4










A′-5










A′-6










A′-7










A′-8










A′-9










A′-10










A′-11










A′-12










Surfactant (B)










[Anionic










Surfactant]










B-1
6.0









B-2


3.0


1.0




B-3










[Nonionic










Surfactant]










B-4










B-5

3.0

3.0


3.0



B-6




6.0





B-7







10.0


B-8










B-9










B-10










Ion-
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Exchanged










Water










Total
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0


State of










Biofilm










Inhibition










P. aeruginosa
D
D
D
D
D
D
D
D


S. marcescens
D
D
D
D
D
D
D
D


K. pneumoniae
D
D
D
D
D
D
D
D







Comparative

Comparative
Comparative
Comparative
Comparative
Comparative



Comparative
Product
Comparative
Product
Product
Product
Product
Product



Product 9
10
Product 11
12
13
14
15
16





Component










(A′)










R′O-(EO)n-H










A′-1










A′-2










A′-3










A′-4
1.0
1.0








A′-5


1.0
1.0






A′-6




1.0
1.0




A′-7






1.0
1.0


A′-8










A′-9










A′-10










A′-11










A′-12










Surfactant (B)










[Anionic










Surfactant]










B-1










B-2










B-3










[Nonionic










Surfactant]










B-4










B-5
3.0

3.0

3.0

3.0



B-6










B-7

10.0








B-8







3.0


B-9



3.0






B-10





10.0




Ion-
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Exchanged










Water










Total
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0


State of










Biofilm










Inhibition










P. aeruginosa
D
D
C
C
D
D
D
D


S. marcescens
D
D
C
C
D
D
D
D


K. pneumoniae
D
D
C
C
D
D
D
D






















TABLE 1-5








Comparative
Comparative
Comparative
Comparative
Comparative
Comparative



Product 17
Product 18
Product 19
Product 20
Product 21
Product 22





Component (A′)








R′O-(EO)n-H








A′-1








A′-2








A′-3








A′-4








A′-5








A′-6








A′-7








A′-8
1.0
1.0






A′-9


1.0
1.0




A′-10




1.0
1.0


A′-11








A′-12








A′-13








A′-14








Surfactant (B)








[Anionic








Surfactant]








B-1
6.0







B-2


3.0





B-3




3.0



[Nonionic








Surfactant]








B-4








B-5

3.0

3.0

3.0


B-6








B-7








B-8








B-9








B-10








Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance


Water








Total
100.0
100.0
100.0
100.0
100.0
100.0


State of Biofilm








Inhibition








P. aeruginosa
D
D
D
D
D
D


S. marcescens
D
D
D
D
D
D


K. pneumoniae
D
D
D
D
D
D






Comparative
Comparative
Comparative
Comparative
Comparative
Comparative



Product 23
Product 24
Product 25
Product 26
Product27
Product28





Component (A′)








R′O-(EO)n-H








A′-1








A′-2








A′-3








A′-4








A′-5








A′-6








A′-7








A′-8








A′-9








A′-10








A′-11
1.0
1.0






A′-12


1.0
1.0




A′-13




1.0
1.0


A′-14








Surfactant (B)








[Anionic








Surfactant]








B-1
3.0

3.0





B-2








B-3








[Nonionic








Surfactant]








B-4








B-5

3.0

3.0




B-6




3.0



B-7








B-8





3.0


B-9








B-10








Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance


Water








Total
100.0
100.0
100.0
100.0
101.0
102.0


State of Biofilm








Inhibition








P. aeruginosa
D
D
D
D
D
D


S. marcescens
D
D
D
D
D
D


K. pneumoniae
D
D
D
D
D
D















Comparative
Comparative




Product 29
Product 30






Component (A′)





R′O-(EO)n-H





A′-1





A′-2





A′-3





A′-4





A′-5





A′-6





A′-7





A′-8





A′-9





A′-10





A′-11





A′-12





A′-13





A′-14
1.0
1.0



Surfactant (B)





[Anionic





Surfactant]





B-1





B-2





B-3





[Nonionic





Surfactant]





B-4





B-5





B-6





B-7





B-8





B-9
3.0




B-10

6.0



Ion-Exchanged
Balance
Balance



Water





Total
100.0
100.0



State of Biofilm





Inhibition





P. aeruginosa
C
C



S. marcescens
D
D



K. pneumoniae
C
C









Example 2
Test For Reduction of Biofilm Production in Large-Volume Plastic Cup
Pseudomonas (Pseudomonas aeruginosa NBRC 13275) was pre-cultured using soybean-casein digest agar [SCD Agar Medium; manufactured by Nihon Pharmaceutical Co., Ltd.] at 37 degrees Celsius for 24 hours. The colonies grown on the medium were scraped off and suspended in 10 mM sterile phosphate buffer (pH 7.2). The resultant suspension was washed by centrifuging twice at 5,000×g for 15 minutes at 10 degrees Celsius, and then suspended again in 10 mM sterile phosphate buffer (pH 7.2) to prepare a bacterial suspension in which the bacterial concentration was adjusted to 1.0 as the absorbance at 600 nm (OD600=1.0). Thereafter, 100 mL of Mueller-Hinton Broth [manufactured by Nippon Becton Dickinson, Co., Ltd.] and each of 15 species of the testing drugs selected from Example 1 were introduced into a 200-mL sterile screw cup [manufactured by EIKENKIZAI CO., LTD.] and mixed thoroughly. The concentration of the component (A) or component (A′) was adjusted to 5 ppm, 10 ppm, 50 ppm, 100 ppm or 500 ppm, and then 0.1 mL of the bacterial suspension prepared as described above was inoculated thereinto. Furthermore, as a control, a test section was simultaneously provided in which bacteria were inoculated as described above in Mueller-Hinton Broth with no added drugs.
These were statically cultured at 37 degrees Celsius, and at the designated time points of 1 day, 2 days, 3 days and 5 days after the initiation of culture, the number of bacterial cells was measured, and the biofilm formed inside the cup was observed by visual evaluation. Subsequently, the culture solutions were centrifuged at 10,000×g for 30 minutes at 5 degrees Celsius, and the respective precipitates were removed, dried in a vacuum desiccator for 24 hours, and weighed, with the weight thus measured being taken as the weight of the biofilm produced in the culture solution. Furthermore, the state of the biofilm formation was graded such that the state in which no biofilm was confirmed in the cup was graded A; the state in which the formation of a biofilm started inside the cup at the air-liquid interface was graded B, and the state in which a biofilm was formed to extend from the air-liquid interface down to the culture solution was graded C.

The results are presented in Table 2-1-1 to Table 2-4-5.











TABLE 2-1-1









Control
Example product 1









Concentration of Component (A)











—
5 ppm
10 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1
32.4
B
8.0
20.6
B
7.9
18.3
B
8.1


Day of


Culture


After 2
49.9
C
8.2
23.1
B
8.3
20.4
B
8.0


Days of


Culture


After 3
51.3
C
8.4
28.4
B
8.1
28.1
B
7.9


Days of


Culture


After 5
63.9
C
8.5
31.9
B
8.6
30.9
B
8.2


Days of


Culture












Example product 1



Concentration of Component (A)











50 ppm
100 ppm
500 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1
10.1
A
7.8
11.0
A
7.9
10.5
A
7.8


Day of


Culture


After 2
17.6
B
8.0
16.8
B
7.9
15.8
B
7.9


Days of


Culture


After 3
26.1
B
8.1
20.8
B
8.1
19.9
B
7.8


Days of


Culture


After 5
29.7
B
8.0
28.2
B
8.3
24.1
B
8.0


Days of


Culture


















TABLE 2-1-2









Example product 4



Concentration of Component (A)










5 ppm















Vis-








ual

10 ppm
50 ppm
100 ppm
500 ppm
























Judg-


Visual


Visual


Visual


Visual




Amount
ment
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of



of
of
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-



Biofilm
Bio-
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial



(mg)
film
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*


























After
2.5
A
7.5
1.7
A
7.6
1.7
A
7.7
1.8
A
7.9
1.9
A
7.4


1 Day


of


Culture


After
4.9
A
7.8
2.8
A
7.9
2.8
A
7.5
2.9
A
7.8
3.1
A
7.6


2 Days


of


Culture


After
20.7
B
7.8
9.8
A
8.1
4.3
A
7.8
3.8
A
7.8
4.5
A
7.5


3 Days


of


Culture


After
31.0
B
7.8
27.1
B
7.9
13.7
B
7.6
7.9
A
7.6
6.4
A
7.8


5 Days


of


Culture


















TABLE 2-1-3









Example product 9



Concentration of Component (A)










5 ppm















Vis-








ual

10 ppm
50 ppm
100 ppm
500 ppm
























Judg-


Visual


Visual


Visual


Visual




Amount
ment
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of



of
of
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-



Biofilm
Bio-
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial



(mg)
film
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*


























After
2.6
A
7.6
1.8
A
7.5
1.8
A
7.5
1.9
A
7.3
1.9
A
7.4


1 Day


of


Culture


After
4.9
A
7.4
3.6
A
7.8
3.0
A
7.4
3.4
A
7.5
2.7
A
7.4


2 Days


of


Culture


After
19.8
B
7.8
9.8
A
7.8
4.5
A
7.6
3.8
A
7.5
3.8
A
7.8


3 Days


of


Culture


After
33.2
B
7.9
32.6
B
7.4
13.2
A
7.5
6.3
A
7.7
5.2
A
7.6


5 Days


of


Culture


















TABLE 2-1-4









Example product 12



Concentration of Component (A)










5 ppm















Vis-








ual

10 ppm
50 ppm
100 ppm
500 ppm
























Judg-


Visual


Visual


Visual


Visual




Amount
ment
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of



of
of
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-



Biofilm
Bio-
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial



(mg)
film
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*


























After
2.8
A
7.4
1.8
A
7.6
1.9
A
7.4
2.1
A
7.2
2.0
A
7.8


1 Day


of


Culture


After
5.6
A
7.9
3.2
A
7.5
2.8
A
7.8
2.6
A
7.6
3.1
A
7.6


2 Days


of


Culture


After
23.6
B
7.8
9.9
A
7.6
5.6
A
7.6
6.8
A
7.5
4.5
A
7.7


3 Days


of


Culture


After
36.1
B
7.8
31.1
B
7.9
14.4
B
7.5
10.1
A
7.4
7.9
A
7.5


5 Days


of


Culture


















TABLE 2-1-5









Example product 14



Concentration of Component (A)










5 ppm















Vis-








ual

10 ppm
50 ppm
100 ppm
500 ppm
























Judg-


Visual


Visual


Visual


Visual




Amount
ment
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of



of
of
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-



Biofilm
Bio-
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial



(mg)
film
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*


























After
2.4
A
7.6
1.9
A
7.8
1.7
A
7.7
1.8
A
7.3
1.8
A
7.6


1 Day


of


Culture


After
5.5
A
7.9
3.5
A
7.8
3.5
A
7.9
3.7
A
7.7
3.5
A
7.8


2 Days


of


Culture


After
28.7
B
7.5
10.0
A
7.6
5.1
A
7.6
5.2
A
7.6
4.3
A
7.5


3 Days


of


Culture


After
35.2
B
7.6
30.2
B
7.6
11.1
A
7.8
7.1
A
7.8
5.6
A
7.4


5 Days


of


Culture





*Log10CFU/mL















TABLE 2-2-1









Example product 15



Concentration of Component (A)










5 ppm















Vis-








ual

10 ppm
50 ppm
100 ppm
500 ppm
























Judg-


Visual


Visual


Visual


Visual




Amount
ment
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of



of
of
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-



Biofilm
Bio-
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial



(mg)
film
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*


























After
3.1
A
7.4
1.8
A
7.1
1.7
A
7.4
1.9
A
7.4
1.7
A
7.3


1 Day


of


Culture


After
6.2
A
7.6
3.8
A
7.6
3.1
A
7.9
3.4
A
7.3
3.1
A
7.4


2 Days


of


Culture


After
27.6
B
7.9
10.7
A
7.6
6.3
A
7.4
5.7
A
7.5
4.7
A
7.2


3 Days


of


Culture


After
35.8
B
7.8
32.1
B
7.6
11.1
B
7.8
10.8
A
7.8
8.0
A
7.1


5 Days


of


Culture


















TABLE 2-2-2









Example product 19



Concentration of Component (A)










5 ppm















Vis-








ual

10 ppm
50 ppm
100 ppm
500 ppm
























Judg-


Visual


Visual


Visual


Visual




Amount
ment
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of



of
of
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-



Biofilm
Bio-
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial



(mg)
film
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*


























After
18.7
B
7.8
18.0
B
8.0
11.2
A
7.5
10.7
A
7.6
12.1
A
8.0


1 Day


of


Culture


After
22.9
B
8.1
22.1
B
7.8
18.6
B
8.3
16.6
B
7.9
16.4
B
7.7


2 Days


of


Culture


After
27.6
B
8.3
25.9
B
7.4
25.3
B
8.2
23.7
B
8.4
21.0
B
7.9


3 Days


of


Culture


After
37.2
B
8.4
29.8
B
7.9
28.1
B
8.4
29.5
B
8.4
26.4
B
7.8


5 Days


of


Culture


















TABLE 2-2-3









Example product 23



Concentration of Component (A)










5 ppm















Vis-








ual

10 ppm
50 ppm
100 ppm
500 ppm
























Judg-


Visual


Visual


Visual


Visual




Amount
ment
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of



of
of
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-



Biofilm
Bio-
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial



(mg)
film
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*


























After
3.2
A
7.1
2.9
A
7.5
2.2
A
7.2
2.4
A
7.9
1.9
A
7.2


1 Day


of


Culture


After
7.3
A
7.5
8.1
A
7.5
3.8
A
7.6
5.3
A
7.8
5.8
A
7.4


2 Days


of


Culture


After
22.1
B
7.6
18.6
B
7.1
7.1
A
7.8
6.9
A
7.6
10.1
A
7.6


3 Days


of


Culture


After
29.8
B
7.7
22.6
B
7.2
19.0
B
7.8
17.7
B
7.7
16.7
B
7.1


5 Days


of


Culture


















TABLE 2-2-4









Example product 26



Concentration of Component (A)










5 ppm















Vis-








ual

10 ppm
50 ppm
100 ppm
500 ppm
























Judg-


Visual


Visual


Visual


Visual




Amount
ment
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of



of
of
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-



Biofilm
Bio-
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial



(mg)
film
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*


























After
5.4
A
6.9
3.1
A
7.3
2.8
A
7.2
2.6
A
7.3
2.4
A
7.3


1 Day


of


Culture


After
6.9
A
7.4
5.1
A
7.7
4.6
A
7.3
5.1
A
7.5
4.6
A
7.4


2 Days


of


Culture


After
30.2
B
7.6
18.6
B
7.7
10.2
A
7.4
11.1
A
7.7
10.8
A
7.6


3 Days


of


Culture


After
38.7
B
7.4
33.1
B
7.8
20.7
B
7.6
19.1
B
7.6
16.4
B
7.6


5 Days


of


Culture


















TABLE 2-2-5









Example product 33



Concentration of Component (A)










5 ppm















Vis-








ual

10 ppm
50 ppm
100 ppm
500 ppm
























Judg-


Visual


Visual


Visual


Visual




Amount
ment
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of



of
of
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-



Biofilm
Bio-
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial



(mg)
film
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*


























After
19.3
B
7.7
8.7
A
7.6
5.3
A
7.4
4.8
A
7.3
3.5
A
7.2


1 Day


of


Culture


After
22.7
B
7.8
20.6
B
7.3
10.4
A
7.1
9.8
A
7.5
6.6
A
7.3


2 Days


of


Culture


After
28.6
B
7.6
25.4
B
7.4
18.8
B
7.6
14.9
B
7.4
12.9
B
7.2


3 Days


of


Culture


After
38.4
B
7.8
34.8
B
7.5
20.5
B
7.4
22.6
B
7.8
24.5
B
7.5


5 Days


of


Culture





*Log10CFU/mL















TABLE 2-3-1









Example product 35



Concentration of Component (A)










5 ppm















Vis-








ual

10 ppm
50 ppm
100 ppm
500 ppm
























Judg-


Visual


Visual


Visual


Visual




Amount
ment
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of



of
of
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-



Biofilm
Bio-
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial



(mg)
film
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*


























After 
2.1
A
7.6
2.1
A
7.9
1.9
A
7.8
2.0
A
7.5
1.7
A
7.9


1 Day


of


Culture


After
5.9
A
7.8
3.6
A
7.8
3.3
A
7.8
3.6
A
7.7
3.2
A
7.7


2 Days


of


Culture


After
30.1
B
7.7
9.9
A
7.8
5.3
A
7.7
5.8
A
7.6
4.7
A
7.8


3 Days


of


Culture


After
34.8
B
7.7
30.5
B
7.7
10.8
A
7.7
7.3
A
7.7
5.2
A
7.6


5 Days


of


Culture


















TABLE 2-3-2









Example product 38



Concentration of Component (A)










5 ppm















Vis-








ual

10 ppm
50 ppm
100 ppm
500 ppm
























Judg-


Visual


Visual


Visual


Visual




Amount
ment
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of
Amount
Judg-
No. of



of
of
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-
of
ment
Bac-



Biofilm
Bio-
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial
Biofilm
of
terial



(mg)
film
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*


























After
3.2
A
7.4
1.9
A
7.2
1.6
A
7.5
1.9
A
7.5
1.6
A
7.4


1 Day


of


Culture


After
6.5
A
7.5
3.9
A
7.2
3.0
A
7.9
3.3
A
7.4
3.3
A
7.3


2 Days


of


Culture


After
25.8
B
7.5
10.7
A
7.4
6.5
A
7.8
5.2
A
7.5
4.9
A
7.4


3 Days


of


Culture


After
35.1
B
7.8
33.2
B
7.5
11.8
B
7.7
10.6
A
7.9
8.5
A
7.4


5 Days


of


Culture


















TABLE 2-3-3









Example product 42



Concentration of Component (A)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
3.1
A
7.8
2.0
A
7.3
1.9
A
7.6


of Culture


After 2 Days
8.2
A
7.8
3.5
A
7.5
3.5
A
7.8


of Culture


After 3 Days
26.1
B
7.7
11.0
A
7.6
6.7
A
7.6


of Culture


After 5 Days
37.6
B
7.7
30.8
B
7.5
11.5
B
7.5


of Culture












Example product 42



Concentration of Component (A)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
2.5
A
7.1
1.8
A
7.6



of Culture



After 2 Days
3.2
A
7.5
2.9
A
7.6



of Culture



After 3 Days
5.4
A
7.4
4.6
A
7.5



of Culture



After 5 Days
10.3
A
7.3
8.2
A
7.3



of Culture



















TABLE 2-4-1









Comparative Product 3



Concentration of Component (A′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
28.5
B
8.1
27.3
B
7.6
25.3
B
7.9


of Culture


After 2 Days
43.6
C
7.6
42.9
C
8.3
49.7
C
8.2


of Culture


After 3 Days
53.4
C
7.9
51.8
C
8.2
53.8
C
8.0


of Culture


After 5 Days
63.1
C
8.3
60.8
C
8.3
63.4
C
7.9


of Culture












Comparative Product 3



Concentration of Component (A′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
27.3
B
7.9
23.6
B
8.1



of Culture



After 2 Days
48.6
C
8.5
45.7
C
8.2



of Culture



After 3 Days
57.3
C
8.5
50.6
C
8.0



of Culture



After 5 Days
61.7
C
8.4
57.1
C
7.9



of Culture



















TABLE 2-4-2









Comparative Product 11



Concentration of Component (A′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
22.3
B
8.1
29.4
B
7.8
24.2
B
7.7


of Culture


After 2 Days
42.3
C
7.8
41.6
C
8.2
48.2
C
8.1


of Culture


After 3 Days
56.1
C
8.1
53.4
C
8.2
52.9
C
8.3


of Culture


After 5 Days
60.8
C
8.1
62.6
C
8.1
61.7
C
8.2


of Culture












Comparative Product 11



Concentration of Component (A′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
23.4
B
7.8
21.7
B
8.2



of Culture



After 2 Days
41.6
C
8.2
47.3
C
8.1



of Culture



After 3 Days
53.4
C
8.4
52.4
C
8.0



of Culture



After 5 Days
60.7
C
8.3
58.6
C
8.3



of Culture



















TABLE 2-4-3









Comparative Product 13



Concentration of Component (A′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
25.3
B
8.0
24.3
B
7.8
21.7
B
8.0


of Culture


After 2 Days
48.7
C
8.0
49.0
C
8.2
48.5
C
8.2


of Culture


After 3 Days
52.4
C
8.3
50.6
C
8.1
52.3
C
8.1


of Culture


After 5 Days
61.4
C
8.1
61.6
C
8.3
60.4
C
8.0


of Culture












Comparative Product 13



Concentration of Component (A′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
26.2
B
7.9
20.7
B
8.3



of Culture



After 2 Days
42.8
C
8.1
41.7
C
8.1



of Culture



After 3 Days
56.8
C
8.2
55.3
C
8.4



of Culture



After 5 Days
64.5
C
8.3
63.7
C
8.5



of Culture



















TABLE 2-4-4









Comparative Product 18



Concentration of Component (A′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
31.3
B
7.6
34.2
B
7.9
35.2
B
8.2


of Culture


After 2 Days
45.8
C
8.0
49.7
C
8.4
43.5
C
8.1


of Culture


After 3 Days
60.1
C
8.2
58.1
C
8.4
57.6
C
8.3


of Culture


After 5 Days
63.5
C
8.1
63.1
C
8.2
60.9
C
8.4


of Culture












Comparative Product 18



Concentration of Component (A′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
25.4
B
8.0
26.1
B
8.2



of Culture



After 2 Days
45.2
C
8.2
49.9
C
8.1



of Culture



After 3 Days
56.8
C
8.3
56.4
C
8.3



of Culture



After 5 Days
64.2
C
8.2
62.3
C
8.1



of Culture



















TABLE 2-4-5









Comparative Product 26



Concentration of Component (A′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
33.3
B
8.2
32.8
B
7.9
30.7
B
8.1


of Culture


After 2 Days
44.6
C
8.1
47.3
C
7.8
46.7
C
8.2


of Culture


After 3 Days
57.3
C
8.1
56.1
C
8.1
52.9
C
8.3


of Culture


After 5 Days
62.1
C
8.3
67.3
C
8.0
63.4
C
8.1


of Culture












Comparative Product 26



Concentration of Component (A′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
30.4
B
7.8
34.6
B
8.2



of Culture



After 2 Days
49.5
C
8.1
50.2
C
8.3



of Culture



After 3 Days
56.1
C
8.3
56.8
C
8.3



of Culture



After 5 Days
63.8
C
8.5
64.0
C
8.3



of Culture







*Log10CFU/mL






Example 3
Test for Inhibition of Biofilm Formation in Silicone Tube
Pseudomonas (Psuedomonas aeruginosa NBRC 13275) and Klebsiella (Klebsiella pneumoniae ATCC13883) were precultured using soybean-casein digest agar [SCD Agar Medium; manufactured by Nihon Pharmaceutical Co., Ltd.] at 37 degrees Celsius for 24 hours. Example product 9, Example product 14 and Example Product 30 used in Example 1 and Comparative Product 3 and Comparative Product 26 were respectively diluted in 1 L of Mueller-Hinton Broth [manufactured by Nippon Becton Dickinson, Co., Ltd.] so that the concentration of the component (A) or component (A′) was 25 ppm or 100 ppm. A loop of the bacterial colonies on the agar medium described above was inoculated into each of the dilutions, and the culture solution suspended with bacteria were circulated in a silicone tube (inner diameter 5 mm, outer diameter 7 mm) manufactured by ARAM CORPORATION at 30 degrees Celsius, using a Masterflex metering pump system (system model No. 7553-80, Head No. 7016-21) manufactured by Cole-Parmer Instrument Company). Furthermore, the circulation of the culture solution was performed at a flow rate of 50 to 60 mL/min. Also, as a control, a test section was simultaneously provided in which bacteria were inoculated in Mueller-Hinton Broth with no added drugs.
The biofilm formation inside the silicone tube was observed by visual evaluation, and at the same time, the number of bacterial cells in the culture solution was measured. The state of the biofilm formation was graded such that the state in which no biofilm was formed was graded A, the state in which biofilm formation was commenced, with slight coloration on the surface of the silicone tube, was graded B, and the state in which biofilm formation was obvious was graded C.
The results are presented in Table 3-1-1 to Table 3-2-3. Table 3-1-1 to Table 3-1-3 present the results obtained with Pseudomonas (Pseudomonas aeruginosa NBRC 13275), while Table 3-2-1 to Table 3-2-3 present the results obtained with Klebsiella (Klebsiella pneumoniae ATCC 13883).







TABLE 3-1-1







Pseudomonas aeruginosa NBRC13275











Control
Example product 9
Example product 14









Concentration of Component (A)













—
25 ppm
100 ppm
25 ppm
100 ppm


















State of
No. of
State of
No. of
State of
No. of
State of
No. of
State of
No. of



Biofilm
Bacterial
Biofilm
Bacterial
Biofilm
Bacterial
Biofilm
Bacterial
Biofilm
Bacterial



Formation
Cells*
Formation
Cells*
Formation
Cells*
Formation
Cells*
Formation
Cells*





















After 1 Day of
B
7.9
A
7.3
A
7.1
A
7.1
A
7.3


Culture


After 3 Days of
C
8.5
A
7.9
A
7.6
A
7.6
A
7.5


Culture


After 5 Days of
C
8.7
A
7.8
A
7.9
A
7.9
A
7.6


Culture


After 7 Days of
C
8.6
B
7.9
A
7.8
B
7.8
A
7.9


Culture


After 14 Days of
C
8.3
B
7.6
B
7.8
B
7.8
A
7.7


Culture





*Log10CFU/mL















TABLE 3-1-2









Example product 30










25 ppm
100 ppm












State of
No. of
State of
No. of


Concentration of
Biofilm
Bacterial
Biofilm
Bacterial


Component (A)
Formation
Cells*
Formation
Cells*





After 1 Day of
A
7.3
A
7.2


Culture


After 3 Days of
A
7.8
A
7.5


Culture


After 5 Days of
A
7.8
A
7.7


Culture


After 7 Days of
A
7.7
A
7.6


Culture


After 14 Days of
A
7.8
A
7.5


Culture



















TABLE 3-1-3









Comparative Product 3
Comparative Product 26









Concentration of Component (A′)












25 ppm
100 ppm
25 ppm
100 ppm
















State of
No. of
State of
No. of
State of
No. of
State of
No. of



Biofilm
Bacterial
Biofilm
Bacterial
Biofilm
Bacterial
Biofilm
Bacterial



Formation
Cells*
Formation
Cells*
Formation
Cells*
Formation
Cells*



















After 1 Day of Culture
B
7.7
B
7.7
B
7.8
B
7.8


After 3 Days of
C
8.3
C
8.2
C
8.4
C
8.2


Culture


After 5 Days of
C
8.5
C
8.7
C
8.5
C
8.4


Culture


After 7 Days of
C
8.5
C
8.4
C
8.4
C
8.8


Culture


After 14 Days of
C
8.6
C
8.5
C
8.4
C
8.7


Culture





*Log10CFU/mL













TABLE 3-2-1







Klebsiella pneumoniae ATCC13883











Control
Example product 9
Example product 14









Concentration of Component (A)













—
25 ppm
100 ppm
25 ppm
100 ppm


















State of
No. of
State of
No. of
State of
No. of
State of
No. of
State of
No. of



Biofilm
Bacterial
Biofilm
Bacterial
Biofilm
Bacterial
Biofilm
Bacterial
Biofilm
Bacterial



Formation
Cells*
Formation
Cells*
Formation
Cells*
Formation
Cells*
Formation
Cells*





















After 1 Day of
B
8.2
A
7.6
A
7.5
A
7.9
A
7.7


Culture


After 3 Days of
C
8.9
A
8.0
A
7.9
A
7.8
A
7.9


Culture


After 5 Days of
C
8.6
A
8.1
A
8.1
A
8.0
A
7.9


Culture


After 7 Days of
C
8.8
B
8.0
A
8.3
B
8.1
A
8.2


Culture


After 14 Days of
C
8.6
B
8.0
A
8.2
B
8.2
A
8.0


Culture
















TABLE 3-2-2







Klebsiella pneumoniae ATCC13883









Example product 30



Concentration of Component (A)










25 ppm
100 ppm












State of
No. of
State of
No. of



Biofilm
Bacterial
Biofilm
Bacterial



Formation
Cells*
Formation
Cells*















After 1 Day of
A
7.6
A
7.8


Culture


After 3 Days of
A
7.8
A
7.8


Culture


After 5 Days of
A
7.9
A
7.9


Culture


After 7 Days of
A
8.2
A
7.8


Culture


After 14 Days of
A
8.1
A
7.9


Culture



















TABLE 3-2-3









Comparative Product 3
Comparative Product 26









Concentration of Component (A′)












25 ppm
100 ppm
25 ppm
100 ppm
















State of
No. of
State of
No. of
State of
No. of
State of
No. of



Biofilm
Bacterial
Biofilm
Bacterial
Biofilm
Bacterial
Biofilm
Bacterial



Formation
Cells*
Formation
Cells*
Formation
Cells*
Formation
Cells*



















After 1 Day of Culture
B
7.9
B
7.8
B
8.0
B
7.9


After 3 Days of Culture
C
8.1
C
8.1
C
8.5
C
8.1


After 5 Days of Culture
C
8.6
C
8.1
C
8.3
C
8.3


After 7 Days of Culture
C
8.4
C
8.3
C
8.4
C
8.2


After 14 Days of Culture
C
8.5
C
8.1
C
8.5
C
8.2





*Log10CFU/mL






In the case of using the products according to the present invention, it was confirmed that biofilm formation in silicone tubes could be remarkably inhibited. With regard to the measurement of the number of bacterial cells in the culture solutions tested simultaneously, the control, the example products, and the comparative products all resulted in bacterial growth. Thus, it is suggested that the biofilm formation is inhibited not by bactericidal or antimicrobial action.
Example 4
Verification for Ability to Inhibit Biofilm Formation and Bactericidal Power by Combinations of Alcohol or Adduct of Alcohol and Ethylene Oxide, and Surfactant
Using the compounds (A-2), (A-3) and (A-7) as the component (A), and the compounds (B-1), (B-5), (B-11) and (B-12) as the component (B), the composition of Example products 47 to 54 and Comparative Products 31 to 34 as indicated in Table 4 were prepared.
Pseudomonas (Pseudomonas aeruginosa NBRC 13275) was pre-cultured using soybean-casein digest agar [SCD Agar Medium; manufactured by Nihon Pharmaceutical Co., Ltd.] at 37 degrees Celsius for 24 hours. The colonies grown on the medium were scraped off and suspended in 10 mM sterile phosphate buffer (pH 7.2). The resultant suspension was washed by centrifuging twice at 5,000×g for 15 minutes at 10 degrees Celsius, and then suspended again in 10 mM sterile phosphate buffer (pH 7.2) to prepare a bacterial suspension in which the bacterial concentration was adjusted to 1.0 as the absorbance at 600 nm (OD600=1.0). Thereafter, 99 mL of Mueller-Hinton Broth [manufactured by Nippon Becton Dickinson, Co., Ltd.] was introduced into a 200-mL sterile screw cup [manufactured by EIKENKIZAI CO., LTD.], and at the same time, 1 mL each of the prepared biofilm formation inhibitor compositions was introduced thereto, the content of the cup being thoroughly mixed. The concentration of the component (A) was adjusted to 150 ppm, and then 0.1 mL of the bacterial suspension prepared as described above was inoculated thereinto.
Component (A)
(A-2) C10 alcohol [KALCOL 1098, manufactured by Kao Corporation]
(A-3) C12 alcohol [KALCOL 2098. manufactured by Kao Corporation]
(A-7) Adduct of C12 alcohol and 3 moles of ethylene oxide (NIKKOL BL-3SY, manufactured by Nikko Chemicals, Ltd., R′=C12 alkyl, p=3)
Component (B) surfactant [the number within the parentheses represents the average number of added moles of ethylene oxide]
(B-1) Sodium lauryl sulfate [EMAL 0, manufactured by Kao Corporation]
(B-5) Polyoxyethylene (12) lauryl ether [EMULGEN 120, manufactured by Kao Corporation]
(B-11) Polyoxyethylene (10) oleic acid ester [EMANON 4110, manufactured by Kao Corporation]
(B-12) Polyoxyethylene (25) hydrogenated castor oil [EMANON CH25, manufactured by Kao Corporation]
Using these culture solutions, static culture was initiated at 37 degrees Celsius. Immediately after, and at the designated time points of 3 hours and 24 hours after the contact with the biofilm formation inhibitor, the number of the bacterial cells in each of the culture solutions was measured, and at 12 hours and 24 hours after the initiation of culture, each of the culture solutions was centrifuged at 10,000×g for 30 minutes at 5 degrees Celsius. The precipitate was removed, dried in a vacuum desiccator for 24 hours, and weighed, with the weight thus measured being taken as the weight of the biofilm produced in the culture solution.

The results are presented in Table 4.

















TABLE 4











Example
Example
Example
Example
Example
Example






product
product
product
product
product
product






47
48
49
50
51
52





Inhibitor
Blended
(A)
A-2
1.5
1.5


1.5


Composition
Components

A-3


1.5
1.5

1.5



(wt %)

A-7




(B)
B-1

9

1.5

0.8





B-5
4.5

4.5

1.0





B-11





B-12

9.0

1.5

0.8















Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance



Water




Total
100
100
100
100
100
100













(A)/(B)
1/3
1/6
1/3
1/1
1.5/1
1.9/1















No. of Bacterial
Immediately
P. aeruginosa
8.08
8.08
8.08
8.08
8.08
8.08


Cells*
After Contact
K. pneumoniae
8.18
8.18
8.18
8.18
8.18
8.18



After 3 Hours
P. aeruginosa
8.23
7.51
8.16
7.6
8.13
7.79




K. pneumoniae
8.26
7.39
8.3
7.81
8.24
8.02



After 24 Hours
P. aeruginosa
8.67
8.35
8.2
8.52
8.71
8.53




K. pneumoniae
9.12
8.64
9.03
8.47
9.01
8.97


Amount of
After 12 Hours
P. aeruginosa
5.5
4.7
5.6
4.4
7.1
5.8


Biofilm (mg)

K. pneumoniae
3.4
3.0
3.4
2.8
6.8
9.3



After 24 Hours
P. aeruginosa
7.1
5.2
6.1
4.8
10.5
13.9




K. pneumoniae
6.3
6.3
4.2
5.1
16.3
17.2























Example
Example
Comp.
Comp.
Comp.
Comp.






product
product
product
product
product
product






53
54
31
32
33
34





Inhibitor
Blended
(A)
A-2




1.5


Composition
Components

A-3



(wt %)

A-7
1.5
1.5




(B)
B-1





9





B-5
10.0


4.5





B-11

1.5


0.305





B-12




0.305















Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance



Water




Total
100
100
100
100
100
100













(A)/(B)
1/6.7
1/1
—
—
2.5/1
—















No. of Bacterial
Immediately
P. aeruginosa
8.08
8.08
8.08
8.08
8.08
8.08


Cells*
After Contact
K. pneumoniae
8.18
8.18
8.18
8.18
8.18
8.18



After 3 Hours
P. aeruginosa
7.86
8.09
8.4
8.36
5.72
7.42




K. pneumoniae
8.03
8.16
8.38
8.41
4.58
7.58



After 24 Hours
P. aeruginosa
7.76
8.21
9.11
9.03
8.32
8.86




K. pneumoniae
8.32
8.59
9.73
9.74
8.92
9.43


Amount of
After 12 Hours
P. aeruginosa
4.9
6.8
20.6
18.2
10.1
12.5


Biofilm (mg)

K. pneumoniae
3.8
8.7
13.8
13.4
9.2
11.3



After 24 Hours
P. aeruginosa
6.2
11.2
68.1
57.2
68.9
53.7




K. pneumoniae
6.7
13.8
50.3
43.1
43.3
46.1





*Log10CFU/mL






As indicated in Table 4, it was found that when the ratio of the component (A) and the component (B) is set at 2 or less, and preferably 1/1 to 1/10, the effect of inhibiting biofilm formation on a long-term basis is manifested (Example products 47 to 54), but the composition having less amount of the component (B) (the ratio (A)/(B) is 2.5, Comparative Product 33) or the composition having only the component (B) (Comparative Product 32, Comparative Product 34) do not exhibit any biofilm formation inhibitory effect.
It also appeared that there was no correlation between the bactericidal effect and the biofilm formation inhibitory effect.
(2) Examples Using Compounds Represented by Formula (2)
Example 5
Blending of Biofilm Formation Inhibitor Composition, and Verification of Capability to Inhibit Biofilm Formation
Component (A) [Formula (2): EO represents an ethyleneoxy group, and m and n each represent the average number of added moles]




(A-21) C8 amine [FARMIN 08D, manufactured by Kao Corporation, R2=C8 alkyl, m+n=0]
(A-22) C10 amine [decylamine, manufactured by Wako Pure Chemical Industries, Ltd., R2=C10 alkyl, m+n=0]
(A-23) C12 amine [FARMIN 20D, manufactured by Kao Corporation, R2=C12 alkyl, m+n=0]
(A-24) C12 amine hydrochloride [dodecylamine hydrochloride, manufactured by Wako Pure Chemical Industries, Ltd., R2=C12, m+n=0]
(A-25) Cocoamine [FARMIN CS, manufactured by Kao Corporation, R2=C8-14 alkyl (coconut composition), m+n=0]
(A-26) Cocoamine acetate [ACETAMIN 24, manufactured by Kao Corporation, R2=C8-14 alkyl (coconut composition), m+n=0]
(A-27) Adduct of cocoamine and 2 moles of ethylene oxide [AMIET 102, manufactured by Kao Corporation, R2=C8-14 alkyl (coconut composition), m+n=2]
(A-28) Adduct of cocoamine and 5 moles of ethylene oxide [AMIET 105, manufactured by Kao Corporation, R2=C8-14 alkyl (coconut composition), m+n=5]
Component (A′) [Formula (2): EO represents an ethyleneoxy group, and m and n each represent the average number of added moles]




(A′-21) C3 amine [propylamine, manufactured by Wako Pure Chemical Industries, Ltd., R2′=C3 alkyl, m+n=0]
(A′-22) C3 amine hydrochloride [propylamine hydrochloride, manufactured by Wako Pure Chemical Industries, Ltd., R2′=C3 alkyl, m+n=0]
(A′-23) C6 amine [hexylamine, manufactured by Wako Pure Chemical Industries, Ltd., R2′=C6 alkyl, m+n=0]
(A′-24) C6 amine hydrochloride [hexylamine hydrochloride, manufactured by Wako Pure Chemical Industries, Ltd., R2′=C6 alkyl, m+n=0]
(A′-25) Hydrogenated tallow amine [FARMIN 86T, manufactured by Kao Corporation, R2′=C16, C18 alkyl, m+n=0]
(A′-26) C18 amine [FARMIN 80V, manufactured by Kao Corporation, R2′=C18 alkyl, m+n=0]
(A′-27) Oleylamine [FARMIN 0, manufactured by Kao Corporation, R2′=C18 alkenyl, m+n=0]
(A′-28) Adduct of hydrogenated tallow amine and 2 moles of ethylene oxide [AMIET 302, manufactured by Kao Corporation, R2′=C16, C18 alkyl, m+n=2]
(A′-29) Adduct of hydrogenated tallow amine and 20 moles of ethylene oxide [AMIET 320, manufactured by Kao Corporation, R2′=C16, C18 alkyl, m+n=20]
Component (B) surfactant [the number within the parentheses represents the average number of added moles of ethylene oxide]
<Anionic Surfactants>
(B-1) Sodium lauryl sulfate [EMAL 0, manufactured by Kao Corporation] (B-2) Sodium polyoxyethylene (2) lauryl ether sulfate [EMAL 20C, manufactured by Kao Corporation; active ingredient 25% by weight]
(B-3) Sodium polyoxyethylene (4.5) lauryl ether acetate [KAO AKYPO RLM45-NV, manufactured by Kao Corporation; active ingredient 24% by weight]
<Nonionic Surfactants>
(B-4) Polyoxyethylene (6) lauryl ether [EMULGEN 106, manufactured by Kao Corporation]
(B-5) Polyoxyethylene (12) lauryl ether [EMULGEN 120, manufactured by Kao Corporation]
(B-6) Lauryl glucoside [MYDOL 12, manufactured by Kao Corporation]
(B-7) Decyl glycerin monocaprylate [SY-GLYSTER MCA750, manufactured by SAKAMOTO YAKUHIN KOGYO CO., LTD.]
(B-8) Sorbitan monolaurate [RHEODOLSP-L10, manufactured by Kao Corporation]
(B-9) Polyoxyethylene (6) sorbitan monolaurate [RHEODOL TW-L106, manufactured by Kao Corporation]
(B-10) Polyoxyethylene (20) sorbitan monolaurate [RHEODOL TW-L120, manufactured by Kao Corporation]
Active ingredients were blended on a weight basis such that the concentration of the component (A) or component (A′) was fixed at 1% by weight, while the concentration of the component (B) was selected from 1% by weight, 3% by weight, 6% by weight and 10% by weight, with the balance being ion-exchanged water. The blend was diluted using Mueller-Hinton Broth [manufactured by Nippon Becton Dickinson Co., Ltd.] to a concentration of 100 ppm of the component (A) or component (A′). The dilution was taken in an amount of 2 mL and placed on a 24-well microplate [manufactured by ASAHI TECHNO GLASS CORPORATION].
Pseudomonas (Pseudomonas aeruginosa NBRC 13275), Serratia (Serratia marcescens NBRC 12648), and Klebsiella (Klebsiella pneumoniae ATCC 13883) were each pre-cultured at 37 degrees Celsius for 24 hours using soybean-casein digest agar [SCD Agar Medium; manufactured by Nihon Pharmaceutical Co., Ltd.], to form colonies. A very small amount of bacterial clusters from the colonies formed was inoculated into each of the above-described test solutions on the microplate, using a sterilized bamboo skewer. The inoculated solution was incubated at 37 degrees Celsius for 48 hours, then the culture solution was discarded, and the state of formation of the biofilm adhering on the microplate wall was observed by visual evaluation. The state of the biofilm was graded such that the state in which the biofilm covered 0 to less than 20% of the microplate wall surface was graded A; the state in which the biofilm covered 20% or more to less than 40% of the wall surface was graded B; the state in which the biofilm covered 40% or more to less than 60% of the wall surface was graded C; and the state in which the biofilm covered 60% or more of the wall surface was graded D. The results are presented in Table 5-1 to Table 5-4.















TABLE 5-1








Example
Example
Example
Example
Example
Example
Example
Example



product
product
product
product
product
product
product
product



55
56
57
58
59
60
61
62





Component


(A)


A-21
1.0
1.0
1.0


A-22



1.0
1.0
1.0
1.0
1.0


A-23


A-24


Surfactant (B)


[Anionic


Surfactant]


B-1
3.0


3.0


B-2


B-3


[Nonionic


Surfactant]


B-4




3.0


B-5

3.0



6.0


B-6






3.0


B-7


B-8


B-9







6.0


B-10


3.0


Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Water



Total
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0


State of


Biofilm


Inhibition


P. aeruginosa
B
B
B
B
A
A
A
A


S. marcescens
B
B
B
B
A
A
A
A


K. pneumoniae
B
B
B
B
A
A
A
A






Example
Example
Example
Example
Example
Example
Example
Example



product
product
product
product
product
product
product
product



63
64
65
66
67
68
69
70





Component


(A)


A-21


A-22


A-23
1.0
1.0
1.0
1.0
1.0


A-24





1.0
1.0
1.0


Surfactant (B)


[Anionic


Surfactant]


B-1


B-2
3.0


B-3





3.0


[Nonionic


Surfactant]


B-4

6.0


B-5


3.0



6.0


B-6


B-7



3.0


B-8







3.0


B-9


B-10




3.0


Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Water



Total
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0


State of


Biofilm


Inhibition


P. aeruginosa
B
A
A
A
A
B
A
A


S. marcescens
B
A
A
A
A
B
A
A


K. pneumoniae
B
A
A
A
A
B
A
A
























TABLE 5-2








Example
Example
Example
Example
Example
Example
Example
Example



product
product
product
product
product
product
product
product



71
72
73
74
75
76
77
78





Component


(A)


A-25
1.0
1.0
1.0
1.0
1.0


A-26





1.0
1.0
1.0


A-27


A-28


Surfactant (B)


[Anionic


Surfactant]


B-1
6.0


B-2





3.0


B-3


[Nonionic


Surfactant]


B-4

6.0


B-5


3.0



6.0


B-6







3.0


B-7



3.0


B-8


B-9


B-10




3.0


Ion-
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Exchanged


Water



Total
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0


State of


Biofilm


Inhibition


P. aeruginosa
B
A
A
A
A
B
A
A


S. marcescens
B
A
A
A
A
B
A
A


K. pneumoniae
B
A
A
A
A
B
A
A






Example
Example
Example
Example
Example
Example
Example
Example



product
product
product
product
product
product
product
product



79
80
81
82
83
84
85
86





Component


(A)


A-25


A-26
1.0


A-27

1.0
1.0
1.0
1.0


A-28





1.0
1.0
1.0


Surfactant (B)


[Anionic


Surfactant]


B-1


B-2





3.0


B-3

6.0


[Nonionic


Surfactant]


B-4


3.0


B-5






3.0


B-6


B-7







3.0


B-8



3.0


B-9
3.0


B-10




3.0


Ion-
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Exchanged


Water



Total
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0


State of


Biofilm


Inhibition


P. aeruginosa
A
B
A
A
A
B
B
B


S. marcescens
A
B
A
A
A
B
B
B


K. pneumoniae
A
B
A
A
A
B
B
B
























TABLE 5-3








Comparative
Comparative
Comparative
Comparative
Comparative
Comparative
Comparative
Comparative



Product
Product
Product
Product
Product
Product
Product
Product



35
36
37
38
39
40
41
42





Component


(A′)


A□-21
1.0
1.0
1.0


A□-22



1.0
1.0
1.0


A□-23






1.0
1.0


A□-24


A□-25


Surfactant (B)


[Anionic


Surfactant]


B-1
3.0


B-2



3.0


6.0


B-3


[Nonionic


Surfactant]


B-4


B-5

3.0


B-6




6.0


B-7


3.0


B-8







3.0


B-9


B-10





3.0


Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Water










Total
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0


State of


Biofilm


Inhibition


P. aeruginosa
D
D
D
D
D
D
D
D


S. marcescens
D
D
D
D
D
D
D
D


K. pneumoniae
D
D
D
D
D
D
D
D




















Comparative
Comparative
Comparative
Comparative
Comparative
Comparative
Comparative




Product
Product
Product
Product
Product
Product
Product




43
44
45
46
47
48
49







Component



(A′)



A□-21



A□-22



A□-23
1.0



A□-24

1.0
1.0
1.0



A□-25




1.0
1.0
1.0



Surfactant (B)



[Anionic



Surfactant]



B-1




3.0



B-2



B-3

6.0



[Nonionic



Surfactant]



B-4



B-5


6.0


3.0



B-6



B-7



3.0



B-8



B-9
3.0



B-10






3.0



Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance
Balance



Water










Total
100.0
100.0
100.0
100.0
100.0
100.0
100.0



State of



Biofilm



Inhibition



P. aeruginosa
D
D
D
D
D
D
D



S. marcescens
D
D
D
D
D
D
D



K. pneumoniae
D
D
D
D
D
D
D























TABLE 5-4








Comparative
Comparative
Comparative
Comparative
Comparative
Comparative



Product
Product
Product
Product
Product
Product



50
51
52
53
54
55





Component (A′)


A□-26
1.0
1.0
1.0


A□-27



1.0
1.0
1.0


A□-28


A□-29


Surfactant (B)


[Anionic


Surfactant]


B-1
3.0


B-2


B-3



3.0


[Nonionic


Surfactant]


B-4


B-5

3.0


B-6




6.0


B-7


B-8





3.0


B-9


3.0


B-10


Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance


Water








Total
100.0
100.0
100.0
100.0
100.0
100.0


State of Biofilm


Inhibition


P. aeruginosa
D
D
D
D
D
D


S. marcescens
D
D
D
D
D
D


K. pneumoniae
D
D
D
D
D
D






Comparative
Comparative
Comparative
Comparative
Comparative
Comparative



Product
Product
Product
Product
Product
Product



56
57
58
59
60
61





Component (A′)


A□-26


A□-27


A□-28
1.0
1.0
1.0


A□-29



1.0
1.0
1.0


Surfactant (B)


[Anionic


Surfactant]


B-1



3.0


B-2
3.0


B-3


[Nonionic


Surfactant]


B-4


B-5




6.0


B-6


B-7

3.0


B-8


B-9





3.0


B-10


3.0


Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance


Water








Total
100.0
100.0
100.0
100.0
100.0
100.0


State of Biofilm


Inhibition


P. aeruginosa
D
D
D
D
D
D


S. marcescens
D
D
D
D
D
D


K. pneumoniae
D
D
D
D
D
D









Example 6
Test for Reduction of Biofilm Production in Large Volume Plastic Cup
Pseudomonas (Pseudomonas aeruginosa NBRC 13275) was pre-cultured at 37 degrees Celsius for 24 hours, using soybean-casein digest agar [SCD Agar Medium; manufactured by Nihon Pharmaceutical Co., Ltd.]. The colonies formed on the medium were scraped off and suspended in 10 mM sterile phosphate buffer (pH 7.2), and then the suspension was washed by centrifuging twice at 5,000×g for 15 minutes at 10 degrees Celsius. The resultant precipitate was again suspended in 10 mM sterile phosphate buffer (pH 7.2) to prepare a bacterial suspension in which the bacterial concentration was adjusted to 1.0 as the absorbance at 600 nm (OD600=1.0). Thereafter, 100 mL of Mueller-Hinton Broth [manufactured by Nippon Becton Dickinson Co., Ltd.] and each of the 15 species of the test drugs selected in Example 5 were introduced into a 200-mL sterilized screw cup [manufactured by EIKENKIZAI CO., LTD.], and thoroughly mixed. The concentration of the component (A) or component (A′) was adjusted to 5 ppm, 10 ppm, 50 ppm, 100 ppm or 500 ppm, and then 0.1 mL of the bacterial suspension prepared as described above was inoculated thereinto. Furthermore, as a control, a test section was simultaneously provided in which the bacteria were inoculated as described above into Mueller-Hinton Broth with no added drugs.
These were statically cultured at 37 degrees Celsius. At the designated time points of 1 day, 2 days, 3 days and 5 days after the initiation of culture, the number of bacterial cells in the culture solution was measured, and the biofilm formed inside the cup was observed by visual evaluation. Subsequently, the culture solution was centrifuged at 10,000×g for 30 minutes at 5 degrees Celsius, and the precipitate was removed, dried in a vacuum desiccator for 24 hours, and weighed, with the weight thus measured being taken as the weight of the biofilm produced in the culture solution. Furthermore, the state of the biofilm formation was graded such that the state in which no biofilm was confirmed in the cup was graded A; the state in which the formation of a biofilm started inside the cup at the air-liquid interface was graded B; and the state in which a biofilm was formed to extend from the air-liquid interface down to the culture solution was graded C.
The results are presented in Table 6-1-1 to Table 6-3-5.










TABLE 6-1-1









Control
Example product 55









Concentration of Component (A)











—
5 ppm
10 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1
32.4
B
8.0
19.8
B
7.8
20.1
B
8.0


Day of


Culture


After 2
49.9
C
8.2
22.6
B
8.2
20.6
B
8.1


Days of


Culture


After 3
51.3
C
8.4
24.1
B
8.3
29.0
B
8.2


Days of


Culture


After 5
63.9
C
8.5
34.6
B
8.5
32.7
B
8.2


Days of


Culture












Example product 55



Concentration of Component (A)











50 ppm
100 ppm
500 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1
11.6
A
7.9
13.2
A
8.0
12.3
A
8.0


Day of


Culture
18.5
B
8.0
17.5
B
8.1
16.0
B
7.8


After 2


Days of


Culture
24.3
B
8.0
20.3
B
8.2
21.7
B
7.9


After 3


Days of


Culture
29.7
B
8.1
26.7
B
8.2
23.6
B
8.1


After 5


Days of


Culture


















TABLE 6-1-2









Example product 59



Concentration of Component (A)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
2.8
A
7.7
1.9
A
7.9
1.8
A
7.9


of Culture


After 2 Days
5.1
A
7.9
3.2
A
8.0
2.5
A
7.8


of Culture


After 3 Days
22.0
B
7.8
9.4
A
8.0
4.6
A
7.9


of Culture


Alter 5 Days
30.8
B
7.7
24.9
B
7.7
10.0
A
7.8


of Culture












Example product 59



Conentration of Component (A)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
2.0
A
8.0
2.1
A
7.7



of Culture



After 2 Days
2.5
A
7.9
3.1
A
7.8



of Culture



After 3 Days
3.1
A
7.8
4.8
A
7.9



of Culture



Alter 5 Days
7.5
A
7.7
7.1
A
7.5



of Culture



















TABLE 6-1-3









Example product 62



Concentration of Component (A)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
3.2
A
7.7
1.6
A
7.7
1.4
A
7.8


of Culture


After 2 Days
5.2
A
7.6
2.8
A
7.9
3.9
A
7.6


of Culture


After 3 Days
19.6
B
7.7
7.9
A
7.8
5.2
A
7.7


of Culture


After 5 Days
31.4
B
8.0
26.8
B
7.9
16.4
B
7.4


of Culture












Example product 62



Concentration of Component (A)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
2.2
A
7.6
1.6
A
7.5



of Culture



After 2 Days
3.5
A
7.7
2.8
A
7.6



of Culture



After 3 Days
4.0
A
7.9
4.6
A
7.5



of Culture



After 5 Days
7.8
A
7.5
4.9
A
7.4



of Culture



















TABLE 6-1-4









Example product 65



Concentration of Component (A)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
3.0
A
7.5
1.7
A
7.8
2.7
A
7.7


of Culture


After 2 Days
6.9
A
8.0
3.6
A
7.9
2.6
A
7.6


of Culture


After 3 Days
21.8
B
7.9
10.1
A
7.5
5.1
A
7.8


of Culture


After 5 Days
40.1
B
7.8
32.7
B
7.8
8.9
A
7.9


of Culture












Example product 65



Concentration of Component (A)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
2.6
A
7.4
2.1
A
7.5



of Culture



After 2 Days
2.6
A
7.5
3.9
A
7.6



of Culture



After 3 Days
5.7
A
7.7
4.7
A
7.8



of Culture



After 5 Days
9.8
A
7.6
8.4
A
7.5



of Culture



















TABLE 6-1-5









Example product 70



Concentration of Component (A)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
3.8
A
7.7
2.9
A
7.6
1.9
A
7.7


of Culture


After 2 Days
6.1
A
7.8
3.1
A
7.7
3.6
A
7.8


of Culture


After 3 Days
25.3
B
7.7
11.6
A
7.6
5.9
A
7.6


of Culture


After 5 Days
31.9
B
7.7
36.8
B
7.8
10.7
B
8


of Culture












Example product 70



Concentration of Component (A)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
1.8
A
7.5
1.6
A
7.6



of Culture



After 2 Days
3.7
A
7.6
3.9
A
7.7



of Culture



After 3 Days
5.0
A
7.4
4.1
A
7.6



of Culture



After 5 Days
6.8
A
7.8
5.7
A
7.8



of Culture







*Log10CFU/mL















TABLE 6-2-1









Example product 72



Concentration of Component (A)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
2.8
A
7.6
1.9
A
7.6
2.0
A
7.5


of Culture


After 2 Days
7.5
A
7.7
2.7
A
7.4
3.8
A
7.6


of Culture


After 3 Days
19.6
B
7.9
8.6
A
7.5
5.9
A
7.5


of Culture


After 5 Days
32.1
B
7.6
26.5
B
7.6
9.9
A
7.9


of Culture












Example product 72



Concentration of Component (A)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
1.8
A
7.3
2.0
A
7.6



of Culture



After 2 Days
6.1
A
7.5
2.9
A
7.5



of Culture



After 3 Days
5.9
A
7.4
4.2
A
7.6



of Culture



After 5 Days
10.2
A
7.7
7.8
A
7.7



of Culture



















TABLE 6-2-2









Example product 74



Concentration of Component (A)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
4.2
A
7.5
2.3
A
7.5
1.8
A
7.6


of Culture


After 2 Days
7.7
A
7.7
4.2
A
7.6
2.9
A
8.0


of Culture


After 3 Days
19.8
B
7.5
10.2
A
7.9
7.5
A
7.9


of Culture


After 5 Days
30.8
B
7.8
36.2
B
7.8
13.7
B
7.7


of Culture












Example product 74



Concentration of Component (A)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
2.6
A
7.9
1.5
A
7.5



of Culture



After 2 Days
3.8
A
7.4
2.7
A
7.4



of Culture



After 3 Days
5.5
A
7.8
4.5
A
7.3



of Culture



After 5 Days
9.9
A
7.8
7.6
A
7.5



of Culture



















TABLE 6-2-3









Example product 77



Concentration of Component (A)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
3.8
A
7.3
2.1
A
7.2
1.8
A
7.5


of Culture


After 2 Days
6.8
A
7.3
7.9
A
7.4
2.7
A
7.7


of Culture


After 3 Days
21.0
B
7.5
17.9
B
7.5
6.8
A
7.6


of Culture


After 5 Days
28.5
B
7.6
20.7
B
7.3
17.6
B
7.8


of Culture












Example product 77



Concentration of Component (A)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
2.0
A
7.6
1.5
A
7.1



of Culture



After 2 Days
5.1
A
7.5
6.4
A
7.6



of Culture



After 3 Days
7.6
A
7.8
8.4
A
7.4



of Culture



After 5 Days
15.7
B
7.9
15.3
B
7.5



of Culture



















TABLE 6-2-4









Example product 82



Concentration of Component (A)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
20.4
B
8.0
11.8
A
7.9
8.9
A
7.8


of Culture


After 2 Days
21.9
B
7.9
21.0
B
8.1
11.3
A
8.2


of Culture


After 3 Days
28.3
B
7.9
23.6
B
8.0
22.2
B
8.3


of Culture


After 5 Days
31.5
B
8.3
30.4
B
7.9
27.1
B
8.0


of Culture












Example product 82



Concentration of Component (A)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
11.2
A
7.7
10.5
A
8.0



of Culture



After 2 Days
15.1
A
7.9
12.6
A
8.1



of Culture



After 3 Days
22.8
B
8.2
22.8
B
8.0



of Culture



After 5 Days
30.0
B
8.1
27.4
B
7.9



of Culture



















TABLE 6-2-5









Example product 84



Concentration of Component (A)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
19.6
B
8.0
19.2
B
7.9
9.8
A
8.0


of Culture


After 2 Days
20.7
B
8.2
23.1
B
7.9
16.4
B
8.0


of Culture


After 3 Days
26.1
B
8.2
27.6
B
8.0
25.5
B
8.0


of Culture


After 5 Days
32.1
B
8.4
30.5
B
8.1
30.2
B
8.3


of Culture












Example product 84



Concentration of Component (A)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
10.6
A
8
9.9
A
8.0



of Culture



After 2 Days
15.7
B
7.9
16.2
B
7.9



of Culture



After 3 Days
24.1
B
8.0
18.5
B
8.0



of Culture



After 5 Days
28.0
B
8.2
27.6
B
8.2



of Culture







*Log10CFU/mL















TABLE 6-3-1









Comparative product 35



Concentration of Component (A′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
29.1
B
8.2
27.3
B
7.8
27.4
B
7.8


of Culture


After 2 Days
45.1
C
8.1
40.6
C
8.2
42.8
C
8.1


of Culture


After 3 Days
50.8
C
7.9
53.7
C
8.1
52.5
C
8.2


of Culture


After 5 Days
63.1
C
8.1
57.4
C
8.1
63.0
C
8.1


of Culture












Comparative product 35



Concentration of Component (A′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
25.0
B
7.6
20.9
B
8.0



of Culture



After 2 Days
46.5
C
8.4
44.4
C
8.1



of Culture



After 3 Days
51.7
C
8.2
51.6
C
8.2



of Culture



After 5 Days
59.7
C
8.3
58.1
C
7.9



of Culture



















TABLE 6-3-2









Comparative product 46



Concentration of Component (A′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
24.3
B
7.8
30.4
B
8.0
28.3
B
7.9


of Culture


After 2 Days
39.7
C
7.9
45.3
C
8.1
45.2
C
8.2


of Culture


After 3 Days
56.2
C
7.9
57.1
C
8.3
51.5
C
8.1


of Culture


After 5 Days
57.9
C
8.1
66.0
C
8.2
62.7
C
8.1


of Culture












Comparative product 46



Concentration of Component (A′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
20.4
B
7.7
23.4
B
8.0



of Culture



After 2 Days
39.8
C
7.9
45.6
C
7.9



of Culture



After 3 Days
46.7
C
8.1
54.5
C
8.0



of Culture



After 5 Days
55.8
C
8.0
59.1
C
8.1



of Culture



















TABLE 6-3-3









Comparative product 49



Concentration of Component (A′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
27.4
B
8.0
20.2
B
7.9
25.6
B
8.2


of Culture


After 2 Days
46.8
C
7.9
46.3
C
8.1
39.9
C
8.1


of Culture


After 3 Days
50.1
C
8.1
46.1
C
8.1
51.0
C
8.2


of Culture


After 5 Days
57.6
C
8.1
62.8
C
8.0
59.4
C
8.1


of Culture












Comparative product 49



Concentration of Component (A′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
23.9
B
8.0
21.6
B
8.1



of Culture



After 2 Days
41.5
C
8.1
34.6
C
8.2



of Culture



After 3 Days
53.4
C
8.1
57.2
C
8.2



of Culture



After 5 Days
64.2
C
8.1
59.3
C
8.3



of Culture



















TABLE 6-3-4









Comparative product 51



Concentration of Component (A′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
28.4
B
7.8
32.4
B
8.1
36.7
B
8.0


of Culture


After 2 Days
46.0
C
7.6
40.9
C
7.9
40.1
C
8.1


of Culture


After 3 Days
58.3
C
8.1
56.7
C
8.3
50.8
C
8.3


of Culture


After 5 Days
66.3
C
8.0
60.7
C
8.1
62.3
C
8.1


of Culture












Comparative product 51



Concentration of Component (A′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
32.5
B
8.0
30.0
B
7.9



of Culture



After 2 Days
44.0
C
8.1
48.7
C
8.0



of Culture



After 3 Days
51.6
C
8.2
55.2
C
8.2



of Culture



After 5 Days
63.4
C
8.1
60.9
C
8.2



of Culture



















TABLE 6-3-5









Comparative product 56



Concentration of Component (A′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
29.5
B
8.0
33.3
B
8.1
31.1
B
8.0


of Culture


After 2 Days
42.1
C
8.2
45.8
C
7.9
45.6
C
8.1


of Culture


After 3 Days
53.4
C
8.1
51.6
C
8.1
51.7
C
8.3


of Culture


After 5 Days
59.1
C
8.5
64.2
C
8.3
61.5
C
8.2


of Culture












Comparative product 56



Concentration of Component (A′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
32.4
B
7.9
28.3
B
8.1



of Culture



After 2 Days
46.2
C
8.1
43.5
C
8.2



of Culture



After 3 Days
59.4
C
8.2
57.2
C
8.1



of Culture



After 5 Days
61.3
C
8.3
66.0
C
8.0



of Culture







*Log10CFU/mL






From the above results, it was found that the example products employing the compounds represented by the Formula (2) can effectively inhibit biofilm formation.
(3) Examples Using the Compounds Represented by Formula (3)
Example 7
Blending of Biofilm Formation Inhibitor Compositions, and Verification of Ability to Inhibit Biofilm Formation
Component (C) R3—SH
(C-31) C8 thiol [1-octanethiol, manufactured by Wako Pure Chemical Industries, Ltd., R3=C8 alkyl]
(C-32) C10 thiol [1-decanethiol, manufactured by Wako Pure Chemical Industries, Ltd., R3=C10 alkyl]
(C-33) C12 thiol [THIOKALCOL 20, manufactured by Kao Corporation, R3=C12 alkyl]
(C-34) C12 thiol (tertiary) [t-dodecanethiol, manufactured by Wako Pure Chemical Industries, Ltd., R3=C12 tertiary alkyl]
Component (C′) R3′-SH
(C′-31) C3 thiol [L-propanethiol, manufactured by Wako Pure Chemical Industries, Ltd., R3′=C3 alkyl]
(C′-32) C3 thiol (secondary) [2-propanethiol, manufactured by Wako Pure Chemical Industries, Ltd., R3′=C3 alkyl]
(C′-33) C6 thiol [1-hexanethiol, manufactured by Wako Pure Chemical Industries, Ltd., R3′=C6 alkyl]
(C′-34) C16 thiol [1-hexadecanethiol, manufactured by Wako Pure Chemical Industries, Ltd., R3′=C16 alkyl]
(C′-35) C18 thiol [1-octadecanethiol, manufactured by Wako Pure Chemical Industries, Ltd., R3′=C18 alkyl]
Component (B) surfactant [the number within the parentheses represents the average number of added moles of ethylene oxide]
<Anionic Surfactants>
(B-1) Sodium lauryl sulfate [EMAL 0, manufactured by Kao Corporation] (B-2) Sodium polyoxyethylene (2) lauryl ether sulfate [EMAL 20C, manufactured by Kao Corporation; active ingredient 25% by weight]
(B-3) Sodium polyoxyethylene (4.5) lauryl ether acetate [KAO AKYPO RLM45-NV, manufactured by Kao Corporation; active ingredient 24% by weight]
<Nonionic Surfactants>
(B-4) Polyoxyethylene (6) lauryl ether [EMULGEN 106, manufactured by Kao Corporation]
(B-5) Polyoxyethylene (12) lauryl ether [EMULGEN 120, manufactured by Kao Corporation]
(B-6) Lauryl glucoside [MYDOL 12, manufactured by Kao Corporation]
(B-7) Decyl glycerin monocaprylate [SY-GLYSTER MCA750, manufactured by SAKAMOTO YAKUHIN KOGYO CO., LTD.]
(B-8) Sorbitan monolaurate [RHEODOL SP-L10, manufactured by Kao Corporation]
(B-9) Polyoxyethylene (6) sorbitan monolaurate [RHEODOL TW-L106, manufactured by Kao Corporation]
(B-10) Polyoxyethylene (20) sorbitan monolaurate [RHEODOL TW-L120, manufactured by Kao Corporation]
The active ingredients were blended on a weight basis such that the concentration of the component (C) or component (C′) was fixed at 1% by weight, while the concentration of the component (B) was selected from 1% by weight, 3% by weight, 6% by weight and 10% by weight, with the balance being ion-exchanged water. The blend was diluted using Mueller-Hinton Broth [manufactured by Nippon Becton Dickinson Co., Ltd.] to a concentration of 100 ppm of the component (C) or component (C′). The dilution was taken in an amount of 2 mL and placed on a 24-well microplate [manufactured by ASAHI TECHNO GLASS CORPORATION].
Pseudomonas (Pseudomonas aeruginosa NBRC 13275), Serratia (Serratia marcescens NBRC 12648), and Klebsiella (Klebsiella pneumoniae ATCC 13883) were each pre-cultured at 37 degrees Celsius for 24 hours using soybean-casein digest agar [SCD Agar Medium; manufactured by Nihon Pharmaceutical Co., Ltd.], to form colonies. A very small amount of bacterial clusters from the colonies formed was inoculated into each of the above-described test solutions on the microplate, using a sterilized bamboo skewer. The inoculated solution was incubated at 37 degrees Celsius for 48 hours, then the culture solution was discarded, and the state of formation of the biofilm adhering on the microplate wall was observed by visual evaluation. The state of the biofilm was graded such that the state in which the biofilm covered 0 to less than 20% of the microplate wall surface was graded A; the state in which the biofilm covered 20% or more to less than 40% of the wall surface was graded B; the state in which the biofilm covered 40% or more to less than 60% of the wall surface was graded C; and the state in which the biofilm covered 60% or more of the wall surface was graded D. The results are presented in Table 7-1 to Table 7-2.















TABLE 7-1








Example
Example
Example
Example
Example
Example
Example
Example



product
product
product
product
product
product
product
product



87
88
89
90
91
92
93
94





Component


(C)


C-31
1.0
1.0
1.0
1.0


C-32




1.0
1.0
1.0
1.0


C-33


C-34


Surfactant (B)


[Anionic


Surfactant]


B-1
2.0


B-2





3.0


B-3


[Nonionic


Surfactant]


B-4




2.0


B-5


B-6

3.0


B-7


6.0


B-8






6.0


B-9



3.0


B-10







3.0


Ion-
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Exchanged


Water



Total
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 


State of


Biofilm


Inhibition


P. aeruginosa
B
B
B
B
A
A
A
A


S. marcescens
B
B
B
B
A
A
A
A


K. pneumoniae
B
B
B
B
A
A
A
A






Example
Example
Example
Example
Example
Example
Example
Example



product
product
product
product
product
product
product
product



95
96
97
98
99
100
101
102





Component


(C)


C-31


C-32


C-33
1.0
1.0
1.0
1.0


C-34




1.0
1.0
1.0
1.0


Surfactant (B)


[Anionic


Surfactant]


B-1


B-2


B-3







6.0


[Nonionic


Surfactant]


B-4
3.0



6.0


B-5

6.0



3.0


B-6


3.0


B-7






3.0


B-8


B-9


B-10



3.0


Ion-
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Exchanged


Water



Total
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 


State of


Biofilm


Inhibition


P. aeruginosa
A
A
A
A
A
A
A
A


S. marcescens
A
A
A
A
A
A
A
A


K. pneumoniae
A
A
A
A
A
A
A
A
























TABLE 7-2








Comparative
Comparative
Comparative
Comparative
Comparative
Comparative
Comparative
Comparative



Product
Product
Product
Product
Product
Product
Product
Product



62
63
64
65
66
67
68
69





Component


(C′)


C-31
1.0
1.0
1.0


C-32



1.0
1.0
1.0


C-33






1.0
1.0


C-34


C-35


Surfactant (B)


[Anionic


Surfactant]


B-1
2.0


B-2



2.0


B-3






2.0


[Nonionic


Surfactant]


B-4


B-5

3.0





6.0


B-6




6.0


B-7


3.0




B-8





3.0


B-9


B-10


Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Water










Total
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 


State of


Biofilm


Inhibition


P. aeruginosa
D
D
D
D
D
D
D
D


S. marcescens
D
D
D
D
D
D
D
D


K. pneumoniae
D
D
D
D
D
D
D
D




















Comparative
Comparative
Comparative
Comparative
Comparative
Comparative
Comparative




Product
Product
Product
Product
Product
Product
Product




70
71
72
73
74
75
76







Component



(C′)



C-31



C-32



C-33
1.0



C-34

1.0
1.0
1.0



C-35




1.0
1.0
1.0



Surfactant (B)



[Anionic



Surfactant]



B-1

3.0



B-2



B-3




3.0



[Nonionic



Surfactant]



B-4



B-5



B-6


3.0



B-7





6.0



B-8



B-9
3.0



B-10



6.0


3.0



Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance
Balance



Water










Total
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 



State of



Biofilm



Inhibition



P. aeruginosa
D
D
D
D
D
D
D



S. marcescens
D
D
D
D
D
D
D



K. pneumoniae
D
D
D
D
D
D
D










Example 8
Test for Reduction of Biofilm Production in Large Volume Plastic Cup
Pseudomonas (Pseudomonas aeruginosa NBRC 13275) was pre-cultured at 37 degrees Celsius for 24 hours, using soybean-casein digest agar [SCD Agar Medium; manufactured by Nihon Pharmaceutical Co., Ltd.]. The colonies formed on the medium were scraped off and suspended in 10 mM sterile phosphate buffer (pH 7.2), and then the suspension was washed by centrifuging twice at 5,000×g for 15 minutes at 10 degrees Celsius. The resultant precipitate was again suspended in 10 mM sterile phosphate buffer (pH 7.2) to prepare a bacterial suspension in which the bacterial concentration was adjusted to 1.0 as the absorbance at 600 nm (OD600=1.0). Thereafter, 100 mL of Mueller-Hinton Broth [manufactured by Nippon Becton Dickinson Co., Ltd.] and each of the 15 species of the test drugs selected from Example 7 were introduced into a 200-mL sterilized screw cup [manufactured by EIKENKIZAI CO., LTD.], and thoroughly mixed. The concentration of the component (C) or component (C′) was adjusted to 5 ppm, 10 ppm, 50 ppm, 100 ppm or 500 ppm, and then 0.1 mL of the bacterial suspension prepared as described above was inoculated thereinto. Furthermore, as a control, a test section was simultaneously provided in which the bacteria were inoculated as described above into Mueller-Hinton Broth with no added drugs.
These were statically cultured at 37 degrees Celsius. At the designated time points of 1 day, 2 days, 3 days and 5 days after the initiation of culture, the number of bacterial cells in the culture solution was measured, and the biofilm formed inside the cup was observed by visual evaluation. Subsequently, the culture solution was centrifuged at 10,000×g for 30 minutes at 5 degrees Celsius, and the precipitate was removed, dried in a vacuum desiccator for 24 hours, and weighed, with the weight thus measured being taken as the weight of the biofilm produced in the culture solution. Furthermore, the state of the biofilm formation was graded such that the state in which no biofilm was confirmed in the cup was graded A; the state in which the formation of a biofilm started inside the cup at the air-liquid interface was graded B; and the state in which a biofilm was formed to extend from the air-liquid interface down to the culture solution was graded C.
The results are presented in Table 8-1-1 to Table 8-2-5.









TABLE 8-1-1









Control



Concentration of Component (C)



—













No of



Amount of
Visual Judgment
Bacterial



Biofilm (mg)
of Biofilm
Cells*














After 1 Day of Culture
33.1
B
7.9


After 2 Days of Culture
48.7
C
8.2


After 3 Days of Culture
50.8
C
8.3


After 5 Days of Culture
62.6
C
8.2


















TABLE 8-1-2









Example product 88



Concentration of Component (C)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
21.3
B
7.8
17.6
B
8.0
11.3
A
7.7


of Culture


After 2 Days
25.4
B
7.8
22.2
B
8.0
18.0
B
7.9


of Culture


After 3 Days
29.6
B
8.2
29.0
B
8.1
28.1
B
8.0


of Culture


After 5 Days
30.7
B
8.1
32.8
B
8.0
30.2
B
7.9


of Culture












Example product 88



Concentration of Component (C)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
10.9
A
7.8
10.3
A
7.8



of Culture



After 2 Days
16.7
B
8.0
15.6
B
8.0



of Culture



After 3 Days
20.7
B
8.2
19.7
B
7.9



of Culture



After 5 Days
28.3
B
8.4
23.9
B
8.0



of Culture



















TABLE 8-1-3









Example product 91



Concentration of Component (C)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
2.5
A
7.7
1.7
A
7.6
1.9
A
7.3


of Culture


After 2 Days
4.8
A
7.5
3.5
A
7.9
2.9
A
7.2


of Culture


After 3 Days
19.7
B
7.9
9.7
A
7.7
4.3
A
7.6


of Culture


After 5 Days
33.1
B
8.0
32.1
B
7.4
13.0
A
7.7


of Culture












Example product 91



Concentration of Component (C)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
1.8
A
7.5
1.8
A
7.5



of Culture



After 2 Days
3.3
A
7.4
2.5
A
7.4



of Culture



After 3 Days
3.7
A
7.3
3.4
A
7.9



of Culture



After 5 Days
6.2
A
7.8
5.5
A
7.8



of Culture



















TABLE 8-1-4









Example product 95



Concentration of Component (C)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
2.2
A
7.6
2.1
A
7.8
1.9
A
7.9


of Culture


After 2 Days
5.3
A
7.8
3.3
A
7.9
3.8
A
7.9


of Culture


After 3 Days
28.1
B
7.6
10.1
A
8.0
5.9
A
7.8


of Culture


After 5 Days
35.0
B
7.4
31.6
B
7.8
13.7
A
7.9


of Culture












Example product 95



Concentration of Component (C)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
1.5
A
7.5
2.0
A
7.8



of Culture



After 2 Days
3.9
A
7.6
3.8
A
7.7



of Culture



After 3 Days
5.5
A
7.9
5.2
A
7.8



of Culture



After 5 Days
7.4
A
7.8
5.9
A
7.9



of Culture



















TABLE 8-1-5









Example product 100



Concentration of Component (C)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
2.7
A
7.7
2.0
A
7.4
2.4
A
7.8


of Culture


After 2 Days
6.4
A
7.9
3.8
A
7.5
2.8
A
7.9


of Culture


After 3 Days
24.8
B
7.6
10.6
A
7.6
6.3
A
7.7


of Culture


After 5 Days
37.9
B
7.8
32.5
B
7.8
17.2
B
7.8


of Culture












Example product 100



Concentration of Component (C)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
2.5
A
7.5
1.8
A
7.4



of Culture



After 2 Days
2.3
A
7.7
3.8
A
7.6



of Culture



After 3 Days
5.9
A
7.4
4.4
A
7.8



of Culture



After 5 Days
12.0
A
7.3
8.2
A
7.8



of Culture







*Log10CFU/mL















TABLE 8-2-1









Comparative product 64



Concentration of Component (C′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
29.1
B
8.2
27.6
B
7.5
26.1
B
8.0


of Culture


After 2 Days
44.7
C
7.8
42.4
C
8.4
49.8
C
8.1


of Culture


After 3 Days
54.2
C
7.8
51.6
C
8.1
53.1
C
8.2


of Culture


After 5 Days
64.2
C
8.1
60.2
C
8.2
63.8
C
8.1


of Culture












Comparative product 64



Concentration of Component (C′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
26.5
B
8.0
23.8
B
8.1



of Culture



After 2 Days
48.9
C
8.4
45.1
C
8.3



of Culture



After 3 Days
58.1
C
8.4
52.1
C
8.2



of Culture



After 5 Days
61.2
C
8.3
56.7
C
8.2



of Culture



















TABLE 8-2-2









Comparative product 66



Concentration of Component (C′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
22.1
B
8.0
30.8
B
7.9
25.1
B
7.8


of Culture


After 2 Days
41.8
C
7.9
40.6
C
8.3
49.5
C
8.0


of Culture


After 3 Days
53.1
C
8.1
54.1
C
8.2
52.6
C
8.2


of Culture


After 5 Days
61.5
C
8.1
68.4
C
8.4
60.9
C
8.2


of Culture












Comparative product 66



Concentration of Component (C′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
22.4
B
7.9
20.6
B
8.1



of Culture



After 2 Days
45.2
C
8.1
42.9
C
8.3



of Culture



After 3 Days
51.8
C
8.3
51.4
C
8.1



of Culture



After 5 Days
62.3
C
8.7
57.4
C
8.4



of Culture



















TABLE 8-2-3









Comparative product 70



Concentration of Component (C′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
27.4
B
7.9
22.3
B
7.9
20.7
B
8.1


of Culture


After 2 Days
45.2
C
8.0
49.8
C
8.1
47.6
C
8.2


of Culture


After 3 Days
51.6
C
8.6
59.6
C
8.2
52.4
C
8.4


of Culture


After 5 Days
60.7
C
8.4
60.7
C
8.3
59.1
C
8.2


of Culture












Comparative product 70



Concentration of Component (C′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
25.3
B
8.0
21.3
B
8.2



of Culture



After 2 Days
46.2
C
8.1
40.8
C
8.2



of Culture



After 3 Days
55.8
C
8.3
52.6
C
8.3



of Culture



After 5 Days
65.0
C
8.3
61.7
C
8.5



of Culture



















TABLE 8-2-4









Comparative product 71



Concentration of Component (C′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
30.2
B
7.9
33.5
B
7.7
33.6
B
8.1


of Culture


After 2 Days
44.7
C
8.1
48.6
C
7.9
45.2
C
8.1


of Culture


After 3 Days
59.2
C
8.3
56.2
C
8.3
56.8
C
8.2


of Culture


After 5 Days
62.5
C
8.2
61.2
C
8.3
61.5
C
8.3


of Culture












Comparative product 71



Concentration of Component (C′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
25.1
B
8.1
25.7
B
8.1



of Culture



After 2 Days
44.3
C
8.2
50.6
C
8.1



of Culture



After 3 Days
58.7
C
8.1
57.0
C
8.2



of Culture



After 5 Days
66.8
C
8.3
62.0
C
8.1



of Culture



















TABLE 8-2-5









Comparative product 75



Concentration of Component (C′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
32.5
B
8.2
31.9
B
8.0
31.5
B
8.0


of Culture


After 2 Days
46.8
C
8.2
47.4
C
7.9
44.7
C
8.1


of Culture


After 3 Days
56.9
C
8.1
55.1
C
8.2
56.8
C
8.2


of Culture


After 5 Days
61.4
C
8.0
68.9
C
8.1
63.4
C
8.1


of Culture












Comparative product 75



Concentration of Component (C′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
32.8
B
7.8
33.9
B
8.1



of Culture



After 2 Days
50.0
C
8.2
49.9
C
8.3



of Culture



After 3 Days
58.3
C
8.5
57.2
C
8.2



of Culture



After 5 Days
65.2
C
8.4
67.7
C
8.2



of Culture







*Log10CFU/mL






From the above results, it was found that the example products containing the compounds represented by the Formula (3) can effectively inhibit biofilm formation on a long-term basis.
(4) Examples Using the Compounds Represented by Formula (4)
Example 9
Blending of Biofilm Formation Inhibitor Compositions, and Verification of Ability to Inhibit Biofilm Formation
Component (C) R40—CO—CH3 
(C-41) C8 alcohol-acetic acid ester [octyl acetate, manufactured by Wako Pure Chemical Industries, Ltd., R4=C8 alkyl]
(C-42) C10 alcohol-acetic acid ester [decyl acetate, manufactured by Wako Pure Chemical Industries, Ltd., R4=C10 alkyl] (C-43) C12 alcohol-acetic acid ester [dodecyl acetate, manufactured by Wako Pure Chemical Industries, Ltd., R4=C12 alkyl]
Component (C′) R4′O—CO—CH3 
(C′-41) C2 alcohol-acetic acid ester [ethyl acetate, manufactured by Wako Pure Chemical Industries, Ltd., R4′=C2 alkyl]
(C′-42) C4 alcohol-acetic acid ester [butyl acetate, manufactured by Wako Pure Chemical Industries, Ltd., R4′=C4 alkyl]
(C′-43) C6 alcohol-acetic acid ester [hexyl acetate, manufactured by Wako Pure Chemical Industries, Ltd., R4′=C6 alkyl]
(C′-44) C16 alcohol-acetic acid ester [hexadecyl acetate, manufactured by Wako Pure Chemical Industries, Ltd., R4′=C16 alkyl]
(C′-45) C18 alcohol-acetic acid ester [octadecyl acetate, manufactured by Wako Pure Chemical Industries, Ltd., R4′=C18 alkyl]
Component (B) surfactant [the number within the parentheses represents the average number of added moles of ethylene oxide]
<Anionic Surfactants>
(B-1) Sodium lauryl sulfate [EMAL 0, manufactured by Kao Corporation]
(B-2) Sodium polyoxyethylene (2) lauryl ether sulfate [EMAL 20C, manufactured by Kao Corporation; active ingredient 25% by weight]
(B-3) Sodium polyoxyethylene (4.5) lauryl ether acetate [KAO AKYPO RLM45-NV, manufactured by Kao Corporation; active ingredient 24% by weight]
<Nonionic Surfactants>
(B-4) Polyoxyethylene (6) lauryl ether [EMULGEN 106, manufactured by Kao Corporation]
(B-5) Polyoxyethylene (12) lauryl ether [EMULGEN 120, manufactured by Kao Corporation]
(B-6) Lauryl glucoside [MYDOL 12, manufactured by Kao Corporation]
(B-7) Decyl glycerin monocaprylate [SY-GLYSTER MCA750, manufactured by SAKAMOTO YAKUHIN KOGYO CO., LTD.]
(B-8) Sorbitan monolaurate [RHEODOL SP-L10, manufactured by Kao Corporation]
(B-9) Polyoxyethylene (6) sorbitan monolaurate [RHEODOL TW-L106, manufactured by Kao Corporation]
(B-10) Polyoxyethylene (20) sorbitan monolaurate [RHEODOL TW-L120, manufactured by Kao Corporation]
The active ingredients were blended on a weight basis such that the concentration of the component (C) or component (C′) was fixed at 1% by weight, while the concentration of the component (B) was selected from 1% by weight, 3% by weight, 6% by weight and 10% by weight, with the balance being ion-exchanged water. The blend was diluted using Mueller-Hinton Broth [manufactured by Nippon Becton Dickinson Co., Ltd.] to a concentration of 100 ppm of the component (C) or component (C′). The dilution was taken in an amount of 2 mL and placed on a 24-well microplate [manufactured by ASAHI TECHNO GLASS CORPORATION].
Pseudomonas (Pseudomonas aeruginosa NBRC 13275), Serratia (Serratia marcescens NBRC 12648), and Klebsiella (Klebsiella pneumoniae ATCC 13883) were each pre-cultured at 37 degrees Celsius for 24 hours using soybean-casein digest agar [SCD Agar Medium; manufactured by Nihon Pharmaceutical Co., Ltd.], to form colonies. A very small amount of bacterial clusters from the colonies formed was inoculated into each of the above-described test solutions on the microplate, using a sterilized bamboo skewer. The inoculated solution was incubated at 37 degrees Celsius for 48 hours, then the culture solution was discarded, and the state of formation of the biofilm adhering on the microplate wall was observed by visual evaluation. The state of the biofilm was graded such that the state in which the biofilm covered 0 to less than 20% of the microplate wall surface was graded A; the state in which the biofilm covered 20% or more to less than 40% of the wall surface was graded B; the state in which the biofilm covered 40% or more to less than 60% of the wall surface was graded C; and the state in which the biofilm covered 60% or more of the wall surface was graded D. The results are presented in Table 9-1 to Table 9-2.




















TABLE 9-1







Exam-

Exam-

Exam-

Exam-








ple
Example
ple
Example
ple
Example
ple
Example
Example
Example
Example
Example



product
product
product
product
product
product
product
product
product
product
product
product



103
104
105
106
107
108
109
110
111
112
113
114




























Component (C)














C-41
1.0
1.0
1.0
1.0


C-42




1.0
1.0
1.0
1.0


C-43








1.0
1.0
1.0
1.0


Surfactant (B)


[Anionic


Surfactant]


B-1

3.0


B-2





3.0


B-3











3.0


[Nonionic


Surfactant]


B-4




3.0



6.0


B-5
3.0








3.0


B-6










3.0


B-7


3.0


B-8






3.0


B-9



6.0


B-10







3.0


Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Water



Total
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 


State of


Biofilm


Inhibition


P. aeruginosa
B
B
B
B
A
A
A
A
A
A
A
A


S. marcescens
B
B
B
B
A
A
A
A
A
A
A
A


K. pneumoniae
B
B
B
B
A
A
A
A
A
A
A
A
























TABLE 9-2








Comparative
Comparative
Comparative
Comparative
Comparative
Comparative
Comparative
Comparative



Product
Product
Product
Product
Product
Product
Product
Product



77
78
79
80
81
82
83
84





Component (C)


C′-41
1.0
1.0
1.0


C′-42



1.0
1.0
1.0


C′-43






1.0
1.0


C′-44










C′-45










Surfactant (B)


[Anionic


Surfactant]


B-1
3.0









B-2



3.0


B-3






6.0



[Nonionic


Surfactant]


B-4


B-5

3.0





6.0


B-6




3.0





B-7





3.0




B-8


3.0


B-9










B-10










Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance
Balance
Balance


Water










Total
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 


State of Biofilm


Inhibition


P. aeruginosa
D
D
D
D
D
D
D
D


S. marcescens
D
D
D
D
D
D
D
D


K. pneumoniae
D
D
D
D
D
D
D
D




















Comparative
Comparative
Comparative
Comparative
Comparative
Comparative
Comparative




Product
Product
Product
Product
Product
Product
Product




85
86
87
88
89
90
91







Component (C)



C′-41



C′-42



C′-43
1.0



C′-44

1.0
1.0
1.0



C′-45




1.0
1.0
1.0



Surfactant (B)



[Anionic



Surfactant]



B-1

6.0



B-2



B-3




3.0



[Nonionic



Surfactant]



B-4



B-5



B-6


3.0



B-7





3.0



B-8



B-9
3.0



B-10



3.0


3.0



Ion-Exchanged
Balance
Balance
Balance
Balance
Balance
Balance
Balance



Water










Total
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 



State of Biofilm



Inhibition



P. aeruginosa
D
D
D
D
D
D
D



S. marcescens
D
D
D
D
D
D
D



K. pneumoniae
D
D
D
D
D
D
D










Example 10
Test for Reduction of Biofilm Production in Large Volume Plastic Cup
Pseudomonas (Pseudomonas aeruginosa NBRC 13275) was pre-cultured at 37 degrees Celsius for 24 hours, using soybean-casein digest agar [SCD Agar Medium; manufactured by Nihon Pharmaceutical Co., Ltd.]. The colonies formed on the medium were scraped off and suspended in 10 mM sterile phosphate buffer (pH 7.2), and then the suspension was washed by centrifuging twice at 5,000×g for 15 minutes at 10 degrees Celsius. The resultant precipitate was again suspended in 10 mM sterile phosphate buffer (pH 7.2) to prepare a bacterial suspension in which the bacterial concentration was adjusted to 1.0 as the absorbance at 600 nm (OD600=1.0). Thereafter, 100 mL of Mueller-Hinton Broth [manufactured by Nippon Becton Dickinson Co., Ltd.] and each of the 15 species of the test drugs selected in Example 9 were introduced into a 200-mL sterilized screw cup [manufactured by EIKENKIZAI CO., LTD.], and thoroughly mixed. The concentration of the component (C) or component (C′) was adjusted to 5 ppm, 10 ppm, 50 ppm, 100 ppm or 500 ppm, and then 0.1 mL of the bacterial suspension prepared as described above was inoculated thereinto. Furthermore, as a control, a test section was simultaneously provided in which the bacteria were inoculated as described above into Mueller-Hinton Broth with no added drugs.
These were statically cultured at 37 degrees Celsius. At the designated time points of 1 day, 2 days, 3 days and 5 days after the initiation of culture, the number of bacterial cells in the culture solution was measured, and the biofilm formed inside the cup was observed by visual evaluation. Subsequently, the culture solution was centrifuged at 10,000×g for 30 minutes at 5 degrees Celsius, and the precipitate was removed, dried in a vacuum desiccator for 24 hours, and weighed, with the weight thus measured being taken as the weight of the biofilm produced in the culture solution. Furthermore, the state of the biofilm formation was graded such that the state in which no biofilm was confirmed in the cup was graded A; the state in which the formation of a biofilm started inside the cup at the air-liquid interface was graded B; and the state in which a biofilm was formed to extend from the air-liquid interface down to the culture solution was graded C.
The results are presented in Table 10-1-1 to Table 10-2-5.









TABLE 10-1-1









Control



—













No. of


Concentration of
Amount of
Visual Judgment
Bacterial


Component (C)
Biofilm (mg)
of Biofilm
Cells*





After 1 Day of Culture
33.1
B
7.9


After 2 Days of Culture
48.7
C
8.2


After 3 Days of Culture
50.8
C
8.3


After 5 Days of Culture
62.6
C
8.2


















TABLE 10-1-2









Example product 103



Concentration of Component (C)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
22.8
B
7.9
18.4
B
8.1
10.8
A
7.8


of Culture


After 2 Days
24.6
B
7.8
23.1
B
8.0
17.6
B
8.0


of Culture


After 3 Days
30.1
B
8.2
29.7
B
8.0
23.4
B
7.9


of Culture


After 5 Days
32.7
B
8.2
31.5
B
7.9
31.7
B
7.8


of Culture












Example product 103



Concentration of Component (C)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
11.0
A
7.9
10.8
A
7.9



of Culture



After 2 Days
15.8
B
7.9
16.8
B
8.0



of Culture



After 3 Days
21.0
B
8.1
20.6
B
7.9



of Culture



After 5 Days
27.6
B
8.2
22.5
B
8.1



of Culture



















TABLE 10-1-3









Example product 107



Concentration of Component (C)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
3.2
A
7.8
2.1
A
7.8
1.9
A
7.6


of Culture


After 2 Days
5.1
A
7.8
3.6
A
7.9
3.8
A
7.5


of Culture


After 3 Days
20.0
B
7.7
9.9
A
7.8
5.1
A
7.7


of Culture


After 5 Days
31.8
B
7.6
33.5
B
7.9
15.0
A
7.9


of Culture












Example product 107



Concentration of Component (C)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
2.0
A
7.6
1.9
A
7.8



of Culture



After 2 Days
3.9
A
7.5
2.8
A
7.6



of Culture



After 3 Days
4.1
A
7.5
3.6
A
7.8



of Culture



After 5 Days
7.2
A
7.9
5.9
A
7.7



of Culture



















TABLE 10-1-4









Example product 112



Concentration of Component (A)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
2.8
A
7.8
2.0
A
7.4
2.6
A
7.9


of Culture


After 2 Days
6.8
A
7.9
3.4
A
7.7
2.9
A
7.8


of Culture


After 3 Days
25.3
B
8.0
11.5
A
7.8
7.1
A
7.7


of Culture


After 5 Days
38.1
B
8.1
35.0
B
7.8
18.3
B
7.6


of Culture












Example product 112



Concentration of Component (A)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
2.2
A
7.6
1.7
A
7.6



of Culture



After 2 Days
2.6
A
7.8
3.6
A
7.4



of Culture



After 3 Days
6.2
A
7.5
4.8
A
7.9



of Culture



After 5 Days
13.8
A
7.4
9.2
A
7.9



of Culture







*Log10CFU/mL















TABLE 10-2-1









Comparative product 78



Concentration of Component (C′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
30.2
B
8.1
28.4
B
7.8
25.1
B
8.0


of Culture


After 2 Days
45.1
C
7.9
41.6
C
8.0
50.0
C
8.2


of Culture


After 3 Days
53.6
C
7.9
50.9
C
7.9
51.4
C
8.0


of Culture


After 5 Days
63.8
C
7.8
61.7
C
8.1
61.6
C
8.3


of Culture












Comparative product 78



Concentration of Component (C′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
25.6
B
7.8
22.4
B
8.0



of Culture



After 2 Days
49.1
C
8.1
44.9
C
8.3



of Culture



After 3 Days
54.6
C
8.1
51.6
C
8.1



of Culture



After 5 Days
59.8
C
8.2
58.4
C
8.2



of Culture



















TABLE 10-2-2









Comparative product 81



Concentration of Component (C′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
20.8
B
7.9
29.7
B
8.0
21.8
B
7.9


of Culture


After 2 Days
40.5
C
7.9
42.1
C
8.2
46.2
C
8.1


of Culture


After 3 Days
52.4
C
8.0
50.7
C
8.1
50.8
C
8.4


of Culture


After 5 Days
63.8
C
8.2
64.3
C
8.1
59.7
C
8.1


of Culture












Comparative product 81



Concentration of Component (C′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
21.5
B
8.1
18.7
B
8.0



of Culture



After 2 Days
43.2
C
8.3
36.8
C
8.1



of Culture



After 3 Days
50.7
C
8.3
49.6
C
8.6



of Culture



After 5 Days
61.4
C
8.5
58.8
C
8.5



of Culture



















TABLE 10-2-3









Comparative product 83



Concentration of Component (C′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
25.6
B
8.0
21.7
B
7.9
19.3
B
7.9


of Culture


After 2 Days
44.7
C
8.1
48.6
C
8.1
42.1
C
8.1


of Culture


After 3 Days
52.4
C
8.3
60.3
C
8.5
50.9
C
8.2


of Culture


After 5 Days
58.6
C
8.2
62.8
C
8.4
58.3
C
8.4


of Culture












Comparative product 83



Concentration of Component (C′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
23.6
B
8.0
20.9
B
8.0



of Culture



After 2 Days
42.7
C
8.2
41.3
C
8.1



of Culture



After 3 Days
53.4
C
8.3
52.4
C
8.1



of Culture



After 5 Days
63.5
C
8.1
64.2
C
8.2



of Culture



















TABLE 10-2-4









Comparative product 87



Concentration of Component (C′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
29.1
B
7.8
32.8
B
7.9
35.8
B
8.0


of Culture


After 2 Days
45.3
C
7.9
46.1
C
7.9
44.6
C
7.9


of Culture


After 3 Days
54.2
C
8.0
55.9
C
8.1
53.1
C
8.1


of Culture


After 5 Days
61.2
C
8.2
60.2
C
8.2
60.7
C
8.3


of Culture












Comparative product 87



Concentration of Component (C′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
29.4
B
8.0
25.7
B
8.2



of Culture



After 2 Days
42.6
C
8.1
50.6
C
8.3



of Culture



After 3 Days
56.1
C
8.3
57.0
C
8.1



of Culture



After 5 Days
66.8
C
8.1
62.0
C
8.2



of Culture



















TABLE 10-2-5









Comparative product 91



Concentration of Component (C′)











5 ppm
10 ppm
50 ppm

















Amount
Visual

Amount
Visual

Amount
Visual




of
Judgment
No. of
of
Judgment
No. of
of
Judgment
No. of



Biofilm
of
Bacterial
Biofilm
of
Bacterial
Biofilm
of
Bacterial



(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*





After 1 Day
32.5
B
8.1
32.1
B
7.9
32.8
B
8.0


of Culture


After 2 Days
44.1
C
8.2
45.2
C
8.1
46.8
C
8.2


of Culture


After 3 Days
56.8
C
8.0
57.6
C
8.0
55.2
C
8.1


of Culture


After 5 Days
62.0
C
8.1
67.4
C
8.3
62.7
C
8.3


of Culture












Comparative product 91



Concentration of Component (C′)










100 ppm
500 ppm
















Amount
Visual

Amount
Visual





of
Judgment
No. of
of
Judgment
No. of




Biofilm
of
Bacterial
Biofilm
of
Bacterial




(mg)
Biofilm
Cells*
(mg)
Biofilm
Cells*







After 1 Day
34.0
B
7.9
32.8
B
8.0



of Culture



After 2 Days
48.3
C
8.1
50.7
C
8.2



of Culture



After 3 Days
51.6
C
8.3
56.4
C
8.1



of Culture



After 5 Days
63.1
C
8.2
65.1
C
8.3



of Culture







*Log10CFU/mL






From the above results, it was found that the example products containing the compounds represented by the Formula (4) can effectively inhibit biofilm formation on a long-term basis.

## Claims

1. A method of inhibiting biofilm formation, which method comprises:
contacting a biofilm formation inhibitor composition with microorganisms, wherein the biofilm formation inhibitor composition comprises the following component (A):
(A) at least one or more selected from compounds represented by Formula (1) and Formula (2) or a salt thereof:




wherein R1 and R2, which are identical or different, each represents an alkyl group or an alkenyl group, which is a straight chain or branched chain having 8 to 14 carbon atoms; EO represents an ethyleneoxy group; p represents an integer from 1 to 5; and m and n are each an average number of added moles, with m+n being a number from 2 to 30; and
(B) a surfactant;
wherein the weight ratio of the component (A) and the component (B), (A)/(B), is not more than 2.

2. The method of inhibiting biofilm formation according to claim 1, wherein the contact between the biofilm formation inhibitor composition and microorganisms is carried out continuously.

3. The method of inhibiting biofilm formation according to claim 1,
wherein the concentration by weight of the component (A) at the time of use of the biofilm formation inhibitor composition is 1 to 10,000 ppm.

4. The method according to claim 1, wherein the component (B) surfactant is at least one or more selected from the group consisting of anionic surfactants, and nonionic surfactants other than the component (A).