JP2003088875A - Method for killing legionella genus bacteria coexisting with amoeba in aqueous system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for killing Legionella genus bacteria coexisting with amoebas in an aqueous system by which Legionella genus bacteria coexistent with amoebas in the aqueous system can be killed and the bacteria can be prevented from growing by using a nonhalogen compound. SOLUTION: The method for killing Legionella genus bacteria coexisting with amoebas in an aqueous system is carried out by adding maleimides to the aqueous system in which amoebas and Legionella genus bacteria coexist.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for erasing Legionella spp. Which coexists with amoeba in an aqueous system. More specifically, the present invention eradicates Legionella spp. Coexisting with amoeba in circulating cooling water of a refrigerating device, cold / hot water system such as circulating hot water in a 24-hour bath, and heat storage water system to prevent its growth. The present invention relates to a method for eradicating Legionella spp. Which coexists with amoeba in a water system capable of producing.

[0002]

2. Description of the Related Art In a refrigerating apparatus used for an air conditioner, a refrigerator, a freezer or the like, circulating water cooled by using an open type cooling tower is often used in order to efficiently perform heat exchange. In such circulating water, microorganisms and the like are likely to enter from the outside and proliferate, which causes a decrease in heat exchange efficiency of the heat exchanger due to slime and the like and causes obstacles such as clogging of the filter, and pathogenic bacteria, particularly Legionella spp. When they multiply and scatter, they cause special pneumonia, such as local illness and Pontiac fever. As a measure against the problem caused by such microorganisms, a method of injecting an antibacterial agent into the circulating water system to suppress the growth of bacteria, physically cleaning and cleaning the inside of the apparatus, or chemically cleaning with a cleaning agent Methods have been used. And, as a bactericide to eradicate Legionella spp, various compounds have been proposed in the past, but many halogen-based compounds, these compounds, in recent years, so that the impact on the environment is regarded as a problem. In recent years, new drugs are required from the viewpoint of safety. In addition, even if a drug having a bactericidal effect in the laboratory was used in an actually operating water system, it was often not always possible to obtain a sufficient effect. In addition, it is known that Legionella spp. In nature naturally parasitize and reproduce even when predated by bacterial predatory protozoa such as amoeba. As a result of the studies by the present inventors, it has been found that conventionally known fungicides have almost no bactericidal effect on Legionella bacteria coexisting with amoeba. Therefore, an effective eradication method for Legionella spp. That coexists with amoeba in the water system is needed. On the other hand, maleimides are known to have antibacterial activity. For example, Japanese Examined Patent Publication No. 39-17546 discloses a agricultural / industrial fungicide containing maleimide or N-carbamoylmaleimide, which is useful for preventing rice blast and for preventing white water slime in the pulp manufacturing process. It is illustrated. Also,
Japanese Patent Laid-Open No. 63-156703 discloses a ship bottom antifouling paint,
2-halo-N- (2 ', 6' used for fishing net antifouling paint etc.
An aquatic biocontrol agent containing a -dialkylphenyl) maleimide has been proposed. However, it has not been known that maleimides are effective in removing Legionella spp. That coexist with Amoeba by adding it to a cooling water system or the like.

[0003]

DISCLOSURE OF THE INVENTION The present invention uses a non-halogen compound to eradicate Legionella spp. Which coexist with amoeba in an aqueous system and coexist with amoeba in an aqueous system capable of preventing its growth. The purpose of the present invention is to provide a method for erasing Legionella spp.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that maleimides have a biocidal activity against amoeba in an aqueous system in which amoeba and Legionella spp. Coexist. It was found that the bacteria of the genus Legionella can be effectively eradicated, and the present invention was completed based on this finding. That is,
The present invention provides (1) a method for eradicating Legionella spp. That coexists with amoeba in an aqueous system, characterized in that maleimides are added to an aqueous system coexisting with Amoeba and Legionella spp., And (2) maleimides Is a compound represented by the general formula [1], wherein the method for disinfecting Legionella spp. Coexisting with amoeba in the water system according to item 1,

[Chemical 2] (However, in the formula, R¹And R²Is hydrogen or 1 to 5 carbon atoms
Is an alkyl group of R ³Is hydrogen, unsubstituted or
C1-C10 alkyl substituted with a droxyl group
Number of carbon atoms substituted with a group, unsubstituted or hydroxyl group
5-8 cycloalkyl groups, unsubstituted or hydroxy
Group substituted with a phenyl group or unsubstituted or hydro
It is a benzyl group substituted with a xyl group. ),
It is something.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION In the method for erasing Legionella spp. Which coexists with Amoeba in the water system of the present invention, maleimides are added to the aqueous system coexist with Amoeba and Legionella spp. The amoeba existing in the water system to which the method of the present invention is applied includes, for example, Acanthamoeba, Amoeba, Vahlkampfia, P.
Examples include elomyxa, Entamoeba, Arcella, Difflugia. Legionella spp. To which the method of the present invention can be applied is a gram-negative pole of a new genus discovered in Philadelphia in 1976 with the occurrence of severe pneumonia of unknown cause, the so-called local military man's disease. It is a monococcal bacterium. At present, many subspecies and serogroups are known in addition to the reference species Legionella pneumophila (11 serogroup), but the method of the present invention can be applied to all of these bacterial species. By applying the method of the present invention, in the circulating cooling water of the refrigerating apparatus, the cold hot water system such as the circulating hot water of the 24-hour bath, the heat storage water system, etc., the Legionella spp. Which coexist with the amoeba is sterilized and its growth is prevented. be able to.

The maleimides used in the present invention are not particularly limited. For example, in addition to maleimide, a compound having a substituent at the nitrogen atom of maleimide, a compound having a substituent at the 2-position and / or 3-position carbon atoms, Examples thereof include compounds having a condensed ring of a maleimide ring and a benzene ring. In the present invention, as the maleimide, the compound represented by the general formula [1] can be preferably used.

[Chemical 3] In the general formula [1], R ¹ and R ² are hydrogen or an alkyl group having 1 to 5 carbon atoms, and R ³ is hydrogen, an unsubstituted or hydroxyl group-substituted alkyl group having 1 to 10 carbon atoms. A cycloalkyl group having 5 to 8 carbon atoms which is unsubstituted or substituted with a hydroxyl group, a phenyl group which is unsubstituted or substituted with a hydroxyl group, or a benzyl group which is unsubstituted or substituted with a hydroxyl group. Examples of the compound represented by the general formula [1] include maleimide, N-methylmaleimide, N-ethylmaleimide, citraconic acid imide, N-methylcitraconic acid imide and N.
-Ethylcitraconic acid imide, 2,3-dimethylmaleic acid imide, N-methyl-2,3-dimethylmaleic acid imide, N-ethyl-2,3-dimethylmaleic acid imide, N- (2-hydroxyethyl) maleimide , N-cyclohexylmaleimide, N-4-hydroxycyclohexylmaleimide, N-phenylmaleimide, Np-hydroxyphenylmaleimide, N-benzylmaleimide and the like. These maleimides are
One kind may be used alone, or two or more kinds may be used in combination.

In the present invention, these maleimides may be used as they are, may be used as a powder or a tablet by blending with a bulking agent and other agents, or may be dissolved in an appropriate solvent to be used as a liquid agent. You can also Water can be preferably used as a solvent in the case of a liquid agent, but a hydrophilic solvent can be used if necessary. Examples of the hydrophilic solvent include alcohols, glycols, amides, glycol esters, esters and the like. In the method of the present invention, there is no particular limitation on the amount of maleimides added to the water system in which Amoeba and Legionella spp. Coexist, and it can be appropriately selected depending on the state of the water system, but usually the concentration of maleimides in water is The amount is preferably 0.1 to 1,000 mg / L, more preferably 0.5 to 100 mg / L, and further preferably 1 to 50 mg / L.
There is no particular limitation on the pH of the aqueous system to be applied, and it can be sufficiently used within the usual pH range of about 3 to 10.
In the method of disinfecting Legionella spp. That coexists with the amoeba of the present invention, a biocide, a microbial growth inhibitor, a corrosion inhibitor, a scale inhibitor, an antifoaming agent, a surfactant, etc. may be used in combination, if necessary. can do. According to the method of the present invention, a non-halogen compound is used to eradicate Legionella spp. That coexist with amoeba in circulating cooling water of a refrigerating device, cold hot water system such as circulating hot water of a 24-hour bath, and heat storage water system,
The proliferation can be effectively prevented.

[0008]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Comparative Example 1 In 1,000 mL of pure water, yeast extract 10.0 g, ACES buffer 10.0 g, L-cysteine monohydrochloride 0.4 g,
Soluble iron pyrophosphate 0.25 g, α-ketoglutarate 1.0
g, 1.5 g of activated carbon powder and 15.0 g of agar, and pH
A BCYE α plate medium of 6.9 was prepared. Legionella pneumophila was inoculated on this plate medium and cultured at 36 ° C. for 2 days. This cultured Legionella bacterium was suspended in a phosphate buffer of pH 7 at a rate of 10 ⁴ cells / mL. This solution is used as a Legionella standard solution. 0.1 mL of this Legionella standard solution was applied to a BCYE α plate medium and cultured at 36 ° C for 5 days, and then the number of viable cells was measured by counting the number of colonies, which was 2.5 × 10 ⁴ cells / mL. there were. Example 1 1 mL of each of the Legionella standard solutions prepared in Comparative Example 1 was placed in three culture flasks. Then, maleimide was added to each flask so that the concentration was 1 mg / L, 5 mg / L or 10 mg / L, and the mixture was cultured with shaking at 37 ° C. for 24 hours, and then each liquid was prepared in the same manner as in Comparative Example 1. The number of viable bacteria in it was measured. When the concentration of maleimide is 1 mg / L, the viable cell count is 3.8 × 10 ³ cells / mL, and the concentration is 5 mg / L and 10 mg.
In the case of / L, the viable cell count was less than 10 ² cells / mL. Example 2 The same operation as in Example 1 was performed except that N-methylmaleimide was used instead of maleimide. When the concentration of N-methylmaleimide is 1 mg / L, the viable cell count is 3.4 × 10 ³ cells / mL, and when the concentration is 5 mg / L and 10 mg / L, the viable cell count is 10 ² cells / mL. Was less than. Comparative Example 2 Instead of maleimide, 5-chloro-2-methyl-4-
10% by weight of isothiazolin-3-one and 2-methyl-4
-Using an aqueous solution containing 4% by weight of isothiazolin-3-one [ROHM & HAACE, KATHON WT],
Examples except that the total concentration of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one was 1 mg / L, 5 mg / L or 10 mg / L. The same operation as 1 was performed. When the concentration of 4-isothiazolin-3-one compound is 1 mg / L, the viable cell count is 2.
It was 1 × 10 ⁴ cells / mL, and when the concentrations were 5 mg / L and 10 mg / L, the viable cell count was less than 10 ² cells / mL. Comparative Example 3 The same operation as in Example 1 was performed except that 2-bromo-2-nitropropane-1,3-diol [general name: bronopol] was used instead of maleimide. 2-bromo-
Concentration of 2-nitropropane-1,3-diol 1 mg / L
, The viable cell count is 2.3 × 10 ⁴ cells / mL, and the concentration is 5 mg.
When the concentration was 10 mg / L, the viable cell count was 6.5 × 10 ³ cells / mL, and when the concentration was 10 mg / L, the viable cell count was less than 10 ² cells / mL.
The results of Examples 1 and 2 and Comparative Examples 1 to 3 are shown in Table 1.

[0009]

[Table 1]

As shown in Table 1, when comparing the viable cell counts when the concentration of each drug added was 1 mg / L, 10 were obtained in Examples 1 and 2 using maleimide or N-methylmaleimide.
^{It is 3} units / mL, and in Comparative Examples 2 to 3 using a conventionally known halogen-based bactericide, since it is 10 ⁴ units / mL, maleimide or N-methylmaleimide does not contain halogen. Nevertheless, it can be seen that it has a bactericidal power superior to that of known halogen-based bactericides against Legionella bacteria. Comparative Example 4 In 950 mL of pure water, 10.0 g of protease peptone, 10.0 g of yeast extract, 20 m of 50% by weight glucose aqueous solution.
L, L-cysteine monohydrochloride 0.95 g, sodium chloride 5.0 g, 0.5 mol / L disodium hydrogen phosphate aqueous solution 20 mL, 0.5 mol / L potassium dihydrogen phosphate aqueous solution 1
Add 0 mL, 0.4 mL / L magnesium chloride aqueous solution 5 mL and 0.4 Mol / L calcium chloride aqueous solution 1 mL, p
A PYGC medium with H of 6.5 was prepared. Inoculate a culture flask containing this PYGC medium with Acanthamoeba and incubate at 30 ° C for 4 days, gently discard the old medium and add a new PYGC medium so as not to peel off the amoeba adhering to the flask. Then, this was inoculated with Legionella bacteria cultured on BCYE α plate medium for 2 days in the same manner as in Comparative Example 1 and cultured at 30 ° C. for 4 days to make a state in which Amoeba and Legionella bacteria coexist. The medium in this culture flask was agitated to remove the amoeba from the wall surface to obtain a culture solution in which the amoeba and Legionella bacteria coexist. When a part of this culture solution was dropped on a hemocytometer and the number of amoeba was measured under a microscope, the number of amoeba was 3.1 × 10 ⁶ cells / mL. In addition, another part of the culture solution was taken out and
After centrifuging at 0 rpm for 20 minutes to destroy the amoeba, 0.1 mL was applied to the BCYE α plate medium, and 36
When the number of Legionella bacteria was measured by counting the number of colonies after culturing at 5 ° C. for 5 days, it was 2.7 × 10 ⁶ cells / mL. The above culture solution in which Amoeba and Legionella bacteria coexist was further cultured at 30 ° C. for 7 days, and the form of Amoeba was examined by microscopic observation. As a result, Amoeba had a normal form and was present as a nutrient. Furthermore, the culture solution is stirred to uniformly disperse the amoeba and Legionella bacteria,
A part of the culture solution was taken out and centrifuged at 10,000 rpm for 20 minutes to destroy the amoeba, and BCYE α
After 0.1 mL was applied to the plate medium and cultured at 36 ° C. for 5 days, the number of viable cells was measured by counting the number of colonies. The result was 2.5 × 10 ⁸ cells / mL. Another part of the culture solution in which Amoeba and Legionella bacteria were uniformly dispersed was inoculated into a fresh PYGC medium as it was, cultured at 30 ° C. for 7 days, and observed under a microscope to examine the live / dead state of Amoeba. Amoeba survived as a vegetative body. Example 3 Three culture flasks containing a culture solution in which Amoeba and Legionella bacteria coexist prepared in the same manner as in Comparative Example 4 were each charged with maleimide so that the concentration was 10 mg / L, 30 mg / L or 100 mg / L. And in the same manner as in Comparative Example 4,
Incubate at 30 ℃ for 7 days, measure the viable cell count, inoculate a part of the culture solution into fresh PYGC medium, and incubate at 30 ℃ for 7 days.
After culturing for one day, the life and death status of amoeba was examined. When the concentration of maleimide is 10 mg / L, the viable cell count is 3.8 x 10 ⁵ cells / mL.
And the amoeba survived as a cyst. When the maleimide concentrations were 30 mg / L and 100 mg / L, the viable cell count was less than 10 ² cells / mL, and the amoeba was dead. Example 4 The same operation as in Example 3 was performed except that N-methylmaleimide was used instead of maleimide. When the concentration of N-methylmaleimide was 10 mg / L, the viable cell count was 3.4 × 10 ⁵ cells / mL, and the amoeba remained cystic. N-
When the concentrations of methylmaleimide were 30 mg / L and 100 mg / L, the viable cell count was less than 10 ² cells / mL, and the amoeba was dead. Comparative Example 5 Instead of maleimide, 5-chloro-2-methyl-4-
10% by weight of isothiazolin-3-one and 2-methyl-4
-Using an aqueous solution containing 4% by weight of isothiazolin-3-one [ROHM & HAACE, KATHON WT],
Examples except that the total concentration of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one was 10 mg / L, 30 mg / L or 100 mg / L. The same operation as 3 was performed. When the concentration of 4-isothiazolin-3-one compound is 10 mg / L, the viable cell count is 2.1 × 10 ⁸ cells / mL, and when the concentration is 30 mg / L, the viable cell count is 8.5 × 10 ⁷ cells. / ML, concentration 100 mg
When it was / L, the viable cell count was 1.2 × 10 ⁷ cells / mL. Amoeba survived as a trophozoite at all concentrations. Comparative Example 6 The same operation as in Example 3 was performed except that 2-bromo-2-nitropropane-1,3-diol [general name: bronopol] was used instead of maleimide. 2-bromo-
Concentration of 2-nitropropane-1,3-diol 10 mg /
When L, the viable cell count is 2.3 × 10 ⁸ cells / mL, and the concentration is 3
At 0 mg / L, the viable cell count is 6.5 × 10 ⁷ cells / mL,
When the concentration is 100 mg / L, the viable cell count is 2.2 x 10 ⁷ cells / mL
Met. Amoeba survived as a trophozoite at all concentrations. Examples 3-4 and Comparative Examples 4-6
The results are shown in Table 2.

[0011]

[Table 2]

As can be seen from Table 2, known bactericides having excellent bactericidal activity against Legionella bacteria cannot effectively eradicate Legionella bacteria in the presence of amoeba. , It is understood that maleimides have a biocidal activity against amoeba and can effectively eradicate Legionella bacteria in a state in which amoeba coexists.

[0013]

EFFECTS OF THE INVENTION Conventional fungicides against Legionella spp. Are poorly effective when Amoeba and Legionella spp. Coexist. Amoeba continues to survive as a trophic body, and Legionella spp. It was According to the method of the present invention, even in an aqueous system in which Amoeba and Legionella spp. Coexist, Amoeba is cystic or dead, and the viable number of Legionella spp. Can be reduced.

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Claims (2)

[Claims] 1. A method for eradicating Legionella spp. Coexisting with Amoeba in an aqueous system, which comprises adding maleimides to an aqueous system coexisting Amoeba and Legionella spp. 2. Maleimides are represented by the general formula [1].
A compound coexisting with amoeba in the water system according to claim 1.
Method for removing Legionella spp. [Chemical 1] (However, in the formula, R¹And R²Is hydrogen or 1 to 5 carbon atoms
Is an alkyl group of R ³Is hydrogen, unsubstituted or
C1-C10 alkyl substituted with a droxyl group
Number of carbon atoms substituted with a group, unsubstituted or hydroxyl group
5-8 cycloalkyl groups, unsubstituted or hydroxy
Group substituted with a phenyl group or unsubstituted or hydro
It is a benzyl group substituted with a xyl group. )