Specimen pretreatment composition for use in testing for Legionella

Abstract

To provide a composition for pretreatment of a sample for inhibiting the growth of contaminants other than Legionella spp. in the sample and accurately detecting Legionella spp., in the testing of Legionella spp.SOLUTION: A composition for the pretreatment of a sample in a test of Legionella spp. comprises an aqueous solution of a dihydrogenphosphate and lower carboxylic acid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition used for pretreatment of a specimen in testing for Legionella spp. [Background technology]

[0002] Legionella bacteria are the causative agents of Legionnaires' disease, which is classified into two types: fulminant pneumonia and transient Pontiac fever.

[0003] Legionella bacteria are environmental bacteria that originally inhabit environments such as soil and freshwater, but they also grow in artificial environmental water such as air conditioning equipment such as cooling towers, water features such as fountains, hot water supply equipment and shower heads in public baths, Jacuzzis, and humidifiers, and are known to cause pneumonia through respiratory tract infection via aerosols. Elderly people, newborns, and immunocompromised patients are considered to be at risk for Legionnaires' disease, and immunocompromised patients in particular are at high risk of developing severe pneumonia and multiple organ failure.

[0004] Legionnaires' disease has become an infectious disease that requires notification under the Act on Prevention of Infectious Diseases and Medical Care for Patients with Infectious Diseases, following the enforcement of the Infectious Diseases Control Law in 1999. For this reason, it is important to isolate and identify the pathogen and to identify the source of infection, and isolation and culture from clinical specimens such as sputum, bronchoalveolar lavage fluid, endotracheal aspirate, and pleural effusion, as well as from environmental specimens such as cooling tower water, bathtub water, and hot water supply water.

[0005] Specific testing methods for Legionella are described in the "New Legionnaires' Disease Prevention Guidelines" (Non-Patent Document 1), "Testing Methods for Legionella in Bath Water, etc. at Public Baths" (Ministry of Health, Labor and Welfare, No. 0919-1, September 19, 2019) (Ministry of Health, Labor and Welfare URL: https: / / www.mhlw.go.jp / content / 11130500 / 000549548.pdf, [searched June 24, 2021]) (Non-Patent Document 2), and Pathogen Detection Manual: Category 4 Infectious Disease, Legionnaires' Disease, Revised September 1, 2020 (National Institute of Infectious Diseases URL: https: / / www.niid.go.jp / niid / images / lab-manual / Legionella20200904.pdf, [searched June 24, 2021]) (Non-Patent Document 3).

[0006] Non-selective or selective isolation media are used to isolate and culture Legionella bacteria, but clinical and environmental samples may contain contaminating bacteria such as bacteria and fungi other than Legionella. Especially when the contaminating bacteria are very numerous, even selective media cannot suppress their growth, making sample pretreatment necessary. Pretreatment involves adding an HCl·KCl buffer solution to clinical or environmental samples, resulting in an acid treatment to kill contaminating bacteria other than Legionella.

[0007] However, acid treatment using HCl·KCl buffer solution has the problem that it is not possible to sufficiently sterilize contaminating bacteria in the sample, and it is not possible to accurately isolate and culture Legionella. For this reason, efforts have been made to improve the pretreatment solution used in acid treatment (Patent Document 1, Non-Patent Document 4), but the above guidelines and manuals have not yet been revised to replace HCl·KCl buffer solution. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2005-237275 [Non-patent literature]

[0009] [Non-Patent Document 1] "New Legionnaires' Disease Prevention Guidelines" Supervised by the Planning Division of the Ministry of Health, Labor and Welfare's Public Health Bureau, Published by the Building Management Education Center (Foundation) First edition published in November 1999 [Non-patent document 2] "Testing Methods for Legionella in Bath Water, etc. in Public Baths" Notification of the Director of the Public Health Division, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, No. 0919-1, September 19, 2019 [Non-patent document 3] Pathogen Detection Manual: Category 4 Infectious Disease, Legionnaires' Disease (Revised September 1, 2020) [Non-patent document 4] Journal of Infectious Diseases, Vol. 76, 1010-1015, 2002 Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a composition for use in pretreatment of specimens, particularly acid treatment, in testing for Legionella spp., which suppresses the growth of contaminating bacteria other than Legionella in environmental specimens or clinical specimens and enables accurate detection of Legionella. [Means for solving the problem]

[0011] As a result of intensive research conducted by the present inventors to achieve the above-mentioned object, they discovered a pretreatment composition for specimens to be used for testing Legionella bacteria, which can be used as a pretreatment solution for acid treatment as a method of specimen pretreatment, and which can improve the accuracy of isolation and culture of Legionella bacteria from specimens while sufficiently suppressing the growth of contaminating bacteria other than Legionella bacteria, without affecting the growth of Legionella bacteria, and thus completed the present invention.

[0012] That is, the present invention comprises the following configurations. Item 1. A composition for pretreatment of a specimen for testing Legionella bacteria, comprising an aqueous solution of dihydrogen phosphate and a lower carboxylic acid. Item 2. The pretreatment composition according to Item 1, wherein the dihydrogen phosphate salt comprises potassium dihydrogen phosphate salt. Item 3. The pretreatment composition according to Item 1 or 2, wherein the lower carboxylic acid includes citric acid, tartaric acid, or a combination thereof. Item 4. The pretreatment composition according to any one of Items 1 to 3, wherein the concentration of the dihydrogen phosphate is 0.015 M or more. Item 5. The pretreatment composition according to Item 4, wherein the concentration of the dihydrogen phosphate is 0.4 M or less. Item 6. The pretreatment composition according to any one of Items 1 to 3, wherein the concentration of the dihydrogen phosphate is 0.02M to 0.2M. Item 7. The pretreatment composition according to any one of Items 1 to 6, wherein the concentration of the lower carboxylic acid is 0.0015 M or more. Item 8. The pretreatment composition according to Item 7, wherein the concentration of the lower carboxylic acid is 0.04M or less. Item 9. The pretreatment composition according to any one of Items 1 to 6, wherein the concentration of the lower carboxylic acid is 0.002M to 0.02M. Item 10. The pretreatment composition according to any one of Items 1 to 9, wherein the pH of the composition is 2.2 or less. Item 11. The pretreatment composition according to any one of Items 1 to 10, wherein the Legionella bacteria are derived from a clinical specimen. Item 12. A method for pretreating a specimen in a Legionella test, comprising treating the specimen with the pretreatment composition according to any one of Items 1 to 11. [Effects of the Invention]

[0013] According to the present invention, by using a pretreatment composition containing an aqueous solution of dihydrogen phosphate and a lower carboxylic acid for acid treatment of a specimen, Legionella bacteria can be accurately detected from the specimen in the isolation and culture for Legionella bacteria testing. [Brief explanation of the drawings]

[0014] [Figure 1]A graph showing the change in pH when 0.1M NaOH is added to a pretreatment composition according to an embodiment of the present invention (◯: 0.2M KH2PO4, △: 0.15M KH2PO4, *: 0.1M KH2PO4, □: 0.05M KH2PO4, ▲: 0.02M KH2PO4, ◆: 0.01M KH2PO4) and an HCl·KCl buffer solution (■). [Figure 2] Photographs showing the suppression of contaminating bacteria and the growth of Legionella colonies in a culture medium containing a pretreated specimen when a pretreatment composition according to an embodiment of the present invention is added to a specimen positive for Legionella. [Figure 3] This photograph shows the growth of Pseudomonas aeruginosa as a contaminant in a medium containing a pretreated sample when HCl·KCl buffer was added to the same positive sample as in Figure 2. DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, embodiments of the present invention will be described in detail. As used herein, the term "Legionella" refers to bacteria belonging to the genus Legionella. Preferred examples of bacteria belonging to the genus Legionella include, but are not limited to, Legionella pneumophila. As used herein, contaminating bacteria other than Legionella bacteria refer to any bacteria other than Legionella bacteria contained in a specimen. Examples of contaminating bacteria other than Legionella bacteria include, but are not limited to, bacteria of the genus Acinetobacter, Enterobacter, Escherichia, Pseudomonas, Serratia, Staphylococcus, and Stenotrophomonas. Examples of bacteria of the genus Acinetobacter include, but are not limited to, Acinetobacter baumannii. Examples of bacteria of the genus Enterobacter include, but are not limited to, Enterobacter cloacae. Examples of bacteria of the genus Escherichia include, but are not limited to, Escherichia coli. Examples of bacteria of the genus Pseudomonas include, but are not limited to, Pseudomonas aeruginosa. Examples of bacteria of the genus Serratia include, but are not limited to, Serratia marcescens. Examples of bacteria of the genus Staphylococcus include, but are not limited to, Staphylococcus aureus. Examples of bacteria of the genus Stenotrophomonas include, but are not limited to, Stenotrophomonas maltophila.

[0016] In the present invention, the dihydrogen phosphate aqueous solution refers to an aqueous solution of dihydrogen phosphate, such as potassium dihydrogen phosphate (KH2PO4), sodium dihydrogen phosphate (NaH2PO4), ammonium dihydrogen phosphate ((NH4)H2PO4), etc., but is not limited to these.

[0017] The lower limit of the concentration of the dihydrogen phosphate aqueous solution in the pretreatment composition is preferably 0.015 M or more, more preferably 0.016 M or more, more preferably 0.017 M or more, more preferably 0.018 M or more, more preferably 0.019 M or more, and more preferably 0.020 M or more, in terms of the buffering ability to maintain the pH of the pretreatment composition at a low level. The upper limit of the concentration of the dihydrogen phosphate aqueous solution in the pretreatment composition is preferably 0.4 M or less, more preferably 0.35 M or less, more preferably 0.3 M or less, more preferably 0.25 M or less, and more preferably 0.2 M or less, in terms of no further buffering effect being obtained even if the concentration is increased above this level. The concentration of the aqueous dihydrogen phosphate solution in the pretreatment composition is preferably 0.02M to 0.2M from the viewpoints of buffering action and suppressing contaminating bacteria other than Legionella bacteria.

[0018] In the present invention, a lower carboxylic acid refers to a carboxylic acid having 1 to 6 carbon atoms. A carboxylic acid having 1 to 6 carbon atoms refers to a carboxylic acid having a total of 1 to 6 carbon atoms in one molecule, including the carbon atoms in the carboxylic acid moiety. A saturated carboxylic acid is sometimes referred to as an organic acid. The lower carboxylic acid may be a monocarboxylic acid, a dicarboxylic acid, or a hydroxy acid. Examples of monocarboxylic acids that can be used include formic acid and acetic acid, dicarboxylic acids such as adipic acid and succinic acid, and hydroxy acids such as citric acid, lactic acid, and tartaric acid, but are not limited to these. When the lower carboxylic acid is a fatty acid, it may be a saturated fatty acid or an unsaturated fatty acid. In a preferred embodiment, the lower carboxylic acid includes citric acid, acetic acid, lactic acid, tartaric acid, formic acid, or a combination thereof. In a more preferred embodiment, the lower carboxylic acid includes citric acid, tartaric acid, or a combination thereof.

[0019] The lower limit of the concentration of the lower carboxylic acid in the pretreatment composition is preferably 0.0015 M or more, more preferably 0.0016 M or more, more preferably 0.0017 M or more, more preferably 0.0018 M or more, more preferably 0.0019 M or more, and more preferably 0.0020 M or more, in terms of the growth inhibitory effect on contaminating bacteria other than Legionella. The upper limit of the concentration of the lower carboxylic acid in the pretreatment composition is more preferably 0.04 M or less, more preferably 0.035 M or less, more preferably 0.03 M or less, more preferably 0.025 M or less, and more preferably 0.02 M or less, in terms of the effect of inhibiting the growth of contaminating bacteria other than Legionella even if the concentration is increased above this level. The concentration of the lower carboxylic acid in the pretreatment composition is preferably 0.002M to 0.02M in order to inhibit the growth of contaminating bacteria other than bacteria of the genus Legionella, while not impairing the growth of bacteria of the genus Legionella.

[0020] In the present invention, the pH of the pretreatment solution used in the acid treatment as a sample pretreatment method, i.e., the pretreatment composition used in the test for Legionella bacteria, is preferably pH 2.2 or less, more preferably pH 1.7 or less, in order to suppress the growth of contaminating bacteria other than Legionella bacteria. Furthermore, the pH is preferably pH 1.3 or more so as not to affect the growth of Legionella bacteria. From the viewpoint of not inhibiting the growth of Legionella bacteria and suppressing the growth of contaminating bacteria other than Legionella bacteria, it is preferable to set the pH of the pretreatment composition of the present invention in the range of pH 1.3 or more and 1.7 or less.

[0021] In the present invention, specimens include clinical specimens such as sputum, bronchoalveolar lavage fluid, endotracheal aspirate, pleural effusion, pericardial fluid, cerebrospinal fluid, and blood; water samples such as cooling tower water, bathtub water, and hot water supply water; and environmental specimens such as wipe samples from water faucets and shower heads.

[0022] The concentrations and pH of the components of the pretreatment composition used in Legionella testing of the present invention can be adjusted by adjusting the ratio to the sample volume. When using the pretreatment composition under conditions in which 1 mL of the composition is added to 1 mL of sample, the concentrations and pH of the components are as described above. However, if acid treatment is to be performed without increasing the volume, a higher concentration of dihydrogen phosphate can be used. For example, when acid treatment is performed using one-tenth the sample volume, a pretreatment composition of the present invention may be prepared and used, comprising an aqueous solution of dihydrogen phosphate at a concentration of approximately 0.2 M to 2 M and a lower carboxylic acid at a concentration of approximately 0.02 M to 0.2 M. The pretreatment composition of the present invention may be added to a sample to obtain a sample pretreatment solution having a pH of 1.3 to 2.2, preferably 1.3 to 1.7, and a dihydrogen phosphate concentration of 0.05 M to 1.0 M. This case is also included in the present invention. [Example]

[0023] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0024] Example 1 A preliminary experiment was carried out to examine the influence of the concentration of KH2PO4 used in the pretreatment composition of the present invention on the inhibitory effect against contaminating bacteria.

[0025] (1) Pretreatment solution Eight different concentrations of KH2PO4 alone, used in the pretreatment composition of the present invention, were prepared for the experiment as pretreatment solutions: 0.01 M / L, 0.02 M / L, 0.04 M / L, 0.06 M / L, 0.08 M / L, 0.1 M / L, and 0.2 M / L. For comparison, an HCl·KCl buffer solution (prepared by mixing a 0.2 mol / L HCl solution with a 0.2 mol / L KCl solution to a pH of 2.2) was used.

[0026] (2) Experimental method Staphylococcus aureus EKN2088 was used as the test bacterium. Dilution of test bacteria (10 3cfu / mL) was mixed with 1 mL of the pretreatment solution in (1) and allowed to stand at room temperature for 5 minutes. Then, 100 μL of the obtained pretreated sample was smeared onto tryptocanthenic medium and cultured at 37°C overnight.

[0027] (3) Results KH2PO4 solutions alone, at concentrations ranging from 0.01 M / L to 0.2 M / L, produced lower colony counts of test bacteria compared with HCl·KCl buffer, demonstrating their inhibitory effect on test bacteria. This is likely due to the buffering effect of KH2PO4, which maintains the pH of the pretreatment solution low. HCl·KCl buffer did not exhibit any inhibitory effect on test bacteria (colony count: 182). The inhibitory effect on test bacteria increased with increasing KH2PO4 concentration. However, even at the highest concentration of 0.2 M / L, colonies of test bacteria were observed, and complete inhibition of test bacteria was not achieved. In the following examples, unless otherwise noted, the KH2PO4 concentration in the pretreatment composition was 0.2 M / L, which exhibits the highest inhibitory effect on test bacteria.

[0028] Example 2 An experiment was conducted to examine the influence of the concentration of the lower carboxylic acid used in the pretreatment composition of the present invention on the inhibitory effect against contaminating bacteria.

[0029] (1) Pretreatment solution The pretreatment solution used in the pretreatment composition of the present invention had a KH2PO4 concentration of 0.2M / L, and five different concentrations of lower carboxylic acids (citric acid, acetic acid, lactic acid, tartaric acid, and formic acid) were added at the same time: 0.002M / L, 0.01M / L, 0.02M / L, and 0.04M / L. HCl·KCl buffer was used as a control.

[0030] (2) Experimental method The same experimental method as in Example 1 was used.

[0031] (3) Results As shown in Tables 1 to 5, citric acid, acetic acid, lactic acid, tartaric acid, and formic acid all showed significant inhibitory effects when the concentration was 0.002 M / L or higher, even after standing at room temperature for 5 or 10 minutes. The numbers in the tables indicate the colony counts of the test bacteria. This confirms that the concentrations of citric acid, acetic acid, lactic acid, tartaric acid, and formic acid used in the present invention are preferably 0.002 M / L or higher. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0032] Example 3 An experiment was conducted to examine the influence of the concentration of KH2PO4 used in the pretreatment composition of the present invention on the inhibitory effect against contaminating bacteria.

[0033] (1) Pretreatment solution As the pretreatment liquid, the concentration of KH2PO4 used in the pretreatment composition of the present invention was set to 0.02 M / L, and the lower carboxylic acid added at the same time was citric acid.

[0034] (2) Experimental method The same experimental method as in Example 1 was used.

[0035] (3) Results As shown in Table 6, even when the concentration of KH2PO4 was 0.02M / L, a significant inhibitory effect was observed after leaving the solution at room temperature for both 5 and 10 minutes. The numbers in the table indicate the number of colonies of the test bacteria. [Table 6]

[0036] Example 4 Experiments were conducted to examine the inhibitory effect of the pretreatment composition of the present invention on various bacteria (bacteria other than those of the genus Legionella, so-called contaminating bacteria).

[0037] (1) Pretreatment solution The following pretreatment solutions were prepared and used in the experiments: a pretreatment composition of the present invention, KH2PO4·citric acid (0.2M KH2PO4, 0.02M citric acid (pH 1.7), HCl·KCl buffer solution (0.2M HCl, 0.2M KCl (pH 2.2)), acidic phosphate buffer solution (0.2M M H3PO4, 0.2M KH2PO4 (pH 2.2)), and McIlvaine buffer solution (0.2M citric acid, 0.2M Na2HPO4 (pH 2.2)).

[0038] (2) Experimental method The test bacteria used were Staphylococcus aureus EKN2088, Escherichia coli EKN704, Pseudomonas aeruginosa EKN2609, Enterobacter cloacae EKN5261, and Stenotrophomonas maltophila A403. Bacterial dilutions of each test bacterium (10 6 , 10 4 cfu / mL) and 1 mL of the pretreatment solution in (1) were mixed, and the mixture was left to stand at room temperature for 10 minutes. 6 , 10 4 100 μL of the mixture (cfu / mL) was smeared onto tryptocanth medium and cultured at 37°C overnight.

[0039] (3) Results As shown in Table 7, in the pretreatment composition of the present invention, no growth was observed for any of the five bacterial species, regardless of the inoculum concentration. In other words, no colonies of the inoculum were observed. In the HCl·KCl buffer solution, acidic phosphate buffer solution, and McIlvaine buffer solution, the growth of all five bacterial species was observed at a bacterial concentration of 10% in the pretreated specimen. 4 In the case of the pretreated specimen with a bacterial concentration of 10 6In the case of cfu / mL, no growth was observed in one or two bacterial species, while several to 10 or more colonies were observed in the remaining bacterial species.

[0040] It was confirmed that the pretreatment composition of the present invention exhibited superior inhibitory effects against all five bacterial species compared to HCl·KCl buffer, acidic phosphate buffer, and McIlvaine buffer.

[0041] [Table 7]

[0042] Example 5 An experiment was conducted to examine the inhibitory effect of the pretreatment composition of the present invention on various bacteria (bacteria other than Legionella species, so-called miscellaneous bacteria) depending on the pH.

[0043] (1) Pretreatment solution Four pretreatment solutions were prepared using the pretreatment composition of the present invention, KH2PO4·citric acid (0.2M KH2PO4, 0.02M citric acid), at pH 1.7, 1.8, 2.0, and 2.2. The pH of each treatment solution was adjusted using phosphoric acid. For comparison, an HCl·KCl buffer solution was used.

[0044] (2) Experimental method The test bacteria used were Serratia marcescens EKN217, E. coli EKN704, S. maltophila A403, S. aureus EKN2088, E. cloacae EKN5261, and Acinetobacter baumannii EKN62, and the evaluation was carried out in the same manner as in Example 1 (2). However, the treatment conditions after mixing the bacterial dilution solution and the pretreatment solution were set at two different levels: leaving the solution at room temperature for 5 and 10 minutes. The obtained pretreated samples (bacterial concentration 10 4 cfu / mL) were tested.

[0045] (3) Results Table 8 shows the results after treatment at room temperature for 5 minutes, and Table 8 shows the results after treatment at room temperature for 10 minutes. Table 8 shows that when treated at room temperature for 5 minutes with the pretreatment composition of the present invention, S. marcescens grew only at pH 2.2 and did not grow at pH 2.0 or below, while S. maltophila grew up to pH 2.0 and did not grow at pH 1.8 or below, indicating that growth of these two bacterial species tended to be inhibited as the pH decreased. The remaining four bacterial species did not grow at pH 2.2 or below. In the HCl·KCl buffer solution, E. cloacae did not grow, but growth was observed for all five other bacterial species.

[0046] As shown in Table 9, when treated at room temperature for 10 minutes, no growth was observed for any of the five bacterial species at pH 2.2 or below when using the pretreatment composition of the present invention. In the HCl·KCl buffer, no growth was observed for S. maltophila, E. cloacae, and A. baumannii, but growth was observed for all of the other four bacterial species. In this example, it was confirmed that the pretreatment composition of the present invention exhibited superior inhibitory effects against bacteria other than Legionella species at all four pH levels, and in proportion to the decrease in pH and treatment time, compared to HCl·KCl buffer solution.

[0047] [Table 8]

[0048] [Table 9]

[0049] Example 6 An experiment was conducted to examine the effect of the pretreatment composition of the present invention on the growth of Legionella bacteria.

[0050] (1) Pretreatment solution The following pretreatment solutions were prepared and used in the experiments: pretreatment compositions of the present invention: KH2PO4·citric acid (0.2M KH2PO4, 0.02M citric acid, pH 1.7), KH2PO4·tartaric acid (0.2M KH2PO4, 0.02M tartaric acid, pH 1.7), KH2PO4·acetic acid (0.2M KH2PO4, 0.02M acetic acid, pH 1.7), and KH2PO4·lactic acid (0.2M KH2PO4, 0.02M lactic acid, pH 1.7); and comparative compositions: HCl·KCl buffer (0.2M HCl, 0.2M KCl, pH 2.2), acidic phosphate buffer (0.2M MHClPO4, 0.2M KH2PO4, pH 2.2), and McIlvaine buffer (0.2M citric acid, 0.2M Na2HPO4, pH 2.2).

[0051] (2) Experimental method The test bacteria used was Legionella pneumophila EKN3680. Dilutions of each test bacterium (10 3 cfu / mL) was mixed with 1 mL of pretreatment solution of each pH, ​​and the mixture was left to stand at room temperature for 10 minutes. The pretreated sample was then smeared onto BCYEα medium using a cotton swab and cultured at 37°C for eight days.

[0052] (3) Results As shown in Table 10, in the pretreatment solutions other than McIlvaine buffer, growth of Legionella pneumophila was confirmed by the fifth day after the start of culture. In particular, rapid growth of Legionella pneumophila was observed in citric acid and tartaric acid. On the other hand, in McIlvaine buffer, growth of Legionella pneumophila was not confirmed by the sixth day after the start of culture. In Table 10, + indicates that at least one colony was observed, and - indicates that no colonies were observed. [Table 10]

[0053] Example 7 An experiment was conducted to examine the effect of the pH of the pretreatment composition of the present invention on the growth of Legionella bacteria.

[0054] (1) Pretreatment solution Nine pretreatment solutions were prepared using the pretreatment composition of the present invention, KH2PO4·citric acid (0.2M KH2PO4, 0.02M citric acid), at pH 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.1, and 2.2. The pH of each treatment solution was adjusted using phosphoric acid. HCl·KCl buffer was used for comparison.

[0055] (2) Experimental method Four strains of Legionella pneumophila (in-house stock strains, strain numbers: EKN3680, EKN3678, EKN6080, A387) were used as test bacteria. Dilutions of each test bacterium (10 4 cfu / mL) was mixed with 1 mL of pretreatment solution of each pH, ​​and the mixture was left to stand at room temperature for 10 minutes. The pretreated sample was then smeared onto BCYEα medium using a cotton swab and cultured at 37°C for three days.

[0056] (3) Results As shown in Table 11, at pH 2.2 to pH 1.6, all test strains showed the growth of more than 300 colonies over the entire surface of the medium. At pH 1.5 to pH 1.3, the number of colonies tended to decrease, but at pH 1.5, one strain showed the growth of approximately 100 colonies, and at pH 1.3, three strains showed the growth of approximately 30 colonies.

[0057] [Table 11]

[0058] Example 8 An experiment was conducted to examine the buffering capacity of the pretreatment composition of the present invention.

[0059] (1) Pretreatment solution The pretreatment solution used in the experiment was 0.5 mL of 0.1 M NaOH added to the KH2PO4 used in the pretreatment composition of the present invention. For comparison, an HCl·KCl buffer solution was used.

[0060] (2) Experimental method To 50 mL of each pretreatment solution prepared in (1), 0.5 mL of 0.1 M NaOH was added and the pH was measured repeatedly to examine the change in pH.

[0061] (3) Results As shown in Figure 1, the pH of the pretreatment composition KH2PO4·citric acid (pH 2.2) of an embodiment of the present invention increased by only 0.2 when 10 mL of 0.1 M NaOH was added, whereas the pH of the HCl·KCl buffer exceeded pH 3.5 when 3.0 mL of 0.1 M NaOH was added, and exceeded pH 9.4 when 4.5 mL of 0.1 M NaOH was added.

[0062] Example 9 An experiment was conducted to examine the pretreatment effect of the pretreatment composition of the present invention on actual specimens.

[0063] (1) Pretreatment solution The pretreatment solution used was the pretreatment composition of the present invention, KH2PO4·citric acid (pH 1.7), and as a control, an HCl·KCl buffer solution.

[0064] (2) Experimental method Five sputum samples from patients with Legionnaires' disease (hereafter referred to as Legionella-positive samples) and four sputum samples from healthy individuals (hereafter referred to as Legionella-negative samples) were used as actual samples in the experiment. 1 mL of each pretreatment solution prepared in (1) was mixed with 1 mL of the actual sample and allowed to stand at room temperature for 10 minutes. 50 μL of the resulting pretreated sample was then streaked onto BCYEα medium and cultured at 37°C for six days.

[0065] (3) Results In all five positive samples, when the pretreatment composition KH2PO4·citric acid (pH 1.7) of the present invention was used, no growth of contaminating bacteria was observed, and sufficient growth of Legionella colonies was confirmed in all samples by the third to sixth day of incubation (Figure 2 shows an example of the growth of Legionella colonies). On the other hand, when HCl·KCl buffer was used, contaminating bacteria were not sufficiently suppressed, and by the third day of incubation, they had grown over the entire surface of the medium, and no Legionella growth was observed (Figure 3 shows the growth of Pseudomonas aeruginosa as a contaminating bacterium in the same positive sample as Figure 2). In all four negative samples, when the pretreatment composition KH2PO4·citric acid (pH 1.7) of the present invention was used, no growth of contaminating bacteria was observed, and no Legionella growth was observed even after the third day of incubation. On the other hand, the HCl·KCl buffer solution was unable to sufficiently suppress the growth of contaminating bacteria, and within three days of culture, contaminating bacteria had grown over the entire surface of the medium. [Industrial Applicability]

[0066] The pretreatment composition of the present invention can be used as a pretreatment composition for specimens such as clinical specimens or environmental specimens in isolation and culture methods for Legionella testing, thereby suppressing the growth of bacteria other than Legionella contained in the specimen without affecting the growth of Legionella, thereby enabling more accurate detection of Legionella.

Claims

1. A composition for pretreatment of a specimen for testing Legionella bacteria, comprising an aqueous solution of dihydrogen phosphate and a lower carboxylic acid, the dihydrogen phosphate salt includes at least one selected from the group consisting of potassium dihydrogen phosphate salt, sodium dihydrogen phosphate salt, and ammonium dihydrogen phosphate salt; The concentration of dihydrogen phosphate in the pretreatment composition is 0.01 M or more and 0.4 M or less, and A composition, wherein the concentration of the lower carboxylic acid in the pretreatment composition is 0.0015M or more and 0.04M or less.

2. 2. The pretreatment composition of claim 1, wherein the dihydrogen phosphate salt comprises potassium dihydrogen phosphate salt.

3. 3. The pretreatment composition of claim 1, wherein the lower carboxylic acid comprises citric acid, tartaric acid, or a combination thereof.

4. 4. The pretreatment composition according to claim 1, wherein the concentration of dihydrogen phosphate in the pretreatment composition is 0.015 M or more.

5. 4. The pretreatment composition according to claim 1, wherein the concentration of dihydrogen phosphate in the pretreatment composition is 0.02M to 0.2M.

6. 6. The pretreatment composition according to claim 1, wherein the concentration of the lower carboxylic acid in the pretreatment composition is 0.002M to 0.02M.

7. A pretreatment composition described in any one of claims 1 to 5, wherein the concentration of dihydrogen phosphate in the pretreatment composition is 0.015 M or more and 0.4 M or less, and the concentration of lower carboxylic acid in the pretreatment composition is 0.002 M or more and 0.04 M or less.

8. A pretreatment composition as described in claim 7, wherein the dihydrogen phosphate salt includes potassium dihydrogen phosphate salt.

9. 9. The pretreatment composition of claim 1, wherein the pH of the composition is 2.2 or less.

10. The pretreatment composition according to claim 1 , wherein the Legionella bacteria are derived from a clinical specimen.

11. A method for pretreating a specimen for Legionella bacteria testing, comprising treating the specimen for Legionella bacteria testing with the pretreatment composition according to any one of claims 1 to 10.

12. A method for pretreating a test specimen for Legionella bacteria testing, comprising a step of mixing a test specimen for Legionella bacteria with a pretreatment composition for the test specimen for Legionella bacteria, the pretreatment composition comprises an aqueous solution of dihydrogen phosphate and a lower carboxylic acid; the dihydrogen phosphate salt includes at least one selected from the group consisting of potassium dihydrogen phosphate salt, sodium dihydrogen phosphate salt, and ammonium dihydrogen phosphate salt; the concentration of dihydrogen phosphate in the mixture of the test specimen and the pretreatment composition obtained in the mixing step is 0.005 M or more and 0.2 M or less; and the concentration of the lower carboxylic acid in the mixture of the test specimen and the pretreatment composition obtained in the mixing step is 0.00075 M or more and 0.02 M or less; method.

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