JP2001169799A - Method for separating.detecting bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for separating.detecting a bacterium, by which the target bacterium can accurately, rapidly and simply be selected.separated from a specimen containing contaminated bacteria having similar properties. SOLUTION: This method for selectively separating.detecting the bacterium, is characterized by culturing bacteria containing the bacterium on a flat plate culture medium containing a compound X-Y (X is a dactericidal, antibacterial or bacteriostatic substance acting on the target bacterium and/or contaminated bacteria; Y is a substrate acted by enzymes derived from bacteria except the target bacterium and is bound to a site where X is inactivated).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for selectively separating and detecting a target microorganism to be detected. Particularly, it is suitable for a method for selectively separating and detecting pathogenic Escherichia coli O157.

[0002]

2. Description of the Related Art In recent years, food poisoning-like patients with diarrhea occur throughout the year, and it is important to accurately determine whether the symptoms are bacterial food poisoning or viral diarrhea. . For example, in the case of bacterial food poisoning, detection or isolation of food poisoning-causing bacteria is indispensable for determining treatment policies, elucidating the route of infection, and preventing food poisoning. Several methods have been used to determine the presence of specific bacteria in a specimen sample. One method is to isolate and identify the causative organism by culturing using a medium suitable for the target organism.Biochemical and serological properties of suspicious colonies can be obtained by directly culturing a specimen sample or by separating and culturing from a pre-enriched culture solution. There is a method for identification based on As other methods, methods such as an ELISA method and immunochromatography based on immunologically detecting a specific bacterium-specific antigen, and a PCR method for amplifying and detecting specific DNA of a specific bacterium have been adopted. I have. Although the immunological method and the PCR method are advantageous in terms of quickness, the results of the separation culture method are indispensable for the final identification and judgment of the causative bacteria because of the possibility of contamination with dead bacteria.

[0003] In order to isolate bacteria from clinical specimens or foods, a medium that is advantageous for the growth of the target bacteria is prepared by taking advantage of its characteristics such as auxotrophy, sensitivity to antibiotics, etc., and response to oxygen. Then, the cells are selectively cultured in an appropriate environment to form colonies. For example, Pseudomonas aeruginosa
a) can be grown by using only acetamide as a carbon source to be added to the medium, but bacteria that cannot use the acetamide do not grow, so that Pseudomonas aeruginosa can be easily and selectively separated. In addition, an appropriate amount of an antibacterial, bactericidal or bacteriostatic substance, for example, an antibiotic (such as novobiocin,
When cefexime, tellurite, etc.) are added to the medium, Escherichia coli is less sensitive to these substances than other bacteria, so that Escherichia coli can be selected and separated and cultured.

[0004] In order to distinguish the bacteria thus separated,
His biological, genetic, biochemical, and serological properties are being investigated. For example, in order to distinguish pathogenic Escherichia coli O157 from Escherichia coli of another serotype, the agglutination reaction by pathogenic Escherichia coli O157 factor serum and the productivity of β-glucuronidase which is a specific enzyme are examined. Pathogenic E. coli O157 is β-
Escherichia coli other than pathogenic Escherichia coli O157 is basically β-glucuronidase positive, although it is negative for glucuronidase. Therefore, by examining the presence or absence of this enzyme activity, it is possible to determine whether the isolated bacteria is pathogenic Escherichia coli O157 or other Escherichia coli.

As a reagent for confirming this specific enzyme, a conjugate of an enzyme substrate and a chromophore, for example, 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, ρ-nitrophenyl-β-D- Glucuronide, 4-methylumbelliferyl-β-D-glucuronide and the like. When these are decomposed by an enzyme (for example, β-glucuronidase), a dye that produces color or fluoresces under ultraviolet light is generated. Also, if the enzyme substrate bound to the chromophore changes,
Another specific enzyme can be confirmed. When these are added to a plate medium, the formed colonies themselves are colored, so that it is possible to distinguish pathogenic Escherichia coli O157 from other bacteria with other serotypes on the medium. If the enzyme substrate is sugar, the acid produced by its metabolism is detected, and if the specific enzyme is decarboxylase, the produced amine is detected by the color change of the pH indicator to discriminate the bacteria.

[0006]

However, the method for selective cultivation of bacteria by utilizing sensitivity to antibiotics or the like is not suitable for the biochemical properties (eg, metabolic mechanism and cell components) among species. Since the difference is based on the difference, the difference in properties between the target bacterium and the other bacterium must be large, and it is impossible when only the species and serotype are different. On the other hand, the specific enzyme has a certain difference depending on the species and serotype, and although the target bacterium and the other bacterium can be distinguished on the plate medium, the number of colonies formed on one plate medium is reduced. There is also a limit, and if there are many contaminating bacteria, the selection and isolation and culture of the target bacteria cannot be performed. Use a highly specific selective agent to select a small number of target bacteria from a sample containing a majority of contaminating bacteria with similar properties, such as only the species and serotype differ from the target bacteria. The purpose of the present invention was to establish a method for enabling separation and culturing, and as a result of intensive studies, the present application was completed.

[0007]

The object of the present invention is to provide a compound X-
Y (X is a substance having a bactericidal, antibacterial, or bacteriostatic property acting on the target bacterium and / or contaminating fungi; Y is a substrate on which a fungal enzyme other than the target bacterium acts; The method can be achieved by a method for selectively separating and detecting a target bacterium, which comprises culturing the bacterium on a plate medium containing the active bacterium. Further, the present invention relates to a compound XY (X is a substance having a bactericidal, antibacterial or bacteriostatic property acting on a target bacterium and / or contaminating fungi, and Y is a fungus derived from a fungus other than the target bacterium. A plate medium for selectively separating and detecting a target bacterium, wherein Y is a substrate and Y is bound to a site where X is inactivated. Hereinafter, the present invention will be described in detail.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The gist of the present invention is to combine a substrate on which a specific enzyme of a bacterium acts with an antibiotic or a synthetic antibacterial substance. This derivative itself does not show antibacterial activity but is subject to degradation by the specific enzyme. Then, a compound that releases an antibiotic having antibacterial activity or a synthetic antibacterial substance and inhibits or suppresses the growth of bacteria is added to a plate medium, and the bacteria are cultured. As a result, bacteria that have this specific enzyme and can degrade this compound are inhibited or suppressed from growing due to released antibiotics and synthetic antibacterials, and colonies are not formed or their size is reduced if they are formed . On the other hand, bacteria which do not have this specific enzyme do not inhibit or inhibit the growth, so that normal colony formation is observed, so that the selection / separation and culture of the target microorganism is facilitated.

[0009] By such a method, for example, the growth of Escherichia coli except pathogenic Escherichia coli O157 can be inhibited or suppressed. The present invention is not limited to this, and has a wide range of application, and Staphylococcus (Staphylococcus) is another bacterium capable of inhibiting or suppressing growth.
occus), Streptococcus pyogenes (St
reptococcus pyogenes), Streptococcus pneumoniae (Streptococcus)
us pneumoniae), Corynebacterium
Diphtheria (Corynebacterium dip)
htheriae), Bacillus anthracis (Bac
Gram-positive bacteria such as illus anthracis and Clostridium, Neisseria gonorrhea (Neisseria gonorr).
rhoeae), Neisseria meningitidis (Nei
sseria meningitidis, Haemophilus influenzae
fluenzae), Klebsiella pneumoniae (K
levsiella pneumoniae, Shigella, Salmonella
la), Serratia Marses Sense (Serratia)
marcescens), Proteus (Proteu)
s), Enterobacter
r), Citrobacter, Pseudomonas
aeruginosa), Bacteroides (Bact)
eroides), Mycoplasma, Mycobacterium (Mycoplasma)
cosp., spirochetes (Spiro)
chaeta), Treponem (Treponem)
a), Rickettsiae, Chlamydiae, Candida (Candi)
da) and the like.

Specific enzymes that can be used to differentiate bacteria include various aminopeptidases such as L-pyroglutamate aminopeptidase, phosphatases, β-D
-Galactosidase, β-D-glucuronidase, caprylate esterase, deoxyribonuclease, and the like, and the types of bacteria, specific enzymes, and substrates thereof are known. For example, L-pyroglutamic acid aminopeptidase is a specific enzyme of Streptococcus pyogenes or Neisseria gonorrhoea, and its substrate is pyroglutamic acid. Β-D-glucuronidase is a specific enzyme of Escherichia coli and its substrate is glucuronic acid. In particular, the enzyme activity can be suitably used for separation and detection of pathogenic Escherichia coli O157. [Thompson, J. et al. S. , O.
S. Hodge, A .; A. Borczyk: J. Cl
initial Microbiol. 28 (10) 21
65-2168 (1990)]

The compound XY of the present invention is a substance having a bactericidal, antibacterial or bacteriostatic property in which X acts on a target bacterium and / or contaminating fungi, and Y is an enzyme derived from a fungus other than the target bacterium. A working substrate. For example, X is an antibiotic,
It is a compound having a substrate Y (for example, a saccharide) of the specific enzyme bound thereto. Antibiotics that can be used as X are known, and specifically include kanamycin, neomycin, paromomycin, ribostamycin, ribidomycin, butyrosine, fortimicin, amikacin, gentamicin, sisomicin, dibekacin, tobramycin, streptomycin, spectinomycin , Apramycin, chloramphenicol and the like. (Nobuo Tanaka, Shoshiro Nakamura, "Summary of Antibiotics, Third Edition, Supplement")
The University of Tokyo Press)

[0012] Y of compound XY, that is, a substance which becomes a substrate, may be a known substrate of the above-mentioned specific enzyme, for example, L-pyrrolidone carboxylylic acid, phosphoric acid, D-galactose, D-glucuronic acid, caprylic acid, Deoxyribonucleic acid and the like. The antibacterial substance and the substrate are appropriately selected depending on the characteristics of the target bacterium, and the compound XY is formed by bonding the substrate to a site where the antibacterial substance is inactivated by a covalent bond such as a peptide bond, an ester bond or a glycoside bond. Obtainable.

In preparing compound XY, it is necessary to bind Y to a specific site of X, which loses its antibacterial activity when chemically modified by a conventionally known operation. In each of the above antibiotics, the site which can inactivate the antibacterial property is known. For example, kanamycin has carbon number 2 'in its structural formula,
Modification of any of 2 ″, 3, 3 ′, 4 ′ and 6 ′ loses antibacterial activity. Chloramphenicol loses its antibacterial activity when it is modified at the position of carbon number 1 or 3 in its structural formula. [J. D. Davis, R .; R
ownd: Science, 176, 758, (197
2)]

The compound XY thus obtained may be added as a composition, for example, when preparing an agar medium, or may be dissolved in an organic solvent such as methanol, purified water or a mixture thereof to form a plate medium. May be added directly or indirectly by infiltrating into filter paper or the like. A method in which bacteria collected from a specimen are smeared on an agar medium containing compound XY directly or indirectly by a conventional method and cultured at about 37 ° C. for about 18 to 48 hours can be exemplified. When contaminants other than the target bacterium are present, when the enzyme of the contaminant reacts with the substrate substance, the activity of the deactivated antibacterial substance is restored (expressed), the antibacterial action is activated, and resistance to this Bacteria that do not have no growth. If the target bacterium has resistance, only the target bacterium is cultured and multiplied, so that the target bacterium can be selectively separated and detected. In addition, when the antibacterial substance is not only contaminating bacteria but also the target bacterium has no resistance to the antibacterial substance, the antibacterial substance released by the decomposition of the compound XY by the specific enzyme of the contaminating bacterium is contained in the medium containing agar. There is a possibility that the growth of the target bacterium may be inhibited or suppressed by spreading in the inside, but the action of the released antibacterial substance stops physiological activities including the production of the enzyme of the contaminating bacterium. The concentration of the antibacterial substance did not increase more, and the concentration of the antibacterial substance decreased as the migration distance increased, and did not inhibit or suppress the growth of the target bacterium, or released the antibacterial substance even if the growth was inhibited or suppressed It is considered that the action is less than that of contaminating bacteria. If the distance between the contaminating bacterium and the target bacterium is extremely small, the target bacterium will also inhibit or suppress the growth, but if a certain distance is secured on the plate medium, the growth will be inhibited. Alternatively, even if suppressed, the bacteria can grow and form colonies, so that only the desired bacteria are cultured and proliferated, or the growth of contaminating bacteria is inhibited or suppressed. Can be detected. This can only be achieved by applying compound XY, which is an important requirement of the present invention, to a plate medium. From such a viewpoint, if an antibacterial substance having a low diffusivity is used as the antibacterial substance used in the present invention, the possibility that the antibacterial substance released by the enzymatic activity of contaminating bacteria inhibits or suppresses the growth of the target bacterium is reduced. It can be even lower.

As the basal medium for culturing bacteria to which the above-mentioned compound XY can be added, any known medium commonly used for culturing bacteria may be used. For example, ordinary agar medium, tryptosome medium, mu- Lart Hinton medium, Heart infusion medium, Brain heart infusion
A John's medium or the like may be used. Any of the well-known components may be added to the basal medium for bacterial culture, if necessary. Examples of such components include antibiotics and vitamins. The bacterial culture medium of the present invention is produced as an agar plate medium containing agar. For example, 1 to 2% by weight of agar and an appropriate amount of purified water with respect to the total weight of each of the above components and the medium except for the compound XY are subjected to high-pressure steam sterilization, and 50 to 60%.
After cooling to about ° C., an aqueous solution of the compound XY is aseptically added, and the resulting medium is dispensed into a sterilized dish, cooled, and solidified to produce the compound. Further, after cooling and solidifying, an aqueous solution of the compound XY is dropped and penetrated on the surface of the culture medium, or the substance contained in a water-absorbing carrier such as filter paper or the like is dried or dried on the culture medium surface. You may add by the method of putting. Alternatively, it may be prepared as a dry powder medium containing each component in advance, and prepared at the time of use. At this time, the compounding amount of the compound XY contained in the culture medium for bacteria is generally 0.0001 to 15.0% by weight, preferably 2.5 to 5.0% by weight based on the total weight of the medium. I just need.

Examples of the subject include all biological samples such as human blood and livestock blood, fluid, feces, urine, etc., foods such as meat and eggs, and samples derived from drinking water and living environment. These can be used as they are, or, if necessary, a processed product (pretreatment) that has undergone steps such as dilution, concentration, homogenization, washing, wiping, filtration, and collection of bacteria using magnetic beads or the like can be used.

[0017]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

[Example 1] Relationship between differences in serotypes and antibacterial activity (A) Isolation and identification of test bacteria Stool of patients suspected of having hemorrhagic Escherichia coli infection is directly smeared on MacConkey sorbitol agar medium (SMAC: Nissui Pharmaceutical) After culturing at 37 ° C. for 18 hours, white colonies were picked. This is triple sugar iron agar (TS
I: BB) and lysine indole mortality agar medium (LIM: Eiken Chemical Co., Ltd.).
After culturing for 8 hours, a strain showing the properties of Escherichia coli was subjected to a toxin-producing test (Escherichia coli verotoxin detection kit: Denka Seiken) and a serum agglutination reaction (pathogenic Escherichia coli immune serum "Seiken": Denka Seiken). As a result, it was found that the toxin type was Stx2 alone and the serotype was O157: H7. Also, as a precautionary measure, as a result of confirming the bacterial species using an intestinal bacteria identification kit (API 20E: BioMérieux), these bacteria were Escherichia coli.
57: H7.

(B) Isolation and detection method Pathogenic Escherichia coli O157: H isolated from the above clinical material
E. coli laboratory stock O1: H7 (JCM
1649: RIKEN Microbial Strain Preservation Facility) was inoculated into a heart infusion medium (Difco) and cultured at 37 ° C. for 7 hours. 1 ml of the culture was spread on a plate medium prepared using a heart infusion agar medium (Difco). Then, the concentration of chloramphenicol-glucuronide (Sigma) obtained by modifying chloramphenicol, an antibacterial substance, with glucuronic acid, a substrate of β-D-glucuronidase, a specific enzyme of E. coli, was 0, 6.4, 12.8, 2
A paper disk (Difco, φ = 6 mm) containing 25 μl of each aqueous solution prepared so as to have 5.6, 50.0, and 100.0 mg / ml was placed thereon, cultured at 37 ° C. overnight, and the growth inhibition circle was determined. The formation was observed. Table 1 shows the results. In the table, "-" means negative and "+" means positive.

[0019]

[Table 1]

The pathogenic Escherichia coli O157: H7 did not produce β-glucuronidase, could not degrade chloramphenicol-glucuronide and did not release chloramphenicol, so that no growth inhibition circle was formed.
1: It is considered that H7 produced β-glucuronidase and decomposed chloramphenicol-glucuronide, so that chloramphenicol was released to form a growth inhibition circle. This chloramphenicol-glucuronide had different effects on E. coli with different serotypes. That is, when a sample containing pathogenic Escherichia coli O157: H7 and Escherichia coli O1: H7 mixed in a medium containing at least 25.6 mg / ml of this compound, the growth of Escherichia coli O1: H7 is suppressed, so that pathogenic Escherichia coli O1: H7 is suppressed. This suggests that O157 can be selectively cultured.

[0021]

Example 2 Effect of the Present Invention in the Presence of Contaminants (A) Isolation and Identification of Test Bacteria E. coli serotype O157: H7 and E. coli serotype O1:
H7 (JCM1649) used in Example 1 was used. (B) Experimental methods in commercial beef mince meat 25g pathogenic E. coli O157: H7 and E. coli O1: H7 overnight culture solution was inoculated each ^105-fold dilution by 0.5 ml, novobiocin pressure modifier Eske as enrichment medium Lichiacoli medium (N-mE
C, Merck) was added, and the final concentration of each bacterium was 13.2 cfu / ml and 21.8 cfu / ml, respectively. After the stomacher treatment, the cells were cultured at 37 ° C. for 18 hours. The final concentration of this bacterial solution was 25.6 mg / ml with a platinum loop.
(2.56% by weight) chloramphenicol
Streaks were applied to a MacConkey agar medium (Difco) prepared by aseptically adding a glucuronide aqueous solution, and cultured overnight at 37 ° C. As a comparative example, the same operation was performed using a MacConkey agar medium containing no chloramphenicol-glucuronide. Then, red colonies were removed from both cultures by 10
Each was selected and subjected to an agglutination reaction using pathogenic Escherichia coli immune serum “Seiken” (Denka Seiken) on a slide glass, and the separation ability of both media was compared based on the detection rate of pathogenic Escherichia coli O157: H7. Table 2 shows the results.

[0022]

[Table 2]

In the medium containing chloramphenicol-glucuronide, pathogenic Escherichia coli O157: H7 could be picked up, but not in the medium not containing it. Therefore, according to the present invention, the probability of selectively separating and detecting a target bacterium (pathogenic Escherichia coli O157: H7) without being affected by contaminants could be increased.

[0024]

According to the present invention, only a small number of contaminants whose properties are similar to those of the target bacterium (eg, pathogenic Escherichia coli O157) differ in the species and serotype from the specimen in which the majority is small. It is possible to select and separate and culture the desired bacteria. In the present invention, the extremely specific compound X-
Since Y can be appropriately selected and used, it has a special effect that target bacteria (eg, pathogenic Escherichia coli O157) can be separated and detected quickly, simply, and with high accuracy.

Claims (4)

[Claims] 1. A compound XY (X is a substance having a bactericidal, antibacterial or bacteriostatic property acting on a target bacterium and / or contaminating fungi, and Y is a substrate on which an enzyme derived from a fungus other than the target bacterium acts. Wherein Y is bound to a site where X is inactivated), wherein the bacteria are cultured on a plate medium containing the same. 2. The method according to claim 1, wherein the target bacterium is pathogenic Escherichia coli O157. 3. The method according to claim 1, wherein X is an antibiotic and Y is a saccharide. 4. A compound XY (X is a substance having a bactericidal, antibacterial or bacteriostatic property acting on the target bacterium and / or contaminating fungi, and Y is a substrate on which an enzyme derived from a fungus other than the target bacterium acts) Wherein Y is bound to a site where X is inactivated.) A plate medium for selectively separating and detecting a target bacterium.