JP6004631B2 - Disc test piece for detecting drug-resistant bacteria - Google Patents

Description

  The present invention relates to a method for detecting drug-resistant bacteria. More specifically, multidrug-resistant Pseudomonas aeruginosa (MDRP), multidrug-resistant Acinetobacter species, vancomycin-resistant enterococci (VRE), vancomycin-resistant yellow grape The present invention relates to a method for detecting drug-resistant bacteria such as vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-intermediate Staphylococcus aureus (VISA), and a disc test piece for detection used therein.

  Pseudomonas aeruginosa (hereinafter referred to as Pseudomonas aeruginosa), Acinetobacter spp. And Enterococcus spp. Infection is rare even if it enters, but it is an opportunistic infectious bacterium that easily develops infection in patients with diseases such as cancer and immunodeficiency, or patients with reduced resistance after surgery. On the other hand, Staphylococcus aureus (hereinafter referred to as Staphylococcus aureus) is a highly pathogenic bacterium.

  In recent years, nosocomial infections due to multi-drug resistant bacteria such as multi-drug resistant Pseudomonas aeruginosa and multi-drug resistant Acinetobacter have increased rapidly, which has become a serious problem at clinical sites in Japan and abroad (Non-Patent Documents 1 and 2). Therefore, a drug susceptibility test has been carried out based on the Japanese Infectious Disease Law, and Pseudomonas aeruginosa that is resistant to three types of drugs, carbapenems such as imipenem, fluoroquinolones such as ciprofloxacin, and aminoglycosides such as amikacin. Is determined to be multi-drug resistant Pseudomonas aeruginosa (Non-patent Document 3). In Japan, multidrug-resistant Acinetobacter is determined to be a multidrug-resistant Acinetobacter in Acinetobacter baumannii and other Acinetobacter species that are resistant to three types of antibacterial agents: carbapenem, fluoroquinolone, and aminoglycoside (Non-Patent Document 4).

  Vancomycin-resistant enterococci are drug-resistant bacteria that are regarded as a problem worldwide as an important cause of nosocomial infection (Non-patent Document 5). Based on the Infectious Disease Law, enterococci (not suppressed by polypeptide drugs such as colistin and quinolone drugs such as nalidixic acid) that have vancomycin resistance are determined to be vancomycin resistant enterococci. On the other hand, vancomycin-resistant Staphylococcus aureus that is resistant to vancomycin and vancomycin-moderately resistant Staphylococcus aureus are also drug-resistant bacteria that are attracting worldwide attention (Non-Patent Document 6). Based on the Japanese Infectious Disease Law, Staphylococcus aureus (not suppressed by polypeptide drugs such as colistin and quinolone drugs such as nalidixic acid) is characterized by vancomycin-resistant Staphylococcus aureus According to the method of Clinical and Laboratory Standards Institute (CLSI), which is an international standard, vancomycin-resistant Staphylococcus aureus and vancomycin moderately resistant Staphylococcus aureus are classified according to the level of vancomycin resistance. Judging.

  As a method for testing drug sensitivity, a disk diffusion method using a disk test piece impregnated with an antibacterial drug has been conventionally performed. In the drug susceptibility test for Pseudomonas aeruginosa, three disc test pieces impregnated with imipenem, ciprofloxacin, and amikacin were placed on an agar medium coated with the test bacteria, and then cultured. It is determined whether or not it is a multidrug-resistant bacterium by measuring a blocking circle formed around the periphery (Non-patent Document 3). On the other hand, in the drug susceptibility test for enterococci, a disc test piece impregnated with vancomycin is placed on an agar medium coated with the test bacteria and cultured, and then the inhibition circle formed around the disc test piece is measured. Thus, it is determined whether or not it is a resistant bacterium.

  For detection of drug-resistant bacteria, conventionally, after culturing a sample using a non-selective medium such as a blood agar medium, colonies that are estimated to be the detection target bacteria are caught from the grown bacteria, and the detection target is detected. It has been carried out by carrying out a pure culture using a medium suitable for culturing fungi, and then conducting a confirmation test and a drug sensitivity test (micro liquid dilution method, disk diffusion method, etc.). However, this method takes a long time of about 72 hours or more until results are obtained, and after isolation culture, all colonies suspected to be detected from several tens to several thousand colonies are fished and each colony is In order to culture in another petri dish medium, it is a method that requires complicated work, skillful technique and a large number of mediums. Furthermore, when the colony is fished, there is a possibility that the drug-resistant bacteria to be detected are caught and missed. Therefore, a method that can detect drug-resistant bacteria easily and accurately in a short time is desired.

  As a simple method for detecting multidrug-resistant Pseudomonas aeruginosa, a screening medium in which three kinds of antibacterial agents, imipenem, ciprofloxacin and amikacin, are added to the medium is disclosed (Patent Document 1). However, in general, a medium to which an antibacterial agent is added as a selective agent has a problem in that the expiration date is short because the titer of the antibacterial agent decreases with time after preparation. Patent Document 1 discloses that a multi-drug resistant Pseudomonas aeruginosa screening medium with imipenem, ciprofloxacin, and amikacin added to the medium is stable until after storage at 4 ° C. for 4 weeks, but longer than that. It is not described that it is stable even when stored for a period.

JP 2010-098950 A

Masatoshi Asari et al., Clinical and Microbiology, 34 (2), 119-123, 2007 Toru Takada, Pathogen Microbe Detection Information (IASR), 31, 197-198, 2010 Tetsuya Matsumoto, Modern Media, 53 (3), 74-79, 2007 Kazukazu Yamane et al., Pathogen Microorganism Detection Information (IASR), 31, 201-202, 2010 Koichi Tanimoto et al., Modern Media, 53 (6), 140-147, 2007 Yumi Anaka et al., Juntendo Medicine, 54 (3), 328-336, 2008

  The present invention has been made in view of the above situation, and provides a method capable of easily and accurately detecting a drug-resistant bacterium in a short time without impairing the stability of an antibacterial agent, and a disc test piece used therefor The purpose is to do.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a disk test piece containing at least three antibacterial agents consisting of a carbapenem antibacterial agent, a fluoroquinolone antibacterial agent and an aminoglycoside antibacterial agent and green Multi-drug resistant Pseudomonas aeruginosa easily and accurately in a short time using a solid medium capable of growing Pseudomonas aeruginosa and Acinetobacter spp. It was found that multidrug resistant bacteria such as multidrug resistant Acinetobacter can be detected. Furthermore, the present inventors have (a) at least two types of antibacterial agents selected from a polypeptide antibacterial agent, a monobactam antibacterial agent, and a quinolone antibacterial agent, and (b) a glycopeptide antibacterial agent. Using a combination of a disk specimen containing three types of antibacterial agents and a solid medium in which enterococci, Staphylococcus aureus, etc. can grow, and simply observing the presence or absence of colonies in the drug diffusion region on the medium The inventors have found that vancomycin-resistant bacteria such as vancomycin-resistant enterococci, vancomycin-resistant Staphylococcus aureus, and vancomycin moderately resistant Staphylococcus aureus can be detected easily and accurately in a short time, and the present invention has been completed.

That is, the present invention has the following configuration.
(1) A method for detecting drug-resistant bacteria using a combination of a disc test piece containing an antibacterial agent and a solid medium in which the detection target bacteria can grow, wherein the disc test piece contains three or more types of antibacterial agents. A method characterized by containing.
(2) The method according to (1), wherein the antibacterial agent comprises at least three kinds consisting of the following (a), (b) and (c).
(A) Carbapenem antibacterial agent (b) Fluoroquinolone antibacterial agent (c) Aminoglycoside antibacterial agent (3) Each carbapenem antibacterial agent 0.001-1,500 μg, Fluoroquinolone antibacterial The method according to (2), wherein the drug is 0.001 to 1,500 μg and the aminoglycoside antibacterial drug is 0.001 to 1,500 μg.
(4) The method according to any one of (1) to (3), wherein the detection target bacterium is a multidrug-resistant bacterium.
(5) Solid medium is blood agar, chocolate agar, Drigalski modified medium (BTB lactose agar), heart infusion agar, brain heart infusion agar, trypsoy agar (SCD agar), normal agar The method according to (4), which is selected from a standard agar medium, a MacConkey agar medium, a DHL agar medium, and a NAC agar medium.
(6) The method according to (1), wherein the antibacterial agent includes at least three kinds of the following (a) and (b).
(A) Two or more types of antibacterial agents selected from polypeptide antibacterial agents, monobactam antibacterial agents and quinolone antibacterial agents (b) glycopeptide antibacterial agents (7) Polypeptide antibacterial agent 0.001-1,500 μg, monobactam antibacterial agent 0.001-1,500 μg, quinolone antibacterial agent 0.001-1,500 μg, glycopeptide antibacterial agent 0.001-1,500 μg (6) The method according to (6).
(8) The method according to (1), (6) or (7), wherein the detection target bacterium is a vancomycin-resistant bacterium.
(9) Solid medium is blood agar medium, chocolate agar medium, Drigalski modified medium (BTB lactose agar medium), heart infusion agar medium, brain heart infusion agar medium, trypsoy agar medium (SCD agar medium), ordinary agar medium Standard agar medium, bile-esculin-azide agar medium, KF streptococcal agar medium, mannitol salt medium, egg yolk mannitol salt medium, egg yolk salt medium, Staphylococcus No. 110 medium and Baird Parker agar medium The method according to (8), wherein the method is selected.
(10) A disc test piece for detecting drug-resistant bacteria, comprising three or more types of antibacterial agents.
(11) The disc test piece according to (10), wherein the antibacterial agent comprises at least three kinds consisting of the following (a), (b) and (c).
(A) Carbapenem antibacterial agent (b) Fluoroquinolone antibacterial agent (c) Aminoglycoside antibacterial agent (12) Each carbapenem antibacterial agent 0.001 to 1,500 μg, fluoroquinolone antibacterial The disc test piece according to (11), wherein the drug is 0.001 to 1,500 μg and the aminoglycoside antibacterial agent is 0.001 to 1,500 μg.
(13) The disc test piece according to any one of (10) to (12), wherein the drug-resistant bacteria are multi-drug resistant bacteria.
(14) The disc test piece according to (10), wherein the antibacterial agent includes at least three kinds of the following (a) and (b).
(A) Two or more kinds of antibacterial agents selected from polypeptide antibacterial agents, monobactam antibacterial agents and quinolone antibacterial agents (b) glycopeptide antibacterial agents (15) Polypeptide antibacterial agent 0.001-1,500 μg, monobactam antibacterial agent 0.001-1,500 μg, quinolone antibacterial agent 0.001-1,500 μg, glycopeptide antibacterial agent 0.001-1,500 μg (14) The disc test piece according to (14).
(16) The disc test piece according to (10), (14) or (15), wherein the drug resistant bacterium is a vancomycin resistant bacterium.
(17) The disc test piece according to any one of (11) to (13) is placed on the solid medium coated with the sample, and after culturing at 20 to 40 ° C. for 15 to 48 hours, A method for determining that a multidrug-resistant bacterium is contained in a sample when the bacterium grows in a drug diffusion region.
(18) The disc test piece according to any one of (14) to (16) is placed on the solid medium coated with the sample, and after culturing at 20 to 40 ° C. for 15 to 48 hours, A method for determining that vancomycin-resistant bacteria are contained in a sample when bacteria grow in the drug diffusion region.

  By using the disc test piece of the present invention in combination with a solid medium in which the detection target bacteria can grow, it is possible to efficiently, easily and accurately perform many tests simply by visually observing the presence or absence of colonies in the drug diffusion region on the medium. Drug-resistant bacteria or vancomycin-resistant bacteria can be detected. Since there is no need to fish a large number of colonies after isolation culture, the time required for detection, labor and operation costs, and reagent costs for the medium can be greatly reduced compared to conventional isolation culture methods. False negatives can be eliminated. Furthermore, the disk test piece of the present invention can be stored for a long time without impairing the stability of the antibacterial agent, and has an effect of being excellent in economic efficiency.

FIG. 1 shows that the disc test piece of the present invention is placed on a sheep blood agar medium, and three-drug resistant Pseudomonas aeruginosa (3R), two-drug resistant Pseudomonas aeruginosa (2R) or sensitive Pseudomonas aeruginosa (S) is cultured. It is a photograph which shows the growth presence or absence (state of a prevention circle) of the microbe in the circumference | surroundings of the disk test piece after performing. (Example 2.A) FIG. 2 shows that after the disc test piece of the present invention is placed on a sheep blood agar medium, E. coli and Klebsiella pneumoniae are mixed with three-drug resistant Pseudomonas aeruginosa (3R) or sensitive Pseudomonas aeruginosa (S). It is a photograph which shows the growth presence or absence (state of a prevention circle) of the microbe in the circumference | surroundings of a disk test piece. (Example 2.B) FIG. 3 shows that the disc test piece of the present invention is placed on a sheep blood agar medium and three-drug resistant Pseudomonas aeruginosa (3R) or susceptible Pseudomonas aeruginosa (S) and Escherichia coli, Klebsiella pneumoniae and Enterococcus faecalis are It is a photograph which shows the growth presence or absence (state of a prevention circle) of the microbe in the circumference | surroundings of the disk test piece after carrying out mixed culture. (Example 2.B) FIG. 4 shows that the disc specimen of the present invention was placed on a sheep blood agar medium, vancomycin-resistant enterococci (Enterococcus faecalis (VRE)) or vancomycin-sensitive enterococci (Enterococcus faecalis), E. coli and Pseudomonas aeruginosa. It is a photograph which shows the growth presence or absence of a microbe in the circumference | surroundings of a disk test piece after carrying out mixed culture of a microbe (state of a prevention circle). (Example 7)

  This specification includes the contents described in the specification of Japanese Patent Application No. 2010-241663, which is the basis of the priority of the present application.

  In the present invention, a disk test piece impregnated with three or more types of antibacterial agents in one test piece is placed on a solid medium coated with a test bacteria-containing sample, and cultured in a short time. Efficient, simple and accurate detection of drug-resistant bacteria is possible.

  The detection target bacterium of the present invention is not particularly limited as long as it is a drug-resistant bacterium that is resistant (not suppressed) to three or more antimicrobial agents. Examples include multidrug resistant Pseudomonas aeruginosa, multidrug resistant Acinetobacter, vancomycin resistant enterococci, vancomycin resistant S. aureus, vancomycin moderately resistant S. aureus, and the like. In the present invention, bacteria showing resistance to vancomycin, such as vancomycin-resistant enterococci, vancomycin-resistant Staphylococcus aureus, and vancomycin moderately resistant Staphylococcus aureus, are referred to as “vancomycin-resistant bacteria”.

  In the present invention, the disc test piece is impregnated with at least three types of antibacterial drugs, but the antibacterial drug system can be set for each drug resistant bacterium to be detected. The antibacterial agent is not particularly limited as long as it belongs to each system, and can be appropriately selected from therapeutic agents or reagents. Furthermore, in the case of the bulk powder, it can be used by dissolving in an appropriate solvent. For example, if the detection target is multi-drug resistant bacteria such as multi-drug resistant Pseudomonas aeruginosa or multi-drug resistant Acinetobacter, impregnate the disc specimen with at least a carbapenem antibiotic, a fluoroquinolone antibiotic, and an aminoglycoside antibiotic . On the other hand, if the detection target is vancomycin-resistant bacteria such as vancomycin-resistant enterococci, vancomycin-resistant Staphylococcus aureus, and vancomycin-moderate Staphylococcus aureus, at least polypeptide antibacterials, quinolone antibacterials and glycopeptide antibacterials A disc test piece, or at least a monobactam antibacterial agent, a quinolone antibacterial agent and a glycopeptide antibacterial agent, or at least a polypeptide antibacterial agent, a monobactam antibacterial agent and a glycopeptide type Impregnate the disc specimen with antimicrobial agent. Specific examples of carbapenem antibacterials, fluoroquinolone antibacterials and aminoglycoside antibacterials that can be used Table 1, specific examples of polypeptide antibacterials, monobactams, quinolones and glycopeptides Is shown in Table 2. These drugs are usually distributed as salts.

  The antibacterial drug content of the disc test piece of the present invention is such that no bacteria sensitive to antibacterial agents grow in the drug diffusion region around the disc test piece after culturing on a solid medium, and the detection target bacteria (antibacterial agent) The amount that only grows. Such an amount is set to an appropriate amount in consideration of the diffusibility of each drug from the solid medium, the culture time (diffusion time), the strength of the antibacterial effect on the detection target bacteria, and the like. For example, when detecting multi-drug resistant bacteria such as multi-drug resistant Pseudomonas aeruginosa and multi-drug resistant Acinetobacter, the content per disc is 0.001 to 1,500 μg of carbapenem antibacterial, fluoroquinolone antibacterial, respectively. 0.001-1,500 μg, aminoglycoside antibacterial agent preferably in the range of 0.001-1,500 μg, carbapenem antibacterial agent 1-1,500 μg, fluoroquinolone antibacterial agent 1-1,500 μg, aminoglycoside Antibacterial drug It is more preferable to set it as the range of 1-1,500 micrograms. On the other hand, when detecting vancomycin-resistant bacteria such as vancomycin-resistant enterococci, vancomycin-resistant Staphylococcus aureus, and vancomycin medium-resistant Staphylococcus aureus, the content per disc is determined to be a polypeptide antibacterial agent 0.001 to 1, respectively. , 500 μg, monobactam antibacterial agent 0.001-1,500 μg, quinolone antibacterial agent 0.001-1,500 μg, glycopeptide antibacterial agent 0.001-1,500 μg, preferably polypeptide antibacterial agent It is more preferable to set it as 1-1,500 micrograms, monobactam type antimicrobial agent 1-1,500 microgram, quinolone type antimicrobial agent 1-1,500 microgram, and glycopeptide antimicrobial agent 1-1500 microgram.

  The disk test piece of the present invention can be further impregnated with a pH buffering agent and other components in addition to the antibacterial agent, if necessary.

  The disc test piece of the present invention is placed and cultured on the surface of a solid medium coated with a sample containing the test bacteria. The sample application method, conditions, and the like can be performed by a method employed in normal separation culture or the like. On the other hand, the culture temperature and time can be appropriately determined according to the diffusibility of the detection target bacteria and the antibacterial drug. The culture temperature can usually be a temperature at which bacteria can grow, specifically 20-40 ° C, more preferably 25-37 ° C, and more preferably 35-37 ° C. More preferably. The culture time is preferably 15 to 48 hours, more preferably 18 to 24 hours.

  By the above culture, each antibacterial drug placed on the surface of the solid medium diffuses on and inside the solid medium. If the test bacterium contained in the sample is a multi-drug resistant bacterium such as multi-drug resistant Pseudomonas aeruginosa or multi-drug resistant Acinetobacter, the present invention contains a carbapenem antibacterial agent, a fluoroquinolone antibacterial agent and an aminoglycoside antibacterial agent It grows in the drug diffusion region around the disk test piece on the solid medium on which the disk test piece is placed to generate colonies. On the other hand, if the test bacteria contained in the sample are vancomycin-resistant bacteria such as vancomycin-resistant enterococci, vancomycin-resistant Staphylococcus aureus, and vancomycin medium-resistant Staphylococcus aureus, polypeptide antibacterials, quinolone antibacterials and glycopeptides It grows in the drug diffusion region around the disc test piece on the solid medium on which the disc test piece of the present invention containing the antibacterial drug is placed, and generates a colony. In addition, if the test bacteria included in the sample are vancomycin-resistant bacteria such as vancomycin-resistant enterococci, vancomycin-resistant Staphylococcus aureus, and vancomycin-medium-resistant Staphylococcus aureus, monobactam antibiotics, quinolone antibiotics, and glycopeptide antibiotics It also grows in the drug diffusion region around the disc test piece on the solid medium on which the disc test piece of the present invention containing an antibacterial drug is placed, and generates a colony. Furthermore, when the test bacteria contained in the sample are vancomycin-resistant bacteria such as vancomycin-resistant enterococci, vancomycin-resistant Staphylococcus aureus, and vancomycin-medium-resistant Staphylococcus aureus, polypeptide antibiotics, monobactam antibiotics and glycopeptides It also grows in the drug diffusion region around the disc test piece on the solid medium on which the disc test piece of the present invention containing the antibacterial agent is placed, and generates a colony.

  The solid medium used in the present invention is not particularly limited as long as it contains nutrients such as a carbon source and a nitrogen source and is capable of growing the detection target bacteria. Medium used for normal isolation culture can be used, blood agar medium, chocolate agar medium, Drigalski modified medium (BTB lactose agar medium), heart infusion agar medium, brain heart infusion agar medium, tryptosoy agar medium Examples include (SCD agar medium), ordinary agar medium, and standard agar medium. The solid medium may further contain a selection agent that suppresses bacteria other than the detection target bacteria. For example, when the detection target is a multidrug-resistant bacterium such as multidrug-resistant Pseudomonas aeruginosa or multidrug-resistant Acinetobacter, McConkey agar medium, DHL agar medium, NAC agar medium, or the like can also be used. On the other hand, when the detection target is vancomycin-resistant enterococci, bile-esculin-azide agar, KF streptococcus agar, or the like can also be used. In addition, when the detection target is vancomycin-resistant Staphylococcus aureus and vancomycin-moderately resistant Staphylococcus aureus, Mannit salt medium, yolk-added Mannit salt medium, Egg York salt medium, Staphylococcus No. 110 medium, Baird Parker An agar medium or the like can also be used.

  As the solidifying agent used in the medium, those usually used such as agar and agarose can be used. Even if the solid medium further contains an enzyme substrate, the action of the present invention is not affected.

  The material of the disc test piece of the present invention is not particularly limited as long as it has an appropriate thickness and can be impregnated with an antibacterial agent, dried, and diffused on the solid medium. In addition, any shape can be used, but it is also possible to use a disk shape, a strip shape, a rectangle shape, or further by pasting it on a support. Some discs are commercially available, but filter paper or the like can be cut into an appropriate shape and size. The size of the disc test piece is not particularly limited, but considering that it is used by placing it on a solid medium in a petri dish having a diameter of about 9 cm, which is usually used for bacterial culture, It is preferable to use a strip shape with a width of 3 to 12 mm and a length of 15 to 80 mm, or a rectangular shape with a side of 5 to 13 mm.

  The sample applied to the solid medium in the present invention is not particularly limited as long as it is a sample that may contain drug-resistant bacteria. Samples include human, other animal living body, environment, food-derived specimens, culture media thereof, and the like.

Examples of the present invention are shown below, but the present invention is not limited to these examples.
The list of test strains used in Examples 2A and 2B is shown in Table 3 below, and the list of test strains used in Examples 3-9 is shown in Table 4.

(Preparation of multi-drug resistant Pseudomonas aeruginosa disc test piece)
Disk test pieces impregnated with meropenem as a carbapenem antibiotic, ciprofloxacin as a fluoroquinolone antibiotic, and amikacin as an aminoglycoside antibiotic were prepared.

(1) Antibacterial content per disc The content of each antibacterial per disc is as shown below.

(2) Preparation of antibacterial drug solution Each antibacterial drug was dissolved in purified water to prepare a solution (solution A) containing meropenem at 500 μg / mL, amikacin at 1,500 μg / mL, and ciprofloxacin at 500 μg / mL. Further, the solution A was diluted to 80% to obtain a solution B, and the solution A was diluted to 50% to obtain a solution C.

(3) Preparation of antibacterial drug-containing disc test piece The solution A, B, C prepared in (2) was impregnated into a filter paper disc having a diameter of 6.35 mm by 20 μL. Thereafter, the disc was dried at 50 ° C. for 60 minutes.

(A. Detection of multidrug resistant Pseudomonas aeruginosa)
The disc test piece prepared in Example 1 was placed on a sheep blood agar medium, and three-drug resistant Pseudomonas aeruginosa, two-drug resistant Pseudomonas aeruginosa, or sensitive Pseudomonas aeruginosa was cultured. The effect of detection of drug-resistant Pseudomonas aeruginosa was examined.

(1) Preparation of solid medium Using the medium components shown below, autoclaved at 121 ° C. for 15 minutes, cooled to 50 ° C., and then added sheep blood. Thereafter, 20 mL of the medium was dispensed into a petri dish and solidified.

(2) Bacterial inoculation and culture Suspend the pre-cultured strain in sterile physiological saline to prepare a 10-fold diluted solution of McFarland No. 1, and put one platinum ear streak on the sheep blood agar medium prepared in (1). did. The disc test piece prepared in Example 1 was placed on the streaked medium, and then cultured at 37 ° C. for 18 hours for determination.

(3) Result The photograph after culture is shown in FIG. 1 as a result. 1 (1) to (3) are obtained by culturing three-drug resistant Pseudomonas aeruginosa (3R) using disc test pieces A to C, respectively, and FIGS. 1 (4) to (6) are disc test pieces A to C, respectively. C-cultured two-drug resistant Pseudomonas aeruginosa (2R), FIGS. 1 (7)-(9) are cultivated sensitive Pseudomonas aeruginosa (S) using disk specimens A-C, respectively. It is. In any of the disc test pieces A to C, the 3-drug resistant Pseudomonas aeruginosa grew in the drug diffusion region around the disc test piece to form a colony, but the 2-drug resistant Pseudomonas aeruginosa and the sensitive Pseudomonas aeruginosa were discs. Growth inhibition was observed around the specimen. From this, it was shown that multidrug-resistant Pseudomonas aeruginosa can be detected by observing the presence or absence of colonies generated around the disk specimen of the present invention.

(B. Detection of multidrug resistant Pseudomonas aeruginosa in mixed culture)
The disc test piece prepared in Example 1 was placed on a sheep blood agar medium, and three-drug resistant Pseudomonas aeruginosa or sensitive Pseudomonas aeruginosa was identified as Escherichia coli (hereinafter referred to as E. coli), Klebsiella pneumoniae. (Klebsiella pneumoniae) and Enterococcus faecalis were mixed and cultured, and the effect of detecting multidrug-resistant Pseudomonas aeruginosa (3-drug resistant Pseudomonas aeruginosa) was examined.

(1) Preparation of solid medium Example 2. Same as A.

(2) Inoculation and culture of fungi Suspension of each pre-cultured strain in sterilized physiological saline to prepare a 10-fold diluted solution of McFarland No. 1, mixed in equal amounts, and sheep blood agar prepared in (1) One platinum loop was drawn on the medium. The disc test piece prepared in Example 1 was placed on the streaked medium, and then cultured at 37 ° C. for 18 hours for determination.

(3) Results Photographs after culture are shown in FIGS. 2 and 3 as results. FIGS. 2 (1) to (3) are mixed cultures of three-drug resistant Pseudomonas aeruginosa (3R), Escherichia coli and Klebsiella pneumoniae using disc test pieces A to C, respectively, FIGS. 2 (4) to (6). Are mixed cultures of sensitive Pseudomonas aeruginosa (S), Escherichia coli and Klebsiella pneumoniae using disc test pieces A to C, respectively. In any of the disk specimens A to C, in the mixed culture with the three-drug resistant Pseudomonas aeruginosa, colonies of Pseudomonas aeruginosa were observed in the drug diffusion region around the disk specimen, but mixed with sensitive Pseudomonas aeruginosa. In culture, no growth of bacteria was observed in the drug diffusion area around the disc specimen.

  On the other hand, FIGS. 3 (1) to (3) are obtained by mixing and culturing three-drug resistant Pseudomonas aeruginosa (3R), Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis using disk test pieces A to C, respectively. 4) to (6) are obtained by mixing and culturing sensitive Pseudomonas aeruginosa (S), Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis using disc test pieces A to C, respectively. In any of the disk specimens A to C, in the mixed culture with the three-drug resistant Pseudomonas aeruginosa, colonies of Pseudomonas aeruginosa were observed in the drug diffusion region around the disk specimen, but mixed with the sensitive Pseudomonas aeruginosa. In culture, no growth of bacteria was observed in the drug diffusion area around the disc specimen. From the above results, it is shown that multidrug-resistant Pseudomonas aeruginosa can be detected from a sample containing a plurality of bacteria by observing the presence or absence of colonies generated around the disk specimen of the present invention. It was done.

(Examination of types and contents of antibacterial agents in detection of multidrug-resistant Pseudomonas aeruginosa)
Change the type and content of carbapenem antibacterial agent, fluoroquinolone antibacterial agent and aminoglycoside antibacterial agent to be impregnated into the disk specimen, and use multidrug resistant Pseudomonas aeruginosa (3-drug resistant Pseudomonas aeruginosa) EKN8118 or EKN8093 The culture was mixed with Escherichia coli and Klebsiella pneumoniae, and the detection effect was examined.

(1) Preparation of disc test piece After each antibacterial solution was prepared, it was mixed and impregnated into a 6.35 mm diameter filter paper disc so that the content per disc was as shown in Table 7. The disk was dried at 50 ° C. for 60 minutes to obtain disks 3-1 to 3-16.

(2) Preparation of solid medium Using the medium components shown below, autoclaved at 121 ° C. for 15 minutes, cooled to 50 ° C., and then added sheep blood. Thereafter, 20 mL of the medium was dispensed into a petri dish and solidified.

(3) Inoculation and culture of bacteria Each pre-cultured strain was suspended in sterile physiological saline and mixed to prepare a mixed bacterial solution of McFarland No. 0.5 diluted 100 to 10,000 times, and prepared in (2) The sheep blood agar was smeared with a cotton swab. The disc test piece prepared in (1) was placed on the smeared medium, and then cultured at 37 ° C. for 18 hours for determination.

(4) Results As shown in Table 7, multi-drug resistant Pseudomonas aeruginosa grew in the drug diffusion region around the disk test piece and produced colonies in any of the disk test pieces. Pneumonier was found to be stunted around the disk specimen. From these results, the detection of multidrug-resistant Pseudomonas aeruginosa according to the present invention in the range of 0.001-1,500 μg content per disc for all carbapenem antibacterial agents, fluoroquinolone antibacterial agents, and aminoglycoside antibacterial agents Was shown to be possible.

(A. Examination of medium types in detection of multidrug resistant Pseudomonas aeruginosa)
The effect of detecting multidrug-resistant Pseudomonas aeruginosa (3-drug resistant Pseudomonas aeruginosa) in mixed culture with E. coli and Klebsiella pneumoniae was examined by changing the type of medium on which the disc test piece was placed.
Inoculation and culture of the bacteria in Examples 4A and 4B and Example 5 shown below were performed in the same manner as in Example 3.

(1) Preparation of a disk test piece After preparing each antibacterial solution, a 6.35 mm diameter filter paper disk was impregnated so that the content per disk was as shown in Table 9, and then the disk Was dried at 50 ° C. for 60 minutes.

(2) Preparation of solid medium Using the medium components shown below, autoclaved at 121 ° C. for 15 minutes. After cooling to 80 ° C., 5% equine defibrinated blood was added and heated with stirring until a chocolate color was achieved. Then, it cooled to 50 degreeC and dispensed 20 mL at a time and solidified.

  Using the medium components shown in the following tables, autoclaved at 121 ° C. for 15 minutes. Then, it cooled to 50 degreeC and dispensed 20 mL at a time and solidified.

  The medium components shown in the following tables were dissolved by heating. Then, it cooled to 50 degreeC and dispensed 20 mL at a time and solidified.

(3) Results As shown in Table 9, in all the examined media, multidrug resistant Pseudomonas aeruginosa grew in the drug diffusion region around the disk specimen and produced a colony, but Escherichia coli, Klebsiella pneumoniae Was found to be stunted around the disk specimen.

(B. Detection of multidrug-resistant Pseudomonas aeruginosa using NAC agar medium)
Using NAC agar medium, we investigated the effect of detection of multidrug-resistant Pseudomonas aeruginosa (3-drug resistant Pseudomonas aeruginosa) in mixed culture with Pseudomonas aeruginosa sensitive to all carbapenems, fluoroquinolones and aminoglycosides It was.

(1) Preparation of disc test piece After preparing each antibacterial solution, impregnation into a filter paper disc having a diameter of 6.35 mm so that the content per disc is as shown in the following table. The disc was dried at 50 ° C. for 60 minutes.

(2) Preparation of solid medium The medium components shown below were dissolved by heating. Then, it cooled to 50 degreeC and dispensed 20 mL at a time and solidified.

(3) Results As shown in Table 20 below, multidrug-resistant Pseudomonas aeruginosa grew in the drug diffusion region around the disk specimen and produced colonies, but carbapenem series, fluoroquinolone series, aminoglycoside series Pseudomonas aeruginosa, sensitive to all drugs, was inhibited from growth around the disk specimen.

(Examination of storage stability of discs for detecting multidrug-resistant Pseudomonas aeruginosa)
Place the disc test piece for detecting multidrug-resistant Pseudomonas aeruginosa after storage for 9 months at 4 ° C on sheep blood agar medium, and multidrug-resistant Pseudomonas aeruginosa (3-drug resistant Pseudomonas aeruginosa) The culture was mixed with Klebsiella pneumoniae and the detection effect was examined.

(1) Production and storage of disk test piece Example 4 A disc test piece was prepared in the same manner as B, and then stored at 4 ° C. for 9 months. In addition, it was confirmed that multi-drug resistant Pseudomonas aeruginosa can be detected by using multi-drug resistant Pseudomonas aeruginosa (3-drug resistant Pseudomonas aeruginosa) at the time of production of the disk test piece produced simultaneously.

(2) Preparation of solid medium The same as in Example 3.

(3) Results As shown in Table 21 below, multidrug-resistant Pseudomonas aeruginosa grew in the drug diffusion region around the disk test piece and produced colonies, but E. coli and Klebsiella pneumoniae Growth inhibition was observed in the surrounding area. From this result, it was shown that the disc test piece of the present invention maintained the effect of detecting multidrug-resistant Pseudomonas aeruginosa even after storage at 4 ° C. for at least 9 months.

(Preparation of disc test piece for detection of vancomycin-resistant enterococci)
Disc test pieces impregnated with colistin as a polypeptide antibacterial agent, nalidixic acid as a quinolone antibacterial agent, and vancomycin as a glycopeptide antibacterial agent were prepared.

(1) Antibacterial content per disc The content of each antibacterial per disc is as shown below.

(2) Preparation of antibacterial drug solution Each antibacterial drug was dissolved in purified water to prepare a solution containing 1,750 μg / mL colistin, 3,000 μg / mL nalidixic acid, and 1,500 μg / mL vancomycin.

(3) Preparation of antibacterial drug-containing disk test piece A filter paper disk having a diameter of 6.35 mm was impregnated with 20 μL of the solution prepared in (2). Thereafter, the disc was dried at 50 ° C. for 60 minutes.

(Detection of vancomycin-resistant enterococci in mixed culture)
The disc test piece prepared in Example 6 was placed on a sheep blood agar medium, and vancomycin-resistant enterococci (Enterococcus faecalis) or vancomycin-sensitive enterococci (Enterococcus faecalis) were mixed and cultured with Escherichia coli and Pseudomonas aeruginosa. The effect of detecting vancomycin-resistant enterococci was examined.

(1) Preparation of solid medium The same as in Example 3.

(2) Inoculation and culture of bacteria Each pre-cultured strain was suspended in sterile physiological saline and mixed to prepare a mixed bacterial solution of McFarland No. 0.5 diluted 100 to 10,000 times. (1) The prepared sheep blood agar medium was smeared with a cotton swab. The disc test piece prepared in Example 6 was placed on the smeared medium and then cultured at 37 ° C. for 18 hours for determination.

(3) Result The photograph after culture is shown in FIG. 4 (1) and (2) as a result. Fig. 4 (1) shows a mixed culture of vancomycin-resistant Enterococcus faecalis (VRE), Escherichia coli and Pseudomonas aeruginosa, and Fig. 4 (2) shows a mixed culture of vancomycin-sensitive Enterococcus faecalis, E. coli and Pseudomonas aeruginosa. It is. In the mixed culture with vancomycin-resistant Enterococcus faecalis, colonies of Enterococcus faecalis were found in the drug diffusion region around the disc specimen, but in the mixed culture with vancomycin-sensitive Enterococcus faecalis, drug diffusion around the disc specimen There was no growth of any fungus in the area. From the above results, it was shown that vancomycin-resistant enterococci can be detected from a sample containing a plurality of bacteria by observing the presence or absence of colonies generated around the disk specimen of the present invention. .

(Investigation of type and content of antibacterial agents in detection of vancomycin-resistant enterococci)
Vancomycin-resistant enterococci in mixed culture with Escherichia coli and Pseudomonas aeruginosa by changing the type and content of polypeptide antibacterial agent, monobactam antibacterial agent, quinolone antibacterial agent and glycopeptide antibacterial agent (Enterococcus faecalis) The effect of detection was investigated.
The bacterial inoculation operation and culture in Examples 8 and 9 shown below were performed in the same manner as in Example 7.

(1) Preparation of disc test piece After each antibacterial solution was prepared, it was mixed and impregnated into a 6.35 mm diameter filter paper disc so that the content per disc was as shown in Table 23. The disc was dried at 50 ° C. for 60 minutes to obtain discs 8-1 to 8-20.

(2) Preparation of solid medium The same as in Example 3.

(3) Results As shown in Table 23, VRE (vancomycin-resistant Enterococcus faecalis) grew in the drug diffusion region around the disk test piece and produced colonies in all the disk test pieces. The growth of Pseudomonas aeruginosa was observed around the disk specimen. From these results, the polypeptide antibacterial agent, monobactam antibacterial agent, quinolone antibacterial agent and glycopeptide antibacterial agent are all resistant to vancomycin according to the present invention in the range of 0.001-1,500 μg per disc. It was shown that enterococci can be detected.

(Examination of medium type in detection of vancomycin-resistant enterococci)
The effect of detecting vancomycin-resistant enterococci (Enterococcus faecalis) in mixed culture with Escherichia coli and Pseudomonas aeruginosa was examined by changing the type of medium on which the disc specimen was placed.

(1) Preparation of disc test piece After preparing each antibacterial solution, the filter paper disc having a diameter of 6.35 mm was impregnated so that the content per disc was as shown in Table 24. Was dried at 50 ° C. for 60 minutes.

(2) Preparation of solid medium In the same manner as in Example 4, chocolate agar medium, Drigalski modified medium (BTB lactose agar medium), heart infusion agar medium, brain heart infusion agar medium, trypsoy agar medium (SCD agar medium) ) Was prepared.

(3) Results As shown in Table 24, VRE (vancomycin-resistant Enterococcus faecalis) grew in the drug diffusion region around the disc specimen and produced colonies in all the examined media. Pseudomonas aeruginosa was found to be stunted around the disk specimen.

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

  The disc test piece of the present invention is used in combination with a solid medium in which the detection target bacteria can grow, and by simply observing the presence or absence of colonies in the drug diffusion region on the medium, the multidrug resistance is easily and accurately observed. Can detect bacteria or vancomycin-resistant bacteria, and can be stored for a long time without impairing the stability of the antibacterial agent. It is possible to greatly reduce the cost of the reagent. Therefore, the method for detecting drug-resistant bacteria of the present invention and the disc test piece for detection used therein are useful in clinical, epidemiological studies, and other wide fields.

Claims (12)

A method for detecting vancomycin-resistant bacteria using a combination of a disc test piece containing an antibacterial agent and a solid medium in which a target microorganism can grow.
The disk test piece includes at least three kinds of the antibacterial agents, and the antibacterial agents consist only of the following (a) and (b).
(A) Two or more kinds of antibacterial agents selected from polypeptide antibacterial agents, monobactam antibacterial agents and quinolone antibacterial agents (b) glycopeptide antibacterial agents The content per disc is
0.001 to 1,500 μg of polypeptide antibacterial agent,
Monobactam antibacterial agent 0.001-1,500 μg,
Quinolone antibacterials 0.001-1,500 μg,
Glycopeptide antibacterial agent 0.001-1,500 μg,
(A) the polypeptide antibacterial agent is colistin or polymyxin,
The monobactam antibacterial is Aztreonam,
The quinolone antibacterial is nalidixic acid, pipemidic acid, norfloxacin, or ciprofloxacin;
(B) The method according to claim 1 , wherein the glycopeptide antibacterial agent is vancomycin or teicoplanin. The method according to claim 1 or 2 , wherein the antibacterial drug is any one of the following drug combinations (1) to (6).
(1) colistin, nalidixic acid, vancomycin (2) polymyxin, pipemidic acid, teicoplanin (3) colistin, norfloxacin, vancomycin (4) colistin, ciprofloxacin, vancomycin (5) aztreonam, nalidixic acid, vancomycin (6) colistin , Aztreonam, vancomycin The detection target bacteria, vancomycin-resistant enterococci, vancomycin-resistant Staphylococcus aureus, and is at least one selected from vancomycin moderate resistant Staphylococcus aureus claim 1, wherein in any one of 3 The method described. The method according to any one of claims 1 to 4 , wherein the disc specimen contains only the antibacterial agent or only the antibacterial agent and a pH buffer. The solid medium is a blood agar medium, chocolate agar medium, Drigalsky modified medium (BTB lactose agar medium), heart infusion agar medium, brain heart infusion agar medium, trypsoy agar medium (SCD agar medium), normal agar medium, standard Selected from agar medium, bile-esculin-azide agar medium, KF Streptococcus agar medium, Mannit salt medium, egg yolk mannitol salt medium, Egg York salt medium, Staphylococcus No. 110 medium and Baird Parker agar medium 6. The method according to any one of claims 1 to 5 , wherein: A disc test piece for detecting vancomycin-resistant bacteria, comprising at least three types of antibacterial agents, wherein the antibacterial agents consist only of the following (a) and (b).
(A) Two or more kinds of antibacterial agents selected from polypeptide antibacterial agents, monobactam antibacterial agents and quinolone antibacterial agents (b) glycopeptide antibacterial agents The content per disc is
0.001 to 1,500 μg of polypeptide antibacterial agent,
Monobactam antibacterial agent 0.001-1,500 μg,
Quinolone antibacterials 0.001-1,500 μg,
Glycopeptide antibacterial agent 0.001-1,500 μg,
(A) the polypeptide antibacterial agent is colistin or polymyxin,
The monobactam antibacterial is Aztreonam,
The quinolone antibacterial is nalidixic acid, pipemidic acid, norfloxacin, or ciprofloxacin;
(B) The disk test piece according to claim 7 , wherein the glycopeptide antibacterial agent is vancomycin or teicoplanin. The disc test piece according to claim 7 or 8 , wherein the antibacterial agent is any one of the following combinations (1) to (6) of the drug.
(1) colistin, nalidixic acid, vancomycin (2) polymyxin, pipemidic acid, teicoplanin (3) colistin, norfloxacin, vancomycin (4) colistin, ciprofloxacin, vancomycin (5) aztreonam, nalidixic acid, vancomycin (6) colistin , Aztreonam, vancomycin The disk test piece according to any one of claims 7 to 9 , wherein the disk test piece contains only the antibacterial drug or only the antibacterial drug and a pH buffer. It contains at least three types of antibacterial agents used for carrying out the method according to any one of claims 1 to 6, and the antibacterial agents consist only of the following (a) and (b): A disc test piece for detecting vancomycin-resistant bacteria.
(A) Two or more kinds of antibacterial agents selected from polypeptide antibacterial agents, monobactam antibacterial agents and quinolone antibacterial agents (b) glycopeptide antibacterial agents The disc test piece according to any one of claims 7 to 10 is placed on a solid medium coated with a sample, and after culturing at 20 to 40 ° C for 15 to 48 hours, in the drug diffusion region around the disc test piece. A method for determining that the sample contains vancomycin-resistant bacteria when the bacteria grow in