JPH07313193A - Exaiviination of drug sensitivity of bacteria - Google Patents

Abstract

PURPOSE:To provide an examination method capable of quickly (within about 4-6hr) estimating the drug sensitivity of bacteria. CONSTITUTION:A medium containing a drug inoculated with a specimen bacteria or a mixture of a specimen bacteria and other bacteria. The increase in NO2<-> or decrease in NO3<-> in the culture system is measured after cultivation and if the increase in NO2<-> or decrease in NO3<-> is observed, the specimen bacteria is judged to be resistant to the drug.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring and evaluating rapid drug sensitivity of bacteria.

[0002] currently being used many chemotherapeutic agents in the pharmaceutical art.
As a result, bacteria become resistant to these drugs, and clinical effects of the drugs cannot be obtained, which makes the bacteria resistant to drugs. Accurate diagnosis of infectious diseases and rapid selection of therapeutic agents are required in the medical field, and rapid bacterial drug susceptibility testing is essential for the selection of such therapeutic agents. In general, medical institutions test the susceptibility of bacteria isolated from clinical materials to drug selection of chemotherapeutic agents to be used.

The purpose of the drug susceptibility test is to accurately select a drug effective for treating an infectious disease in a short time. That is, which drug has an antibacterial effect on the detected causative bacteria,
It is also to measure how much antibacterial activity is,
Accuracy and speed are required.

Conventionally, the following three methods have been widely used as drug sensitivity tests. (1) Disc method The test bacteria are spread on an agar plate, a disc containing a certain concentration of the drug is placed on the plate, and the diameter of the circle in which the growth of bacteria is inhibited by the diffused drug is measured. (2) Liquid medium dilution method A doubling diluted solution of a specific drug is added to a liquid medium, a test bacterium is inoculated into this medium, and the minimum concentration (MIC) of the drug that inhibits the growth of the bacterium is measured. (3) Agar plate dilution method A test medium is inoculated into an agar medium containing a double-diluted solution of a specific drug, and the minimum concentration (MIC) of the drug that inhibits the growth of the bacteria is measured.

All of these methods are based on direct observation of bacterial growth and determination. That is, drug sensitivity is determined by observing how much bacterial growth is inhibited and how much bacterial count is reduced. Therefore, when the culturing time of the test bacteria is included, it takes 2-3 days until drug-sensitive results are obtained, during which time the patient's condition may worsen and, in the worst case, death may occur.

As a method for solving these problems, for example, the method described in JP-A-61-96999 has been proposed. In this method, a culture medium for bacteria is filled with a drug-containing medium, and after culturing a test bacterium, a substrate that serves as an indicator of bacterial growth is added and allowed to react for a certain period of time, and an indicator for the substrate and substrate decomposition products. Alternatively, the presence or absence of bacterial growth is detected by a change in color tone with a color former.

Further, in Japanese Patent Laid-Open No. 62-25998, MI is disclosed.
A method has been proposed in which both C (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) are automatically obtained.

However, either method requires a considerable amount of time before a drug sensitivity result is obtained.

[0009]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a test method capable of rapidly evaluating drug sensitivity of bacteria.
The present inventors have now determined that bacterial drug susceptibility is determined by the bacterial NO ₃
^- to obtain a finding that can be evaluated through the reduction action. The present invention is based on that finding. The bacterial drug susceptibility test method according to the present invention comprises:
It was inoculated with a mixture of test bacteria or test bacteria and other bacteria, cultured, NO 2 in the culture system ^_- increase, or NO
_{3 ^-} decrease measured, NO 2 ^_- increased, or NO 3 ^_- if there is decrease is, those which comprise determining to be resistant to the test bacteria against drug .

According to the present invention, the drug sensitivity of bacteria can be evaluated more rapidly, according to the preferred embodiment, in about 4 to 6 hours, as compared with the conventional method which required 2 to 3 days until the determination. You can In addition, the present invention is also advantageous in that it is possible to evaluate the drug sensitivity of a test bacterium and to select an appropriate drug without separating the test bacterium even when other bacteria are mixed.

[0011]

The process according to the invention DETAILED DESCRIPTION OF THE INVENTION The bacterial drug sensitivity, bacterial NO 3 ^_- is a method of assessing through reducing action. Without being bound by the following theory, the reason why the drug sensitivity of bacteria can be evaluated by the method according to the present invention is considered as follows. By nature, many oxygen-breathing bacteria can also anaerobically develop using nitrates instead of oxygen as hydrogen acceptors. That is, nitrate can be utilized as anaerobic respiration even when oxygen is exhausted as a result of aerobic respiration. And, the reduction product of nitric acid is generally nitrite. Most of the aerobic bacteria have the ability to assimilate nitrates. Here, when a drug is present in the medium and the drug does not suppress the growth of bacteria, NO
_{2 ^-} production, or NO 3 ^_- should assimilating is observed. On the other hand, the case of suppressing the agent is to foster bacterial, or if the bacteria are not present, NO 2 ^_- acidic or NO,
₃ ^-No utilization is observed. Therefore, NO in this culture system
₂ ^- increase, or NO ₃ ^- by measuring the reduction amount, NO ₂ ^-
Increase, or NO 3 ^_- in the case of a decrease, may be able to determine that a resistance that the test bacteria against drug.

[0012] As described above, the method according to the present invention, the ability to assimilate nitrate, i.e. NO 3 ^_- is applicable to any microorganisms having a reducing action. NO 3 ^_- reduction action is the ability to have a number of bacteria, the method according to the invention can be said to be capable applicable for a wide range of bacteria method.

Therefore, the bacterium to which the method according to the present invention can be applied is not particularly limited as long as it has the ability to assimilate nitric acid. For example, Escherichia coli, Enterobacter
aerogenes, Klebsiella pneumoniae, Alcaligenes fa
ecalis, Pseudomonas aeruginosa, Serratia marcescen
s, Sfaphylococcus aureus, Enterococcus faecalis
, Enterobacteriaceae (intestinal bacteria group) and other bacteria can be applied.

Since nitrite is quite harmful,
Nitrate reduction, which accumulates nitrite, does not produce sufficient anaerobic growth. Successful anaerobic growth of fungi using nitrates requires the reduction of nitrites to further non-toxic products, namely N ₂ O, N ₂ or NH ₃ .
However, in the method according to the present invention, these N ₂ O,
The bacterium's drug susceptibility is assessed at a stage prior to reduction to N ₂ or NH ₃ .

Some bacteria produce nitrogen or ammonia from nitrate without passing through nitrite. In such a bacterium, the drug sensitivity is evaluated by measuring the reduction amount of NO ₃ ⁻ . Further, it is considered possible to evaluate the drug sensitivity by measuring the production amounts of these nitrogen and ammonia.

In the concrete implementation of the method according to the present invention, first, a test sample which may contain a test bacterium is collected or prepared. Preparation of test sample is performed by collecting urine, body fluid such as sediment, cerebrospinal fluid, blood, etc. from clinical materials, specifically, patients with infectious diseases, and if necessary diluting it with an appropriate medium. Is preferred. Moreover, in the method according to the present invention, even if the bacterium to be evaluated is present in the sample in a state of being mixed with other bacteria, the drug sensitivity of the entire mixture can be evaluated. As a result, an appropriate drug can be selected. Therefore, the isolation of the test bacteria may not be sufficient. That is, infectious diseases are generally caused by multiple bacteria rather than a single bacterium. According to the method according to the invention, in the case of multi-bacterial infections, agents which are sensitive to bacterial species with a high bacterial population or bacterial species with a fast growth rate are selected and are sufficient.

In the method of the present invention, a medium containing a specific drug is further prepared. This medium may be appropriately selected in consideration of the type of bacteria expected to exist, but preferred specific examples include Mueller-Hinton.
broth (Beef, infusion: 300g / l, Casamino acids: 17.5g /
l, Soluble starch: 1.5g / l), Brain Heart infusion br
oth (Calf brain, infusion: 200g / l, Beef heart, infusi
on: 250g / l, Proteose Peptone: 10g / l, Dextrose: 2g / l
, NaCl: 5g / l, Disodium phosphate: 2.5g / l), Heart i
nfusion broth (Beef beart, infusion: 500g / l Tryptose:
10 g / l, NaCl: 5 g / l) and the like. To such a medium, a drug that is considered to be effective against the test bacterium, specifically, an antibiotic or the like is added. The amount to be added may be appropriately determined in consideration of the test bacterium expected to exist, the type of drug, the culture conditions, etc., but is preferably about 0.1 to 500 μg, more preferably 1 to 50 μg medium per 1 ml of the medium. It is a degree.

Next, the test sample which will contain the above-mentioned test bacteria is inoculated into the medium containing the drug and cultured. The culture conditions may be appropriately determined in consideration of the test bacteria expected to exist, the type of drug, the culture conditions, etc., but according to a preferred embodiment, the culture time is about 2 to 12 hours, more preferably It is possible to evaluate the drug sensitivity with a culture time of about 4 to 6 hours. The culture may be aerobic, but static culture without stirring or aeration is preferred. This is because, as described above, it is generally considered that nitration of bacteria occurs after oxygen is exhausted, so it is preferable to terminate aerobic respiration early.

In the method according to the invention, the NO ₂ ⁻ in the culture system is increased or NO ₃ is increased as a result of the culture of the test bacteria.
^- To determine whether decreased. This increase or decrease may be before or after the culture, and the amount of increase in NO ₂ ⁻ or the decrease in NO ₃ ⁻ at any time during the culture may be measured. The NO ₂ ^- increase of, or NO
_{3 ^-} decrease of can be determined in a conventional manner, preferably measured by using the auto-analyzer. As such an autoanalyzer, a commercially available one can be used.

[0020]

The present invention will now be described in more detail with reference to the following examples, which are merely examples and the present invention is not limited thereto. The following abbreviations are used below. ABK: Arbekacin FOM: Fosfomycin VCM: Vancomycin CFS: Cefsulodin IPM: imipenem

Example 1 21 g of commercially available Mueller Hinton Broth (hereinafter abbreviated as "MHB", manufactured by Difco) was added with 1 liter of water, dissolved by heating, and sterilized at 121 ° C. for 15 minutes. As a pre-culture, 1 ml of E. coli NIHJ JC-2 as a test bacterium was added to 10 ml of M.
An HB medium was inoculated and statically cultivated at 37 ° C. for 20 hours for preparation. For the test, dilute it with MHB medium and
10 ml of the bacterial solution adjusted to have a concentration of 0 ⁷ , 1 × 10 ⁵ cells / ml was used. After adding 0.1 ml of 10 mM KNO ₃ solution thereto, the drug having the concentration shown in the following table was added, and the mixture was incubated at 37 ° C. Sampling with time to remove mycelium (pore size 0.22μ)
M, manufactured by Millipore), and the obtained filtrate was put in a sample cup, and the concentration (μM) of NO ₂ ⁻ and NO ₃ ⁻ was measured by an autoanalyzer (manufactured by Blanc Roube). The measurement result was as shown in Table 1.

[0022]

[Table 1]

Example 2 MRSA (methicillin-resistant Stap) was used as a test bacterium.
hylococcus aureus) 1371, the same test as in Example 1 was performed. The results are as shown in Table 2.

[0024]

[Table 2]

Example 3 Pseudomonas aeruginosa (Pseudo) as a test bacterium
monas aeruginosa) E-2 was used and the same test as in Example 1 was performed. The results are as shown in Table 3.

[0026]

[Table 3]

[Procedure amendment]

[Submission date] June 23, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction content]

However, both methods require a considerable amount of time before the drug sensitivity result is obtained.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0013

[Correction method] Change

[Correction content]

Therefore, the bacterium to which the method according to the present invention can be applied is not particularly limited as long as it has the ability to assimilate nitric acid. For example, Escherichia col.
i, Enterobacter aerogenes,
Klebsiella pneumoniae, Alc
alignes faecalis, Pseudom
onas aeruginosa, Serratia
marcescens, Staphylococcus
aureus, Enterococcus faec
It can be applied to bacteria such as alis and Enterobacteriaceae (group of intestinal bacteria).

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0016

[Correction method] Change

[Correction content]

In the concrete implementation of the method according to the present invention, first, a test sample which may contain a test bacterium is collected or prepared. Preparation of a test sample is performed by collecting urine, sputum, body fluid such as sputum, and blood from a clinical material, specifically, a patient with an infectious disease, and diluting it with an appropriate medium in some cases. preferable. Further, in the method according to the present invention, even if the bacteria to be evaluated are present in the sample in a state of being mixed with other bacteria, the result is obtained by evaluating the drug sensitivity of the entire mixture. It enables selection of an appropriate drug. Therefore, the isolation of the test bacteria may not be sufficient. That is, infectious diseases are generally caused by multiple bacteria rather than a single bacterium. According to the method according to the invention, in the case of multi-bacterial infections, agents which are sensitive to bacterial species with a high bacterial population or bacterial species with a fast growth rate are selected and are sufficient.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0017

[Correction method] Change

[Correction content]

In the method of the present invention, a medium containing a specific drug is further prepared. This medium may be appropriately selected in consideration of the type of bacteria expected to exist, but preferred specific examples include, for example, Mueller
Hinton broth (Beef, infusio
n: 300 g / l, Casamino Acids: 1
7.5 g / l, Soluble Starch: 1.5
g / l), BrainHeart infusion
broth (Calf Brains, infusio
n: 200 g / l, Beef Heart, infus
ion: 250 g / l, Proteose Pepto
ne: 10 g / l, Dextrose: 2 g / l, Na
Cl: 5 g / l, Disodium Phosphat
e: 2.5 g / l), Heart infusion
broth (Beef Heart, infusio
n: 500 g / l Tryptose: 10 g / l, N
aCl: 5 g / l) and the like. To such a medium, a drug that is considered to be effective against the test bacterium, specifically, an antibiotic or the like is added. The amount to be added may be appropriately determined in consideration of the test bacterium expected to exist, the type of drug, the culture conditions, etc., but is preferably about 0.1 to 500 μg, more preferably 1 to 50 μg medium per 1 ml of the medium. It is a degree.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction content]

Next, the test sample which will contain the above-mentioned test bacteria is inoculated into the medium containing the drug and cultured. The culture conditions may be appropriately determined in consideration of the test bacteria expected to exist, the type of drug, the culture conditions, etc., but according to a preferred embodiment, the culture time is about 2 to 12 hours, more preferably It is possible to evaluate the drug sensitivity with a culture time of about 4 to 6 hours. The culture may be aerobic, but static culture without stirring or aeration is preferred. This is because, as described above, it is generally considered that the nitrite utilization of bacteria occurs after oxygen is exhausted, and therefore it is preferable to terminate aerobic respiration early.

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0021

[Correction method] Change

[Correction content]

Example 1 21 g of commercially available Mueller Hinton Broth (abbreviated as "MHB" hereinafter, manufactured by Difco) was dissolved in 1 liter of water by heating, and sterilized at 121 ° C. for 15 minutes. As a pre-culture, the E. coli N
IHJ JC-2 1ase was inoculated into 10 ml of MHB medium, and statically cultured at 37 ° C. for 20 hours for preparation. For the test, this was diluted with MHB medium to give 1 × 10 ⁷ , 1 ×
10 ml of the bacterial solution adjusted to have a concentration of 10 ⁵ cells / ml was used. After adding 0.1 ml of a 15 mM KNO ₃ solution thereto, a drug having a concentration shown in the following table was added and the mixture was incubated at 37 ° C. Sampling over time,
Millex to remove bacteria (pore size 0.22 [mu] M, manufactured by Millipore) and filtered treated, put the resulting filtrate a sample cup, the automatic analyzer (manufactured by Bran Roubaix Co.), NO ₂ ^- - and NO ₃ ^- Was measured (μM). The measurement result was as shown in Table 1.

Claims (2)

[Claims] To 1. A medium containing drug, it was inoculated with a mixture of test bacteria or test bacteria and other bacteria, and cultured, NO 2 in the culture system ^_- increase, or NO 3 ^_- decrease the amount was measured, NO 2 ^_- increased, or NO 3 ^_- in the case of a decrease, the comprising determining that the test bacteria resistant to the drug, drug sensitivity test method of bacteria . 2. The method according to claim 1, wherein the test bacterium is a bacterium that assimilates nitric acid.