KR102096662B1 - Reagent for the selection assay of acinetobacter baumannii like gram negative bacteria and selection assay of acinetobacter baumannii like gram negative bacteria using the same - Google Patents

Abstract

The present invention relates to a reagent composition for a selection assay of Acinetobacter baumannii-like (A. Baumannii-like) gram negative bacteria, and to an A. Baumannii-like gram negative bacteria selection assay method using the reagent composition. The reagent composition and the A. Baumannii-like gram negative bacteria selection assay method using the reagent composition of the present invention have convenience, accuracy, and rapidness, and can be utilized as basic data to present effective treatment methods by rapidly and accurately discriminating multi-drug resistant A. Baumannii infected patients in the clinical medicine field. Thus, reagent composition and the method are the very useful in the diagnostic medicine industry. The reagent composition comprises a colorless oxidase solution and human serum.

Description

Reagent for THE SELECTION ASSAY OF ACINETOBACTER BAUMANNII LIKE GRAM NEGATIVE BACTERIA AND SELECTION ASSAY OF ACINETOBACTER BAUMANNATIVE BACTERIA USING THE SAME}

The present invention relates to an Acinetobacter baumannii Acinetobacter baumannii (Acinetobacter baumannii) similar to gram negative bacteria screening method for using the (Acinetobacter baumannii) Similar Gram screening reagent composition of the negative bacteria and the reagent composition.

Acinetobacter baumannii is an oxidase-negative glucose non-fermenting Gram-negative rod, aerobic and non-motile algae. In particular, A. baumannii ( A. baumannii ) is widely present in nature, such as soil, water, and sewage, and grows well in dry, inanimate environments. Although it is not a normal germ of the human body, it often forms colonies on the patient's skin or respiratory tract and can survive long on the human body surface. Acinetobacter baumannii is an opportunistic strain or immunodeficiency patient with a very low risk of infection to healthy individuals, patients with long-term hospitalization, patients with ventilators and catapults, and severe cases of catheterized pneumonia, meningitis , It is an important causative agent of in-hospital infection that causes urinary tract infection, surgical wound infection, pleural fluid infection, or blood infection. Recently, the infection of carbapenem-resistant Acinetobacter Baumanii ( A. baumannii ) due to high drug resistance is often multi-drug resistant, which is also resistant to other antimicrobial agents, and is very limited in the selection of appropriate therapeutic agents. Due to the spread of resistant bacteria caused by acquired infection, it is regarded as a major problem in infection management worldwide.

Most bacteria normally secrete proteins out of the cytoplasm. These proteins include adhesin, toxins, protease and other toxic substances, and enzymes that are transported from the cytoplasm to the cell surface and are secreted out of the cell are called foreign enzymes. Foreign enzymes function as digestive enzymes that break down nutrients or toxic substances that cause the pathogenicity of bacteria.

Acinetobacter baumannii secretes proteins containing β-lactamases, phospholipases, aminoglycosidemodifying enzymes, etc. and activates efflux pumps, changes in drug binding sites or secretory organs such as T2SS, T5SS, T6SS According to the structural characteristics of the drug by causing drug resistance, it shows a fatal etiology to the host.

Bacterial enzymes are mostly amino acid polymerized proteins and catalyze metabolic reactions. It has specificity for the substrate, and generally exhibits high activity at a temperature ranging from 35 to 40 ° C and a neutral pH. As the type of enzyme, hydrolytic enzymes that catalyze hydrolysis, enzymes that catalyze various decomposition reactions, and oxidative reductases that catalyze oxidation and reduction are known. The reaction of the enzyme is that the protein part of the enzyme is denatured by heat at high temperature, and the three-dimensional structure of the enzyme is deformed or destroyed.

Oxidase is the last electron acceptor in the electron transport system that breaks down sugars to produce energy by the redox potential difference, and causes oxidation of cytochrome reduced by oxygen molecules. The two oxidized cytochrome c generated at this time reacts with the oxidase reagent to make a quinone-based reactant, which is dark purple.

On the other hand, it has been reported that oxidized cytochrome c present in human serum can induce a positive Oxidase reaction when reacted with 1% oxidase reagent in vitro by the above reaction. However, A. baumannii is an oxidase-negative bacterium that does not contain cytochrome c Oxidase, which plays an important role in this reaction.

Multidrug-resistant A. baumannii (MRAB) infection is caused by A. baumannii , which is resistant to carbapenem, aminoglycoside, and fluoroquinolone antibiotics. MRAB infection is diagnosed as a patient only when MRAB is detected in the blood, and is diagnosed as a pathogen holder when MRAB is detected in a clinical sample other than blood.

Infectious diseases of multidrug-resistant Acinetobacter baumannii transmit resistance genes to other bacteria, resulting in increased mortality and prolonged hospital stay. A. baumannii ( A. baumannii ) is a major cause of obtaining multi-drug resistance, OXA-23 production or OXA carbapenemase production by overproduction of OXA-51, OMP (outer membrane protein) changes, and PBP (penicllin) -binding protein) is known. At present, the carbapenem resistance rate of Acinetobacter bacteria in Korea is reported to be over 50%.

In general, carbapenem antibiotics such as imipenem or meropenem are used to treat infectious diseases of Acinetobacter baumannii . However, carbapenem-resistant Acinetobacter Baumanii ( A. baumannii ) is often resistant to most other antibiotics, so treatment is performed by selecting an available antibiotic based on the antimicrobial susceptibility test results. Is limited in antibiotic selection and colistin is administered in this case. Colistin is effective against Gram-negative bacteria, but it is a drug that causes side effects such as kidney toxicity and neurotoxicity. Therefore, a combination therapy of amikacin, tobramycin and amino-glycoside or colistin and rifampicin, and monotherapy of tigecycline may be performed. MRAB infection is currently classified as a designated infectious disease in Korea, and Acintobacter baumannii is managed as a second-risk class of pathogens capable of human infection and prevention and treatment.

Multidrug-resistant bacteria transmit infection through contact, and in hospitals, they spread very quickly and widely between patients and medical staff. Infection control of multi-drug resistant bacteria must comply with the general principles of multidrug resistant bacteria prevention and management, including proper use and restriction of antibiotics, improved hand hygiene, patient isolation and contact caution, disinfection with disinfectants in hospital environments and medical devices, and sterilization of goods. do.

Currently, microbial identification tests are made with the latest automated system, and it is possible to simultaneously identify bacterial species and minimal inhibitory concentration (MIC). In addition, the latest mass spectrometry-based techniques, molecular biological techniques such as polymerase chain reaction (PCR), and sequencing are also used to identify microorganisms. However, it is not easy to adopt and use the latest automation system practically due to economical conditions because it is very expensive to maintain as well as the equipment itself. Therefore, in most hospital laboratories, an automated test method is widely used to separate bacteria using a selective medium, cultivate them sufficiently, and then automatically read biochemical reactions using an automated microbial identification device.

Vitek2, an automated inspection equipment, has the advantages of quick and easy operation of bacterial identification, but it is a system that conducts tests at the same time because it has a card for identification of bacteria and a card for antibiotic susceptibility testing, and then combines the two test results. (Figure 1). Since Vitek2 automatic microbial identification devices must appropriately select the card panel for identification and antibiotic susceptibility testing according to the type of bacteria to be tested, the nature of these devices is the growth of Acinetobacter baumani before the Vitek2 automatic microbial identification device test . baumannii ) should be primarily visually screened for Gram-negative bacteria showing colonies similar to A. baumannii on blood media or MacConkey medium. However, in the selection process with other Gram-negative bacteria exhibiting colony properties similar to A. baumannii , there is a limit to performing only by visual classification (FIG. 2).

As a result, difficulties in selecting an antibiotic susceptibility test (AST) test card for fermentation and non-fermentation bacteria used in the automatic microorganism identification device of the Vitek2 system occur, and economic and time losses due to mismatch continue to occur. In addition, since the automatic microbial identification device presents the scientific name of the bacterial species based on the results of biochemical reactions, there may be a problem that is entirely dependent on the subjective judgment of the tester when the results of the bacterial identification on the automated inspection device are less than reliability. .

KR 10-2017-0004539 A KR 10-1404473 B

The present invention is to solve the problems of the prior art as described above, the problem to be solved in the present invention is a novel Acinetobacter Baumani ( A. baumannii ) similar Gram-negative bacteria screening with convenience, accuracy and speed It is intended to provide a test reagent and a method for screening a gram-negative bacterium similar to A. baumannii using the reagent.

In order to solve the above problems, the present invention provides a reagent composition for screening a Gram-negative bacterium similar to Acinetobater baumannii comprising a colorless oxidase solution and human serum.

The colorless oxidase solution is preferably at a concentration of 0.1 to 20%.

It is preferable that the component content of the reagent composition includes 300 to 500 parts by weight of human serum relative to 100 parts by weight of a colorless oxidase solution.

In addition, the present invention provides a method for screening for Gram-negative bacteria similar to Acinetobater baumannii comprising the following steps:

(1) inducing a quinone-based reddish purple by leaving a reagent mixed with 300 to 500 parts by weight of human serum at 100 parts by weight of a colorless oxidase solution;

(2) adding and incubating the same volume of the test strain suspension to the reagent from which the quinone-based reddish purple was induced;

(3) visually observing the presence or absence of a change in color of the reagent; And

(4) When the color of the reagent is changed to colorless, it is identified as Acinetobater baumannii , and when the color of the reagent maintains a quinone-red purple color, it is identified as Acinetobater baumannii- like Gram-negative bacteria. Step to.

The incubation is preferably performed at room temperature to 37 ℃.

The reagent composition of the present invention and A. baumannii- like Gram-negative bacterium screening method using the reagent composition have convenience, accuracy, and rapidity, and are multi-resistant acinetobacter baumani in the field of clinical medicine ( A. baumannii ) It is a very useful invention in the diagnostic medicine industry because it can be used as basic data to quickly and accurately discriminate patients and present effective treatment methods.

Figure 1 shows the operation flow diagram of the Vitek2 system for identification of microorganisms.
FIG. 2 shows Acintobacter baumannii and A. baumannii like Gram-negative bacteria having similar colony forms.
Figure 3 illustrates a method for using to identify Acinetobacter baumannii (A. baumannii) and Acinetobacter baumannii (A. baumannii) Similar Gram-negative bacteria to a reagent composition of the present invention as a flow chart.
Figure 4 shows the difference in the results of the temperature of the incubation after adding the Acintobacter baumannii ( A. baumannii ) suspension to the reagent composition in the course of the test method of the present invention.
Figure 5 shows the difference between the results of the presence or absence of heat treatment of the suspension prior to adding the Acintobacter baumannii ( A. baumannii ) suspension to the reagent composition in the course of the test method of the present invention.
Figure 6 shows the results when the protease inhibitor was treated by concentration in the suspension prior to the addition of Acinetobacter Baumanii ( A. baumannii ) suspension to the reagent composition in the course of the test method of the present invention.
Figure 7 shows the results of the treatment of EDTA by concentration in the suspension prior to the addition of Acintobacter Baumanii ( A. baumannii ) suspension to the reagent composition in the course of the test method of the present invention.
8 shows the results of evaluating the reducing power by adding dithiothreitol (DTT) to the reagent composition of the present invention by concentration.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention Acinetobacter baumannii (A. baumannii) is developed Acinetobacter baumannii (A. baumannii) screening test reagent using the extracellular protein secretion, and Acinetobacter in clinical through which Bauer Mani ( The process of identifying A. baumannii ) was not only quick and accurate, but also an economically useful test method was developed.

The Vitek2 automatic microbial identification device used to identify A. baumannii must select different identification and card panel for antibiotic susceptibility testing depending on the nature of the test bacteria. However, in Acinetobacter baumannii , some gram-negative and non-fermentative bacteria and colonies and medium properties are not easily distinguished visually. Therefore, due to errors in the screening process that relies on subjective selection when distinguishing A. baumannii from similar bacterial species, the economical waste of cards required for Vitek2 automated testing, the effort of re-examination, and the reporting time therefor Various problems such as delay are frequently occurring. In the present invention, in order to solve this problem, some gram-negative fermentation bacteria and rain that are not easily distinguished by the naked eye due to a reduction induction reaction of a protein enzymatic system secreted out of cells by A. baumannii that exhibits an oxidase negative reaction The aim was to develop a reagent capable of screening fermentation bacteria and Acinetobacter baumannii .

First, the reagent for screening test was prepared by mixing a colorless oxidase reagent and human serum, and when the reagent is left at room temperature for 10 minutes, it changes to a quinone-red purple oxidase positive reaction solution.

When the reagent was reacted with A. baumannii suspension, it was found that the reddish-purple oxidase positive reaction solution changed colorlessly by a reduction induction reaction. Therefore, in the present invention, it was determined that this reduction-inducing reaction was due to the action of an external enzyme secreted by A. baumannii , and the experiment was conducted using this as a differentiation from similar strains.

Such Acinetobacter baumannii (A. baumannii) suspension action of other enzymes present in the suspension, Acinetobacter baumannii (A. baumannii) to confirm that the material involved in the reduction reaction by the induction was verified by extensive .

First, the reaction results according to the temperature conditions during the reduction induction reaction of the experimental strains using reagents were compared. A reduction induction reaction occurred at 37 ° C and room temperature, but a colorimetric reaction at a temperature of 37 ° C occurred more rapidly than the room temperature condition, and the reduction reaction was more pronounced. In addition, in order to understand the degree of reaction according to the heat treatment, A. baumannii suspended liquid was heat treated at 65 ° C. and 80 ° C. for 20 minutes, respectively, and then the reduction induction reaction was confirmed. As a result, a reduction induction reaction did not occur in the experimental group of A. baumannii suspension, which was heat-treated at 65 ° C and 80 ° C for 20 minutes. From the results of this experiment, a substance that reduces the reagent and changes colorlessly can be estimated as a substance having enzymatic activity contained in A. baumannii suspension, and furthermore, heat deterioration causes protein denaturation. It was found that the foreign enzyme was inactivated in the A. baumannii suspension.

Second, as part of another experiment to confirm whether the reducing inducer contained in A. baumannii suspension was a protein component, the color change of the reagent was observed after protease Inhibitor treatment of the bacterial suspension. Reduction-induction reactions were observed at concentrations of 4.17X to 8.33X when A. baumannii suspension was treated according to the complex protease inhibitor concentration, but no reduction-induction reactions occurred in other concentration ranges. From these results, it was possible to prove that the foreign enzyme component having the action of protease was present in the A. baumannii suspension. The Protease Inhibitor cocktail used in the experiment is a complex of four protease inhibitors (AEBSF, Hydrochloride 500 μM, Aprotinin 150 nM, E-64 1 μM, Leupeptin, Hemisulfate 1 μM), and contains serine, cysteine, trypsin-like protease, and esterase. Can be inactivated. Therefore, it could be assumed that the foreign enzyme component in the A. baumannii suspension was also a protease of this kind.

Thirdly, when the A. baumannii suspension was treated by EDTA concentration, a reduction induction reaction could be observed in a concentration range of 3.13 mM to 6.25 mM, but a reduction induction reaction was observed in a higher concentration. Did not. From these results, it could be estimated that the substance acting on the reduction-inducing reaction of A. baumannii suspension is a protein extraenzyme that exhibits metalloprotease properties.

Finally, dithiothreitol, which reduces the disulfide bond of the protein to the -SH group, was diluted in steps and mixed with a reagent to induce a reduction reaction. As a result, a reduction induction reaction was not observed at a concentration of 3.13 mM to 6.25 mM, but a reduction induction reaction was observed at a concentration higher than that. From these results, it can be estimated that the reduction-inducing reaction of the reagent by A. baumannii suspension is a reductase-reducing substance among the Acinetobacter baumannii external enzymes.

Synthesizing the above experimental results, the substance present in A. baumannii suspension, which can induce a reduction reaction of the reagent, is a bacterial foreign enzyme, and concludes that it is an enzyme type that has protease and reductase functions of protein components. Was able to erase.

In order to investigate the clinical applicability of the reagents developed in the present invention, among the bacteria isolated from clinical samples, 141 strains of A. baumannii and non-Asyne identified in the Vitek2 and Microsacn walkway 96 plus automatic microbial identification devices A verification evaluation of reagents was performed on 60 strains of A. baumannii . When screening with Acinetobacter baumannii and non-Acinetobacter baumannii , the kappa coefficient of the test results using automatic microbial identification devices and reagents was 0.92, which is high between the two methods. Concordance was confirmed, and was judged to be available for clinical application.

Re-analysis was performed with the RapID ONE System and the MALDI biotyper equipment for 6 strains that showed inconsistencies in the reagents used in the present invention and automatic microbial identification. The RapID ONE System is used to identify Gram-negative microorganisms including intestinal bacteria, and is a micromethod method that uses a substrate and a chromogenic substrate. And, the MALDI biotyper is an automated microbial identification device based on Matrix-Assisted Laser Desorption Ionization / Time of Flight using a mass spectrometer.

Through further reanalysis, the reagent was able to accurately distinguish Acinetobacter baumannii from ACB. In addition, the reagent was found to be able to distinguish A. lowffii and A. junii , which are not distinguishable by automatic microbial identification, from A. baumannii .

However, 1 strain M. morganii was confirmed by M. morganii as a result of additional tests with Vitek retest and RapID ONE System in automatic microbial identification. This was the only strain that was unable to accurately screen with the reagent of the present invention, a screening test reagent for identification of A. baumannii .

Thus, the findings, Acinetobacter baumannii a reagent of the present invention designed as a screening reagent (A. baumannii) is Acinetobacter baumannii (A. baumannii) and gross properties are similar colonies of gram-negative bacteria of the present invention Compared to the current type of work that relies on subjective screening to distinguish the human eye, it will be possible to accurately screen and contribute to improving the quality of microbial identification work for identification of A. baumannii . Became.

Accordingly, the present invention provides a reagent composition for screening for Gram-negative bacteria similar to Acinetobater baumannii , comprising a colorless oxidase solution and human serum.

The colorless oxidase solution is preferably 0.1 to 20% concentration.

It is preferable that the component content of the reagent composition includes 300 to 500 parts by weight of human serum relative to 100 parts by weight of a colorless oxidase solution.

In addition, the present invention provides a method for screening for Gram-negative bacteria similar to Acinetobater baumannii comprising the following steps:

(1) inducing a quinone-based reddish purple by leaving a reagent mixed with 300 to 500 parts by weight of human serum at 100 parts by weight of a colorless oxidase solution;

(2) adding and incubating the same volume of the test strain suspension to the reagent from which the quinone-based reddish purple was induced;

(3) visually observing the presence or absence of a change in color of the reagent; And

(4) When the color of the reagent is changed to colorless, it is identified as Acinetobater baumannii , and when the color of the reagent maintains a quinone-red purple color, it is identified as Acinetobater baumannii- like Gram-negative bacteria. Step to.

The incubation is preferably performed at room temperature to 37 ℃.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples describe one preferred embodiment of the present invention, and the scope of the present invention is not limited by the matters described in the following examples.

<Example>

1. Materials and Methods

1.1. Collection of test strains

Since August 2017 isolated from the specimens referred to Gangneung Asan Hospital Laboratory Medicine in Gangneung in Gangwon Province and materials until February 2018 A. baumannii 141 strains and isolates non- A. baumannii 60 [Aeromonas hydrophilia (1 strain) Acinetobacter lwoffii (4 strains), A. junii (1 strain), Enterobacter cloacae (4 strains), Escherichia coli (5 strains), Pseudomonas aeruginosa (2 strains), Providencia rettgeri (2 strains), P. stuartii (7 strains) , Klebsiella oxytoca (1 strain), K. pneumoniae (1 strain), Morganella morganii (14 strains), Stenotrophomonas maltophilia (3 strains), Serratia marcescens (14 strains), S. plymuthica (1 strain)] were used in the experiment. . The clinically isolated strain was cultured in MacConkey agar (Asan Pharmaceutical Co., Ltd., Hwaseong, Korea). Each cultured bacterial strain was dried on Vitek2 GN-card (bioMerieux, Marcy I'Etoile, France) on Vitek2 (bioMerieux, Marcy I'Etoile, France) and Dried on Microscan walkway 96 plus (Beckman coulter, West Sacramento, USA) Identification was performed using Gram Negative Combo Panel Type 72 (Beckman coulter, West Sacramento, USA). A. baumannii ATCC19606 was used as a standard strain.

1.2. Inspection reagent manufacturing

(N, N, N, N) -tetramethyl-p-phenylenediamine dihydrochloride (Aolfa Aesar, Massachusetts, USA) was dissolved in DW to prepare a 1% oxidase solution, aliquoted in a certain amount, and stored frozen under light-shielding conditions. Used. Human serum shows normal serum color when visually confirmed among the serum samples discarded after analysis of the patient's diagnostic test, and the biochemical analysis results fall within the normal range. After screening and refrigeration, it was used for the experiment. The reagent was prepared by mixing 20 µl of a colorless 1% oxidase reagent and 80 µl of human serum and incubating it at room temperature for 10 minutes to induce an oxidase positive reaction causing quinone reddish purple. The reagent from which the oxidase positive reaction was induced was named Acidivision. On the other hand, for the induction of the reduction reaction, 100 µl of A. baumannii suspension (McFarland turbidity 3.5) was dispensed into Acidivision reagent and incubated at 37 ° C for 10 minutes, and then the positive oxidase reaction color (quinone-based reddish purple) was negative (colorless system). It was confirmed to change to (Fig. 3).

1.3. A. baumannii  Proof of foreign enzymes

1.3.1. Enzyme activity according to temperature

10 strains of A. baumannii isolated from the clinic, including the standard strain A. baumannii ATCC19606, were cultured in MacConkey medium for 17 hours. A. cultured using sterile physiological saline for 10 baumannii strains made A. baumannii suspension respectively adjusted to 3.5 McFarland turbidity. 100 µl of A. baumannii suspension was added to the reagent, and reacted for 10 minutes at 37 ° C. and room temperature, respectively, and then the color change was observed.

1.3.2. Enzyme activity according to heat treatment

Ten strains of A. baumannii isolated from the clinic, including the standard strain A. baumannii ATCC19606, were prepared as A. baumannii suspension using sterile physiological saline, and then the heat treatment conditions were changed to heat treatment and non-heat treatment groups. In the heat treatment group, the A. baumannii suspension was heat treated at 65 ° C and 80 ° C for 20 minutes, respectively, and the non-heat treatment group was left at room temperature for the same time. After the 65 ℃, after dividing the heat-treated A. 100 A. baumannii ㎕ the baumannii suspension was allowed to stand at room temperature and the suspension each in 80 ℃ and allowed to react 10 minutes color change in the reagent was observed.

1.3.3. Protease inhibitor cocktail treatment

A. baumannii ATCC19606 strains, including the sterilization of the A. baumannii 10 strain isolated from the clinical use of physiological saline A. baumannii suspension after protease inhibitor complex of Protease Inhibitor Cocktail 2 Perfect (quartett, Berlin, Germany) prepared with 100X Dilution step up to 6.25X. 50 μl of the protease inhibitor corresponding to each dilution was mixed with 100 μl of each A. baumannii suspension and reacted for 15 minutes. After completion of the reaction, 100 μl was dispensed into the reagent to determine the concentration level that could not induce an oxidase positive reaction when the reaction was performed, and the effect of the protease inhibitor on the activity of A. baumannii on the external enzyme was estimated.

1.3.4. EDTA treatment

10 strains of clinically isolated A. baumannii , including the standard strain A. baumannii (ATCC19606), were prepared as A. baumannii suspension using sterile physiological saline, and the metalloprotease inhibitor EDTA (DAEJUNG, Siheung, Korea) from 200 mM to 6.25 Diluted stepwise to mM. 100 µl of EDTA dilution corresponding to each dilution was reacted with 100 µl of each A. baumannii suspension and complex mixture for 15 minutes. After the reaction was completed, 100 μl was dispensed into the reagent to determine the concentration level that could not induce an oxidase positive reaction when the reaction was carried out, and the effect of the metalloprotease inhibitor on the external enzyme action of A. baumannii was estimated.

1.4. A. baumannii  Measurement of reducing power of exoenzyme

The reducing agent dithiothreitol (DTT; SIGMA, Steinheim, Germany) was diluted stepwise from 50 mM to 3.125 mM. Reducing power of A. baumannii was estimated by determining the concentration level that does not induce an oxidase negative reaction when assaying by dispensing 100 μl of DTT corresponding to each dilution to a reagent.

1.5. Verification of test strain mismatch

The strains identified as Vitek2 and Microscan walkway 96 plus automated microbial identification targets for clinically isolated strains as reagents are identified as RapID ONE System (REMEL, Lenexa, USA) and MALDI biotyper (BRUKER, Bremen, Germany) equipment was verified with microflex (BRUKER, Bremen, Germany).

1.6. Statistical analysis

Match between test results identified with A. baumannii and non- A. Baumannii and results selected with reagents in automatic microbial identification devices (Vitek2 system, s / w name: Vitek2 compact, Microscan system, s / w name: Lap pro) The degree of evaluation was evaluated by the kappa coefficient using the SPSS (PASW Statistics18; IBM, New York, USA) program, and it was determined that it was significant when the κ coefficient was 0.8 or more.

2. Research Results

2.1. A. baumannii  Reduction reaction by external enzyme

2.1.1. Enzyme activity and inactivation

In order to confirm whether the reduction induction reaction of the reagent was due to the action of the foreign enzyme present in the A. baumannii suspension, only the temperature conditions were set differently when inducing the reduction reaction of the experimental strain with the reagent to compare the reaction results at 37 ° C and room temperature.

As a result of the reaction, a similar reduction reaction was induced at two temperature conditions, but a colorimetric reaction at a temperature of 37 ° C. occurred more rapidly than at room temperature, and the reduction reaction was more pronounced (FIG. 4).

2.1.2. A. baumannii  heat treatment of suspension

A. baumannii is other enzymes contained in the suspension is modified by a heat treatment thereto to A. baumannii suspension to see if the reaction is inhibited induction reduction was carried out according to the experimental group divided by the thermal treatment and the specific heat-treated group. The heat treatment group was heat treated at 65 ° C and 80 ° C for 20 minutes, respectively, and the non-heat treatment group was left at room temperature for the same time. Then, 100 µl of A. baumannii suspension heat-treated at 65 ° C, 80 ° C, and room temperature, respectively, was added to the reagent, reacted for 10 minutes, and then observed for reduction reaction.

As a result, no reduction induction reaction occurred in the experimental group heat-treated at 65 ° C and 80 ° C for 20 minutes. However, in the non-heat treatment group, a complete reduction reaction was induced, and the color of the reagent was changed to colorless (FIG. 5).

2.1.3. Protease inhibitor cocktail treatment result

In order to experimentally prove that the A. baumannii foreign enzyme acts on the reduction-induced reaction of the reagent by A. baumannii suspension, the complex protease inhibitor Protease Inhibitor Cocktail 2 Perfect was diluted stepwise from 100X to 6.25X. 50 μl of the protease inhibitor corresponding to each dilution was mixed with 100 μl of each A. baumannii suspension, and then reacted at room temperature for 15 minutes. After completion of the reaction, 100 μl of this mixture was dispensed into the reagent, and then the concentration level that could not induce an oxidase positive reaction was determined as an assay standard to estimate the effect of the protease inhibitor on the activity of A. baumannii 's external enzyme.

When A. baumannii suspension was treated with complex protease inhibitors by concentration, reduction reactions were observed at concentrations of 4.17X to 8.33X. The results of this experiment showed that the A. baumannii suspension treated with the protease inhibitor did not induce a reduction reaction of the reagent, and the reduction-inducing reaction of the A. baumannii suspension was caused by the action of protease among the foreign enzymes secreted by this bacterium. It was proved (Fig. 6).

2.1.4. EDTA treatment results

A. When exoenzyme with a metalloprotease function acts when inducing a reduction reaction of a reagent with a baumannii suspension, the induction of the reaction should not occur if the enzyme system involved in the reduction induction reaction is inactivated with EDTA, a protease inhibitor. After EDTA, a metalloprotease inhibitor, was serially diluted from 200 mM to 3.13 mM, 100 µl of protease inhibitor EDTA corresponding to each dilution was reacted with 100 µl of A. baumannii suspension and a complex mixture for 15 minutes. Subsequently, 100 µl of the mixed solution was dispensed into the reagent, and then the degree of reduction induction reaction was observed, and the exoenzyme activity of A. baumannii was estimated based on the concentration at which the reagent changed from reddish purple to colorless.

A. When the baumannii suspension was treated by EDTA concentration, a reduction induction reaction was observed in the concentration range of 3.13 mM to 6.25 mM, but a reduction induction reaction was not observed in a higher concentration. From these results, it can be inferred that the protease, a metalloprotease-based foreign enzyme, acts on the reduction-induction reaction of A. baumannii suspension (FIG. 7).

2.2. A. baumannii  Measurement of reducing power of foreign enzymes

Dithiothreitol (DTT), diluted by concentration, was added to the reagent, and then induction of oxidase negative reaction was tested. Dithiothreitol (DTT; SIGMA, Steinheim, Germany), which reduces the disulfide bond of the protein to the -SH group, was diluted stepwise from 50 mM to 3.125 mM. Reducing power of A. baumannii was estimated by determining the concentration level that does not induce an oxidase negative reaction when assaying by dispensing 100 μl of DTT corresponding to each dilution concentration into a reagent.

When the reagents were treated with dithiothreitol concentration, a reduction induction reaction was not observed at a concentration of 3.13 mM to 6.25 mM, but a reduction induction reaction was observed at a concentration higher than that. From these results, it can be inferred that the reduction-inducing reaction of the reagent by A. baumannii suspension is a reduction action of a substance having a reductase function among A. baumannii external enzymes (FIG. 8).

2.3. Isolation of clinical isolates using automated microbial identification devices and acidivision A. baumannii  Selectivity comparison

The A. baumannii strains isolated from the Vitek2 and Microscan walkway 96 plus automatic microbial identification devices and 60 non- A. Baumannii strains were able to quickly and accurately distinguish A. baumannii from bacteria with similar visual characteristics of colonies. Validation evaluation was conducted for the reagents. Reagents were specifically identified in A. baumannii , and it was confirmed that the reduction reaction was induced and changed to colorless. Therefore, it was performed using the clinically possible to target the 60 strains identified to be a strain with non- 141 A. baumannii identified as A. baumannii on the automatic microorganism identification device to verify the reagent and the cross check (Table 1).

[Table 1]

Of the 141 strains identified as A. baumannii in automatic microbial identification devices, 135 strains (96%) were consistent with A. baumannii in screening using reagents. However, six strains Auto microbial identification devices have been identified as A. baumannii, the screening test using the reagent showed non A. baumannii. Of the 60 strains identified as non- A. Baumannii in an automated microbial identification device, 59 strains (98%) were identified as not A. baumannii in a screening test using reagents. However, 1 strain was identified as Morganella morganii in the automatic microbial identification device, but the reagent showed the result of being judged as A. baumannii .

2.4. Test strain mismatch verification result

The strains identified as the 6 strains and A. baumannii is determined that not the A. baumannii on the reagents were added to the RapID ONE System retest analysis and MALDI biotyper device.

In the case of 6 strains that were determined to be not A. baumannii in the reagent, all 6 strains were identified as ACB ( Acinetobacter calcoaceticus -baumannii) in the RapID ONE System, and in the MALDI biotyper, Acinetobacter bereziniae 4 strains, A. pittii 1 strain, A, respectively It was identified as the nosocomialis 1 strain.

One strain identified as A. baumannii among the non- A . Baumannii strains in the reagent was identified as M. morganii in the RapID ONE System, and was consistent with the retest results in an automatic microbial identification device (Table 2).

[Table 2]

Claims (5)

Acinetobater baumannii- like reagent composition for screening for Gram-negative bacteria similar to a colorless oxidase solution and human serum. According to claim 1, wherein the colorless oxidase (oxidase) solution is a reagent composition, characterized in that 0.1 to 20% concentration. According to claim 1, Reagent composition characterized in that it contains 300 to 500 parts by weight of human serum relative to 100 parts by weight of a colorless oxidase solution. Acinetobater baumannii- like Gram-negative bacterium screening method comprising the following steps:
(1) inducing a quinone-based reddish purple by leaving a reagent mixed with 300 to 500 parts by weight of human serum at 100 parts by weight of a colorless oxidase solution;
(2) adding and incubating the same volume of the test strain suspension to the reagent from which the quinone-based reddish purple was induced;
(3) visually observing the presence or absence of a change in color of the reagent; And
(4) When the color of the reagent is changed to colorless, it is identified as Acinetobater baumannii , and when the color of the reagent maintains a quinone-red purple color, it is identified as Acinetobater baumannii- like Gram-negative bacteria. Step to. The method of claim 4, wherein the incubation is performed at room temperature to 37 ° C.

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