KR20220076951A - Reagent Composition, Kit, and Method for the Detection of Gram-Negative Bacteria Acinetobacter Baumannii - Google Patents

Abstract

상기 화학식 1에서,
R¹ 및 R²는, 각각 독립적으로 탄소수 1 내지 5개의 직쇄 또는 분지쇄 알킬이다.The present invention relates to a reagent composition for detecting Gram-Negative bacteria Acinetobactger baumanii , a kit for diagnosing Acinetobacter baumani infection, and a method for detecting Acinetobacter baumanii. According to the present invention, only Acinetobacter baumani can be selectively detected by using a compound represented by the following formula (1):
[Formula 1]

In Formula 1,
R ¹ and R ² are each independently a linear or branched chain alkyl having 1 to 5 carbon atoms.

Description

Reagent Composition, Kit, and Method for the Detection of Gram-Negative Bacteria Acinetobacter Baumannii

The present invention relates to a reagent composition, kit, and detection method for detecting Gram-Negative bacteria Acinetobacter Baumannii .

Globally, due to the misuse of antibiotics, the number of pathogens exhibiting multi-drug resistance is increasing significantly, and the increasing threat of bacteria and drug resistance are constantly harming humans. Accordingly, many organizations, such as the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO), are worldwide recognized as a serious threat to public health. Accordingly, in 2017, the World Health Organization (WHO) identified the six bacterial species that pose the greatest threat to human health [ESKAPE: Enterococcus faecium (EF), Staphylococcus aureus (SA), Klebsiella pneumoniae (KP), Acinetobacter baumannii (AB), Pseudomonas aeruginosa ). (PA) and Enterobacter species (ES)].

Identification of bacterial strains is very important for diagnosis, prevention and treatment of infectious diseases caused by bacteria. Bacterial culture is required for diagnosis and identification of such bacteria, and differential staining is generally performed to identify them. However, a general staining method requires several steps, and misdiagnosis may occur depending on the staining method and readout. Recently, as an alternative to this traditional differential staining technique, fluorescence staining based on dyes or target molecules has been attracting attention. This fluorescence imaging technique can be quantified in real time through bacterial monitoring (survival/death) and is usefully used in various clinical fields due to the high selectivity and sensitivity of fluorescence. Accordingly, it was recognized that there is a need for an improved method for imaging specific bacteria. In particular, it is very necessary to develop a fluorescence-based staining technique for clearly identifying and diagnosing pathogens such as ESKAPE.

One object of the present invention is to provide a reagent composition capable of selectively detecting a specific bacterium.

Another object of the present invention is to provide a diagnostic kit capable of selectively detecting a specific bacterium.

Another object of the present invention is to provide a method for selectively detecting a specific bacterium.

In the present invention, the bacteria-dye combination screening (BDCS) approach, which is a new approach for identifying and diagnosing biofilms caused by target bacteria that can be generated in medical devices (catheters, etc.) as well as specific bacteria, is newly developed. introduced and used. In the present invention, among ESKAPE strains in the BDCS development process, drug-sensitive (in particular, carbapenem-sensitive) Acinetobacter baumannii and drug-resistant (in particular, carbapenem-resistant) Acinetobacter baumannii (CRPA) , carbapenem-sensitive A. baumannii ) was identified as a dye capable of selectively fluorescence imaging, as well as Acinetobacter baumannii bacteria and biofilms capable of selectively fluorescence imaging in the urinary catheter.

Accordingly, according to an aspect of the present invention, a reagent composition for selectively detecting Acinetobacter baumannii , containing a compound represented by the following formula (1) is provided:

[Formula 1]

In Formula 1, R ¹ and R ² are each independently a linear or branched chain alkyl having 1 to 5 carbon atoms.

According to another aspect of the present invention, there is provided a kit for diagnosing infection of Acinetobacter baumannii , comprising the compound represented by Formula 1 above.

According to another aspect of the present invention, there is provided a method for selective detection of Acinetobacter baumannii , comprising the step of contacting the compound represented by Formula 1 with an analyte.

According to an embodiment of the present invention, the selective detection method may further include observing the fluorescence of the analyte.

Since the compound according to the present invention selectively fluoresces only Acinetobacter baumani, it is possible to selectively detect only Acinetobacter baumani among various bacteria. Accordingly, by using the reagent composition, diagnostic kit, and detection method containing the compound of the present invention, only Acinetobacter baumani can be selectively, efficiently, simply and quickly diagnosed and monitored. In particular, since Acinetobacter baumani is a kind of superbacteria and is a major cause of urinary tract infection, it can be used to screen bacteria that cause urinary tract infections or to monitor contamination of medical devices in real time.

1 is a diagram showing the results of bacterial-dye combination screening using BMeS-pA, coumarin 120, and rhodamine 123 for multidrug-resistant Gram-negative and positive bacteria.
Figure 2 is a graph showing the change in bacterial cell membrane zeta potential caused by BMeS-pA (compound of formula 2).
3 is a graph showing absorption and fluorescence spectra of BMeS-pA.
4 is a graph showing the stability test results of BMeS-pA in various conditions such as pH buffer, temperature, light, serum and biometal.
5 is a graph showing the results of the minimum inhibitory concentration test for confirming the antibacterial effect of BMeS-pA.
6 is a diagram showing the results of bacterial biofilm fluorescence imaging by BMeS-pA.
7 is a diagram showing an image showing the fluorescence of the urinary catheter coated with a specific bacterial biofilm by the compound of the present invention.

Hereinafter, the present invention will be described in detail.

The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. Unless defined otherwise, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Throughout the specification, when a part "includes", "contains", or "has" a certain element, it means that other elements may be further included unless otherwise defined.

The present invention relates to a reagent composition for selective detection of Acinetobacter baumannii , characterized in that it contains a compound represented by the following formula (1), Acinetobacter baumannii ) A kit for diagnosing infection, and Acinetobacter baumannii Provided is a method for selective detection of Bacto baumannii ( Acinetobacter baumannii ):

[Formula 1]

In Formula 1, R ¹ and R ² may each independently be a straight-chain or branched chain alkyl having 1 to 5 carbon atoms, and specifically, each independently may be a straight-chain or branched chain alkyl having 1 to 3 carbon atoms.

The compound of Formula 1 may be specifically represented by Formula 2 below:

[Formula 2]

The compound of Formula 1 is a single benzene ring-based fluorescent probe. Benzene is the simplest aromatic hydrocarbon with 6 carbon rings and is one of the most basic structural units for constructing pi-conjugation systems, which are widely used as fluorescent dyes for fluorescence imaging, display, and other light-emitting materials due to their enhanced spectral signal. The compound of Formula 1 of the present invention exhibits high fluorescence in liquid and solid states, and is chemically stable under light irradiation and various acidity conditions. The compound of Formula 1 is a positive charge green fluorescent probe containing two amines (-NH ₂ ), and exhibits absorption at 377 nm and fluorescence at 517 nm.

In an embodiment of the present invention, fluorescence imaging screening was performed on 8 gram-negative bacteria and 4 gram-positive bacteria (including multidrug-resistant bacteria) through the fluorescent probe, ie, the compound of Formula 1 (FIG. 1). Through this, it was confirmed that the compound of Formula 1 of the present invention is selectively fluorescently stained with respect to the Gram-negative bacteria Acinetobacter baumani sensitive and resistant strains (FIG. 1).

Commercially available bacterial fluorescence staining probes can be applied to fluorescence imaging by largely distinguishing gram-negative and gram-positive bacteria, but fluorescent probes capable of fluorescence imaging targeting specific bacteria have not been reported so far. The compound of Formula 1 of the present invention can selectively perform fluorescence imaging of specific bacteria (Gram-negative Acinetobacter baumani sensitive and resistant strains), and can be used in reagent compositions, kits, etc. to selectively detect only the corresponding bacteria. have.

Currently, as fluorescent probes for staining bacteria, SYTO 9, PI (Propidium Iodine), Alexa-Fluoro, and the like are commercially available. However, since these fluorescent probes are multi-aromatic-based phosphors, they have problems such as low solubility in most organic solvents and low photostability due to their large molecular weight and tight binding. The compound of Formula 1 of the present invention has high photostability, thermal stability, stability in serum and acidity in vivo due to the characteristics of the material. Therefore, the compound of the present invention can be applied not only for biological research, but also high-resolution microscopy (super-resolution microscopy) of bacterial fluorescence imaging through this property.

In this regard, in an embodiment of the present invention, the zeta-potential of Acinetobacter baumani (sensitive / resistant strain) cell membrane was investigated by the compound of Formula 1, and the compound of the present invention When stained with, it was confirmed that the zeta potential of the cell membrane of the Acinetobacter baumani strain increased (FIG. 2). At this time, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA, methicillin-resistance S. aureus ) were typically used as a control among gram-positive bacteria that are not stained with BMeS-pA. In an embodiment of the present invention, the optical properties of BMeS-pA were analyzed based on the above results, and as a result, bio-metal; FeCl ₂ , FeCl ₃ , CuCl ₂ , CaCl ₂ , ZnCl ₂ , KCl, NaCl ₂ , MgCl ₂ It was confirmed that there was no specific reaction, pH7.4 buffer, light, temperature (50 ℃) and serum albumin ( It was confirmed that it was stable against bovine serum albumin (BSA) (FIG. 3-4).

According to one embodiment of the present invention, the Acinetobacto baumani selectively detected in the reagent composition, the infection diagnosis kit, and the detection method of the present invention are specifically multi-drug resistant Acinetobacter baumani. , more specifically carbapenem-resistant Acinetobacter baumani.

The Acinetobacter baumani may create a biofilm on a medical device. In particular, the Acinetobacter baumani may exist by forming a biofilm on the urethral catheter by contaminating the urinary catheter. infection may occur. In an embodiment of the present invention, as a result of performing fluorescence imaging through the compound of the present invention on a biofilm formed by specific bacteria (AB/CRAB, SA/MRSA) on the surface of the urethral catheter, which is a medical device, the catheter surface It was confirmed through confocal laser scanning microscopy (CLSM) that the biofilm formed by Bacter baumani was fluorescently stained with the compound of the present invention. On the other hand, it was confirmed that the biofilm formed by Staphylococcus aureus on the catheter surface was not stained with fluorescence (FIGS. 6-7). Accordingly, according to one embodiment of the present invention, the reagent composition of the present invention can be used to selectively detect a biofilm produced by Acinetobacter baumani, wherein the biofilm is specifically a medical device, more specifically may be formed in the urethral catheter. According to another embodiment of the present invention, the selective detection method of Acinetobacter baumani of the present invention comprises contacting the compound represented by Formula 1 as an analyte with a medical device, more specifically, a urinary catheter. can

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Bacterial fluorescence imaging screening

Fluorescence imaging screening was performed on Gram-negative bacteria and Gram-positive bacteria, including multidrug-resistant bacteria, using the compounds of Formulas 2, 3, or 4 below, and the results are shown in FIG. 1 .

[Formula 2]

[Formula 3]

[Formula 4]

The compound of Formula 2 is a compound according to the present invention.

The compound of Formula 3 represents a fluorescent dye coumarin 120 (7-amino-4-methylcoumarin, coumarin 120). Coumarin 120 has a molecular weight of 175.2 g/L, and exhibits absorption at 345 nm and fluorescence at 445 nm.

The compound of Formula 4 represents rhodamine 123 among the fluorescent dyes. Rhodamine 123 has a molecular weight of 287.33 g/L, absorbs light at a wavelength of 512 nm and fluoresces at 531 nm.

In the present invention, the fluorescence imaging characteristics of the compounds of Chemical Formulas 2, 3, and 4 were observed by selecting the gram-negative bacteria Acinetobacter baumani sensitive and resistant strains and the Gram-positive Staphylococcus aureus sensitive and resistant strains. Information on gram-negative and gram-positive bacteria, including multidrug-resistant bacteria, for bacterial-dye combination screening is shown in Table 1 below.

[Table 1]

Specifically, the strain for the bacterial-dye combination screening was inoculated in CA-MHB (Ca ²⁺ Muller Hilton broth) medium, and then cultured at 37° C. for 24 hours. After overnight incubation, centrifuge the bacterial culture at 13,000 rpm for 5 minutes using a 1.5 mL microtube, and discard the supernatant. The bacterial pellet remaining on the bottom of the microtube is washed three times using phosphate buffered saline (PBS, phosphate buffer saline, pH 7.4). After washing, the remaining bacterial pellet is resuspended in phosphate-buffered saline and adjusted to about 1 x 10 ⁸ CFU/mL. The dyes for screening, BMeS-pA, Coumarin 120 and Rhodamine 123, were added to the bacterial suspension at 100 μM, 100 μM, and 15 μM, respectively, and then incubated at 37° C. for 1 hour. After 1 hour of incubation, centrifugation at 13,000 rpm for 5 minutes, discarding the supernatant, and washing thoroughly with phosphate buffered saline (pH 7.4) 3 times to remove the remaining dye. The fluorescence image of the stained bacteria was observed through CLSM, and for this purpose, the washed bacterial pellet was resuspended in phosphate buffered saline and spread on a slide glass. The fluorescence imaging of each dye for bacteria was 405 nm excitation/519 nm emission for BMeS-pA (compound of Formula 2), 405 nm excitation/460 nm emission for Coumarin 120 (compound of Formula 3), and rhodamine 123 (compound of Formula 4). compound) was performed under the conditions of 488 nm excitation/560 nm emission.

As a result of screening bacteria-dye combinations for Acinetobacter baumani sensitive and resistant strains and Staphylococcus aureus susceptible and resistant strains, in the case of Coumarin 120 (compound of Formula 3), fluorescence imaging was not performed in Gram-negative and Gram-positive bacteria, and Rhod In the case of Min (compound of Formula 4), it was confirmed that staining was well performed in both Gram-negative and Gram-positive bacteria without selectivity. On the other hand, in the case of BMeS-p-A (compound of Formula 2), it was confirmed that the sensitive and resistant strains of Acinetobacter baumani of Gram-negative bacteria were selectively fluorescently stained. In addition, as a result of analyzing the fluorescence intensity, it was confirmed that the fluorescence was increased only in Acinetobacter baumani sensitive and resistant strains that were similarly BMeS-p-A selectively ( FIGS. 1a and b ).

Based on this, in order to more clearly characterize the bacterial selective fluorescence imaging of BMeS-p-A, 6 gram-negative bacteria and 2 gram-positive bacteria were additionally subjected to bacterial-dye combination screening in the same manner as above.

As a result, it was confirmed that BMeS-p-A was not capable of fluorescence imaging except for Acinetobacter baumani sensitive and resistant strains of Gram-negative bacteria ( FIG. 1c ).

Example 2. Observation of changes in zeta-potential of bacterial cell membranes by BMeS-p-A

According to the bacterial-dye combination screening result, we decided to select BMeS-p-A with a specific bacterial fluorescence staining ability, and to investigate the interaction between BMeS-p-A and the bacterial cell membrane, the zeta potential of the bacterial cell membrane was measured, and the The results are shown in FIG. 2 .

BMeS-p-A is a positively charged material having two amine groups, and is expected to have high binding affinity to the cell membrane of bacteria having a relatively negative charge. Accordingly, the zeta potential of the bacteria was measured to observe the change in the potential difference of the bacterial cell membrane according to the BMeS-p-A treatment. In this experiment, Acinetobacter baumani sensitive and resistant strains selectively fluorescing to BMeS-p-A were used, and a Gram-positive Staphylococcus aureus sensitive and resistant strain that did not show any fluorescence was used as a control.

More specifically, as in Example 1, the four strains were inoculated in CA-MHB medium, and then cultured at 37° C. for 24 hours. After overnight incubation, the bacterial culture medium is centrifuged at 13,000 rpm for 5 minutes, and the supernatant is discarded. Bacterial residues from which the supernatant has been removed are washed 3 times with phosphate buffered saline (pH 7.4), and then resuspended in phosphate buffered saline to adjust to about 1 x 10 ⁸ CFU/mL. After the bacterial suspension was treated with 100 μM of BMeS-pA, it was incubated at 37° C. for 1 hour. After 1 hour of incubation, the cultured solutions of the four stained bacteria are again centrifuged to remove the supernatant, and then washed three times with phosphate-buffered saline to remove the remaining BMeS-pA. After the washed bacteria were resuspended in sterile water, the charge on the bacterial surface was evaluated by measuring the zeta potential using a Zetasizer Nano ZS90 (Malvern Instruments).

As a result, Acinetobacter baumani sensitive and resistant strains not treated with BMeS-p-A were measured to be -45.2 mV and -46.9 mV, respectively, and Staphylococcus aureus susceptible and resistant strains showed -39.2 mV and -35.5 mV, respectively. was (Fig. 2). After treatment with BMeS-p-A, as a result of measuring the zeta potential of the bacteria, the Acinetobacter baumani sensitive and resistant strain showed an increased potential compared to the group not treated with BMeS-p-A. In contrast, S. aureus susceptible and resistant strains did not observe a significant change in potential when compared to the group not treated with BMeS-p-A.

These results show that Gram-negative bacteria have a slightly higher negative charge than Gram-positive bacteria because they contain lipopolysaccharide (LPS) in their outer membrane. Due to the characteristics of these Gram-negative bacteria, it is thought that they were more highly bound to BMeS-p-A. Therefore, it is considered that BMeS-p-A can be used as a suitable fluorescent dye for selective fluorescence imaging. In addition, the positive charge of the used coumarin 120 containing one amine is not sufficient to label the bacterial surface, and the rhodamine 123 containing two cationic amines does not appear to be suitable in terms of selectivity.

Example 3. Confirmation of absorption / fluorescence characteristics of BMeS-p-A

Absorption and fluorescence graphs were obtained for BMeS-p-A capable of selectively fluorescence imaging of Acinetobacter baumani sensitive and resistant strains in Example 1, and the results are shown in FIG. 3 .

BMeS-p-A was obtained by using a spectrophotometer (UV/Vis spectrophotometer; Agilent Technologies Cary 8454, US) and a fluorometer (SHIMADZU CORP. RF-6000, Japan) to obtain absorption and emission spectra (UV/ Vis absorption and emission spectra) were obtained, and a standard quartz cuvette (Hellma Analytics, Jena, Germany) was used for absorption and fluorescence measurements. The indicated graph shows the absorption and emission fluorescence graphs of BMeS-p-A in a solvent of phosphate buffered saline at pH 7.4. Under these conditions, BMeS-p-A showed the maximum absorbance at 375 nm and the maximum fluorescence in green at 515 nm ( 3).

Example 4. Confirmation of stability under various conditions of BMeS-p-A

In consideration of the further practical application potential of BMeS-p-A, which showed specific bacterial fluorescence imaging results in Example 1, the stability of BMeS-p-A was confirmed under various conditions, and the results are shown in FIG. 4A.

pH 7.4 buffer (culture medium condition), temperature of 50°C (thermal stability), bovine serum albumin (BSA; 35 mg/mL, this concentration is based on human blood serum concentration) and light stability The stability of BMeS-p-A was evaluated under the conditions of UV light (365 nm, 3W) for Specifically, after adding BMeS-p-A (10 μM) to each condition and reacting at room temperature for 60 minutes, the change in fluorescence spectrum was confirmed. The graph indicated in FIG. 4a is shown by comparing the value of 515 nm showing the maximum fluorescence in the fluorescence spectrum obtained at the initial stage and 1 hour after each condition.

As a result, no change in fluorescence was observed due to pH7.4 buffer, light irradiation, and bovine serum albumin, and a slight decrease in fluorescence intensity was observed by light irradiation, but this is not significant and is considered a negligible change in fluorescence.

In addition, in order to evaluate the stability of BMeS-pA for metal ions, FeCl ₂ , FeCl ₃ , CuCl ₂ , CaCl ₂ , ZnCl ₂ , KCl, NaCl ₂ , MgCl ₂ in vivo, the fluorescence intensity change after reaction with metal ions was observed, and the results are shown in FIG. 4B.

As a result, no change in fluorescence of BMeS-p-A by biometal was observed.

Based on these results, it was confirmed again that stable fluorescence characteristics can be exhibited without external interference when conducting fluorescence imaging studies of bacteria.

Example 5. Minimum inhibitory concentration test to confirm the antibacterial effect of BMeS-p-A

The antibacterial effect of BMeS-p-A against 12 types of bacteria, including multidrug-resistant bacteria, was confirmed, and it is shown in FIG. 5 .

To measure the antimicrobial activity against 12 types of bacteria, including multidrug-resistant bacteria, the Minimum Inhibitory Concentration (MIC) was investigated according to the CLSI (clinical and laboratory standard institute, 2016) guidelines. A broth micro-dilution method using a 96-well plate was used, and the same CA-MHB as used in Example 1 was used as the medium. Six standard strains used in this experiment were sold from the strain banks ATCC (american type culture colletion) and CCARM (culture collection of antibiotics resistant microbes), and 6 resistant strains were sold from Asan Medical Center (AMC, Asan Medical Center). received, and information on a total of 12 strains is shown in Table 1.

Specifically, to prepare a bacterial culture solution, 12 strains including multidrug-resistant bacteria were cultured on a blood agar plate at 37° C. for 24 hours. The next day, 12 test strains were suspended using sterile water to McFarland standard turbidity of 0.5 to prepare a culture solution. BMeS-pA was diluted 2-fold in a concentration range of 100 μM to 0.39 μM using CA-MHB and dispensed into 96 well round bottom microplates. The prepared bacterial culture is diluted 1/10 and inoculated at 10 μL in the medium containing BMeS-pA for each concentration (final bacterial concentration 5 x 10 ⁵ CFU/mL). Thereafter, incubated for 20 hours in an incubator at 37° C., the MIC was read by observation with the naked eye, and the results are shown in FIG. 5 .

As a result, it was confirmed that BMeS-p-A did not inhibit the growth of 12 strains including multidrug-resistant bacteria at any concentration. These results mean that BMeS-p-A has no antibacterial activity against the 12 tested species. However, these results also suggest that BMeS-p-A is not toxic to the 12 tested species.

Example 6. Bacterial Biofilm Fluorescent Staining by BMeS-p-A

It was confirmed that BMeS-p-A was capable of fluorescence imaging of the biofilm produced by the strain, and this was done through CLSM observation, and the results are shown in FIG. 6 .

It was attempted to confirm whether the biofilm produced by the Acinetobacter baumani sensitive and resistant strain that was fluorescently stained specifically for BMeS-p-A was fluorescence imaging, and as a control, Staphylococcus aureus sensitive and resistant bacteria as in Example 2 above. The biofilms of the cells were simultaneously fluorescence-stained and analyzed for comparison.

Specifically, for each strain, 2 mL of TSB medium containing 1% glucose is put on a confocal dish from the strain culture medium cultured overnight in TSB medium, and the strain is inoculated with 0.1%. For effective biofilm formation of each strain, it was cultured at 37°C for 48 hours. After that, all of the supernatant is removed, washed three times with phosphate buffered saline, and incubated with 100 μM BMeS-p-A at 37° C. for 1 hour under the same conditions as in Example 1. After the reaction, it was thoroughly washed with phosphate-buffered saline to remove BMeS-p-A and heat-fixed, followed by CLSM observation.

As a result, it was confirmed that BMeS-p-A selectively fluorescently stained the biofilm formed by Acinetobacter baumani sensitive and resistant strains as indicated in FIG. 6 . On the other hand, fluorescence was not observed in the biofilm produced by Staphylococcus aureus susceptible and resistant strains.

Example 7. Application Study of Bacterial Biofilm Fluorescence Imaging by BMeS-p-A

Based on the selective fluorescence staining of the biofilm produced by BMeS-p-A Acinetobacter baumani, it was confirmed whether fluorescence imaging of the biofilm produced by artificially contaminating the urinary catheter, where the formation of the bacterial biofilm is a problem and the results are shown in FIG. 7 .

Acinetobacter baumani sensitive and resistant strains capable of selectively fluorescing BMeS-p-A were used, and Staphylococcus aureus susceptible and resistant strains were used as controls.

Specifically, a sterile ureteral catheter (double pigtail urethral stent) is cut into 2.5 cm and placed in a fluorescence measurement vessel containing 1.0% glucose-containing TSB medium. Bacteria cultured overnight were inoculated with 0.1% (about 1 x 10 ⁷ CFU/mL) and cultured at 37° C. for 48 hours. After incubation, the resulting biofilm was stained in two ways. First, crystal violet was used to check the presence or absence of biofilm formation on the surface of the urinary catheter. Before staining the biofilm on the surface of the urinary catheter, the urinary catheter coated with the biofilm formed by bacteria was carefully washed with phosphate-buffered saline, and then stained in 1.0% crystal violet solution for 10 minutes. After gently washing the stained catheter, the dye was eluted by shaking with 33% glacial acetic acid for 20 minutes. The absorbance of the eluted crystal violet was measured at 600 nm using a microplate reader. Second, BMeS-pA was used for selective biofilm fluorescence imaging. The urinary catheter was stained with BMeS-pA for fluorescence imaging, and was stained in the same manner as in Example 6. Urethral catheters stained with BMeS-pA were observed using confocal fluorescence microscopy (CLSM).

First, as a result of observing the presence or absence of biofilm formation on the surface of the urethral catheter by crystal violet, it was confirmed that all urethral catheter surfaces were stained with crystal violet regardless of the strain type. Through this, it was found that the biofilm was effectively formed by each strain and coated the surface of the urethral catheter (Fig. 7a). On the other hand, as a result of observing the fluorescence image of the urinary catheter by BMeS-p-A, it was found that the urethral catheter coated with a biofilm formed by Acinetobacter baumani sensitive and resistant bacteria can selectively fluorescence imaging by BMeS-p-A. was confirmed (Fig. 7b). On the other hand, fluorescence images could not be observed in the urinary catheter coated with the biofilm formed by the control Staphylococcus aureus.

The description of the present invention stated above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (12)

A reagent composition for selectively detecting Acinetobacter baumannii , containing a compound represented by the following formula (1):
[Formula 1]

In Formula 1,
R ¹ and R ² are each independently a linear or branched chain alkyl having 1 to 5 carbon atoms. According to claim 1,
The reagent composition, characterized in that the compound is represented by the following formula (2).
[Formula 2]

The reagent composition according to claim 1, wherein the Acinetobacter baumannii is multi-drug resistant Acinetobacter baumannii . 4. The method of claim 3,
The reagent composition, characterized in that the Acinetobacter baumannii is carbapenem-resistant Acinetobacter baumannii (carbapenem-resistant Acinetobacter baumannii ). According to claim 1,
Reagent composition, characterized in that for selectively detecting the biofilm (biofilm) produced by the Acinetobacter baumani. 6. The method of claim 5,
The reagent composition, characterized in that the biofilm is formed on a medical device. 7. The method of claim 6,
The reagent composition, characterized in that the medical device is a urinary catheter. A kit for diagnosing infection of Acinetobacter baumannii , comprising a compound represented by the following formula (1):
[Formula 1]

In Formula 1,
R ¹ and R ² are each independently a linear or branched chain alkyl having 1 to 5 carbon atoms. A method for selective detection of Acinetobacter baumannii , comprising the step of contacting a compound represented by the following formula (1) with an analyte:
[Formula 1]

In Formula 1,
R ¹ and R ² are each independently linear or branched alkyl having 1 to 5 carbon atoms. 10. The method of claim 9,
Method characterized in that it further comprises the step of observing the fluorescence of the analyte. 10. The method of claim 9,
The method of claim 1, wherein the analyte is a medical device. 12. The method of claim 11,
The method of claim 1, wherein the medical device is a urethral catheter.

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