KR102595909B1 - Blood culture reagent consisting of a carbon dioxide colorimetric sensor and anaerobic microorganism growth medium - Google Patents

Abstract

The present invention relates to an anaerobic blood culture reagent based on a carbon dioxide colorimetric sensor for diagnosing sepsis. The present invention relates to an anaerobic blood culture reagent that uses a complex of a cation exchange resin and an adsorption resin that can effectively neutralize various antibiotics, and includes a carbon dioxide detection colorimetric sensor. By providing this, it neutralizes various antibiotics that may exist in the blood of sepsis patients who have been pre-treated with antibiotics, thereby facilitating the growth of anaerobic microorganisms that cause sepsis, and can also be useful in diagnosing the causative bacteria of sepsis patients within a short period of time.

Description

Blood culture reagent consisting of a carbon dioxide colorimetric sensor and anaerobic microorganism growth medium}

The present invention relates to a blood culture reagent necessary for the diagnosis of sepsis, including a carbon dioxide colorimetric sensor and an anaerobic microbial growth medium. More specifically, it neutralizes various antibiotics that may be present in the blood when culturing the blood of a sepsis patient, thereby neutralizing the antibiotics that cause sepsis. This relates to an anaerobic blood culture reagent that not only facilitates the growth of anaerobic microorganisms, but can also diagnose the presence or absence of microorganisms using a colorimetric sensor that changes color due to an increase in carbon dioxide secreted by the microorganisms.

Sepsis, which is caused by blood infection caused by microorganisms that invade the human body, is a disease with a mortality rate of 40 to 70%, with 6 million deaths occurring every year worldwide. In Korea, the number of sepsis deaths has been continuously increasing over the past 10 years. This is the reality (Kim Seong-nam et al., 2000, Weekly Health and Disease, Vol. 13, No. 37: 2750-2760). Blood culture is one of the essential requirements for identifying the exact causative bacteria of sepsis in order to use strong, broad-spectrum antibiotics in the early stage of the disease, which is one of the principles of sepsis treatment (Woon-seong Kim, Hyun-jung Lee. 2013, J. Korean Med. Assoc. 56:819-826 ).

Blood culture is the only standard method for rapid and accurate diagnosis of bloodstream infections, and is very important for detecting and identifying causative bacteria and determining antibiotic sensitivity in patients suspected of having sepsis (Aronson, M. D. and Bor, D. H. 1987. Blood cultures. Ann. Intern. Med. 106:246-253). In diagnosing sepsis-causing bacteria, it is more important to determine the presence or absence of microbial growth. Methods for this include dry weight measurement, volume measurement, turbidity measurement, and physiological index method. Due to their universality and easy operation, the dry weight measurement method, volume measurement method, and turbidity measurement method are most widely used to confirm the growth of microorganisms. However, due to the disadvantage of requiring a long working time due to a large number of repetitions, the growth of microorganisms can be performed quickly and accurately. The physiological index method has recently been widely used as a diagnostic method (Shao, J. et al. 2018. Sens. Actuators B Chem. 273:656-663).

The physiological index method utilizes the property that the growth (rate) of microorganisms is accompanied by changes in a series of physiological indices, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), adenosine triphosphate (ATP), and natural actomyosin (NAM). ), carbon dioxide (CO ₂ ) production, oxygen (O ₂ ) consumption, viscosity, transparency and heat production are used as biological indicators (Kocincova, AS et al. 2008. Biotechnol. Bioeng. 100:430-438, Lazcka , O. and Del Campo, F.X. 2007. Biosens. Bioelectrol. 22:1205-1217). In particular, carbon dioxide is one of the main metabolites secreted by microorganisms as they grow, so the carbon dioxide content is an indicator that can reflect the growth state of microorganisms. This characteristic has been used in blood culture since 1990, using colorimetric detection of pH changes based on the reading of carbon dioxide generation due to microbial growth, by installing a carbon dioxide-sensitive receptor at the bottom of the blood culture bottle. has largely led to the development of automatic blood culture systems that can be installed colorimetrically or fluorescently to continuously monitor the growth of microorganisms (Gimenez, M. et al. 2002. Clin. Microbiol. Infect. 8:222-228, Kato, H. et al. 2017. Int. J. Antimicrob. Agents. 50:S256-S257, Horvath LL et al. 2004. J. Clin. Microbiol. 42:115-118, Brecher, ME et al. 2002. Transfusion. 42:774-779, Thorpe, TC et al. 1990. J. Clin. Microbiol. 28:1608-1612).

However, in effective antibacterial treatment, which is an essential factor in the survival of sepsis patients, the resins (cation exchange resin and adsorption resin) and carbon dioxide detection sensors contained in blood culture bottles for rapid isolation and identification of bloodstream infectious bacteria have not been clearly identified to date. Currently, it is the exclusive property of a few specific companies, occupying 90% of the global blood culture bottle market. Accordingly, based on a cation exchange resin and adsorption resin that can neutralize various antibiotics, including ampicillin, a beta-lactam antibiotic, which was the first report of clinical efficacy against Gram-negative bacteria, anaerobic and anaerobic microorganisms are being treated. There is a need to manufacture a new blood culture reagent containing a carbon dioxide colorimetric sensor that can clearly detect growth in a short period of time.

In this regard, Republic of Korea Patent No. 10-2413155 (registration date: 2022.06.21) relates to a microbial growth medium based on a carbon dioxide colorimetric sensor for sepsis diagnosis, which includes a cation exchange resin and an adsorption resin that can effectively neutralize various antibiotics. A microbial growth medium using a composite and including a colorimetric sensor for detecting carbon dioxide is disclosed.

In addition, Republic of Korea Patent No. 10-2405920 (registration date: 2022.05.31.) relates to a microbial growth medium with antibiotic neutralizing ability, which neutralizes various antibiotics that may be present in the blood when culturing the blood of a sepsis patient, thereby preventing the cause of sepsis. A microbial growth medium that can facilitate the growth of these microorganisms is disclosed.

Additionally, Korean Patent Publication No. 10-1999-7007313 (publication date: November 25, 2000) relates to a sensor device and method for detecting microorganisms, including containers; A fixing layer for immobilizing the sample to be tested; Disclosed is a sensor device comprising a sensor layer capable of detecting a detectable amount of change due to the presence of immobilized microorganisms on and/or within a fixed bed.

As such, research has been conducted on microbial growth media with conventional antibiotic neutralizing ability, but research on blood culture reagents containing a carbon dioxide colorimetric sensor and anaerobic microbial growth media useful for sepsis diagnosis according to the present invention is still insufficient.

Accordingly, the present inventors manufactured blood culture reagents by selecting the mixing ratio of a composite resin mixed with a cation exchange resin and an adsorption resin, the raw materials of the carbon dioxide colorimetric sensor, and the type and ratio of the indicator mixed, and the most frequently isolated from sepsis patients. By measuring the antibiotic susceptibility and antibiotic neutralizing ability of each facultative anaerobic ATCC strain, when using the blood culture reagent of the present invention, the anaerobic microbial growth rate is similar to that of the conventional reagent, while maintaining excellent antibiotic susceptibility and antibiotic neutralizing ability. By confirming, the present invention was completed.

Republic of Korea Patent No. 10-2413155 Republic of Korea Patent No. 10-2405920 Republic of Korea Patent Publication No. 10-1999-7007313

The present invention effectively neutralizes various antibiotics that may be present in the blood of sepsis patients who have been pre-treated with antibiotics (using cation exchange resin and adsorption resin), thereby not only facilitating the growth of the corresponding microorganisms, but also increasing the amount of carbon dioxide secreted by microorganisms. We aim to select raw materials and indicators for colorimetric sensors that change color accordingly, verify the function of the colorimetric sensors, and provide anaerobic blood culture reagents containing them.

In one embodiment of the present invention to achieve this problem, a cation exchange resin (sodium vinylbenzensulfonate divinyl benzene polymer, CAS No. 63182-08-1) and an adsorption resin (divinylbenzene-ethylvinylbenzene-styrene copolymer, CAS No. 9052- Provided is an anaerobic blood culture reagent endowed with antibiotic neutralizing ability, including a medium containing the complex of 95-3).

In another embodiment of the present invention, an anaerobic blood culture reagent with antibiotic neutralizing ability is provided, wherein the reagent further includes a colorimetric sensor.

In another embodiment of the present invention, a cation exchange resin (sodium vinylbenzensulfonate divinyl benzene polymer, CAS No. 63182-08-1) and an adsorption resin (divinylbenzene-ethylvinylbenzene-styrene copolymer, CAS No. 9052-95-3) Provided is a reagent for sepsis diagnosis endowed with antibiotic neutralizing ability, including an anaerobic medium containing a complex and a colorimetric sensor.

In another embodiment of the present invention, a sepsis diagnostic kit including the anaerobic blood culture reagent is provided.

In another embodiment of the present invention, 1) mixing blood collected from the patient with the anaerobic blood culture reagent to obtain a mixed solution; 2) cultivating the mixed solution of step 1); and 3) a method of providing information for diagnosing sepsis, including the step of checking the color change after culturing, and determining that it is sepsis when the color of the colorimetric sensor changes from blue to yellow, green, or yellow. to provide.

The present invention uses an anaerobic microbial growth medium containing a complex of a cation exchange resin and an adsorption resin that can effectively neutralize various antibiotics, and provides an anaerobic blood culture reagent containing a colorimetric sensor for detecting carbon dioxide, thereby preventing antibiotic-pretreated sepsis. It has the effect of neutralizing various antibiotics that may be present in the blood of patients, facilitating the growth of anaerobic microorganisms that cause sepsis, and clearly diagnosing the presence or absence of causative bacteria in sepsis patients in a short period of time.

Figure 1 is a graph showing the results of culturing 7 types of ATCC strains in 5 types of anaerobic nutrient media calculated in CFU and converted to Log quantification.
Figure 2 is a graph showing the minimum growth concentration for 5 types of antibiotics according to 7 types of anaerobic ATCC strains.
Figure 3 is a graph showing the results of measuring the neutralizing ability of each individual antibiotic against E. faecalis ATCC 29212 strain in anaerobic blood culture reagent containing composite resin.
Figure 4 is a graph showing the results of measuring the neutralizing ability of each antibiotic against E. faecium ATCC 700221 strain in anaerobic blood culture reagent containing composite resin.
Figure 5 is a graph showing the results of measuring the neutralizing ability of each individual antibiotic against E. coli ATCC 25922 strain in anaerobic blood culture reagent containing composite resin.
Figure 6 is a graph showing the results of measuring the neutralizing ability of each individual antibiotic against K. pneumoniae ATCC 700603 strain in anaerobic blood culture reagent containing composite resin.
Figure 7 is a graph showing the results of measuring the neutralizing ability of each individual antibiotic against S. aureus ATCC 25923 strain in anaerobic blood culture reagent containing composite resin.
Figure 8 is a graph showing the results of measuring the neutralizing ability of each individual antibiotic against S. epidermidis ATCC 12228 strain in anaerobic blood culture reagent containing composite resin.
Figure 9 is a graph showing the results of measuring the neutralizing ability of each individual antibiotic against S. anginosus ATCC 12395 strain in anaerobic blood culture reagent containing composite resin.
Figure 10 shows the raw materials of the carbon dioxide colorimetric sensor (two types of room temperature curing type) mixed with xylenol blue, bromothymol blue, phenolphthalein, and thymol blue at a certain ratio. This is a graph showing the results of measuring the colorimetric change and reflectance value after manufacturing the sensor by adding the indicator prepared by inoculating and culturing the S. aureus ATCC 25923 strain.
Figure 11 shows indicator HXB produced by mixing xylenol blue, bromothymol blue, phenolphthalein, and thymol blue in room temperature curing DY-H11 silicone at a certain ratio. This is a graph showing the results of measuring colorimetric change and reflectance values after fabricating a sensor by adding 0.05% of 1 to 20, inoculating and culturing the S. aureus ATCC 25923 strain.
Figure 12 shows the anaerobic blood culture reagent produced by adding 0.05% of the indicator HXB 20 to room temperature curing type DY-H11 silicone to produce a sensor and adding composite resin, 10% of carbon dioxide, and 70% of nitrogen, respectively, and the off-the-shelf FN from BIOMEIEUX. This is a graph showing the results of measuring the colorimetric change and reflectance values after culturing the PLUS product with the same treatment of S. aureus ATCC 25923 strain, sheep blood, and each antibiotic.

Sepsis is caused by an infection in the blood caused by microorganisms that invade the human body. As the mortality rate continues to increase, interest in treating it is high. In diagnosing sepsis-causing bacteria, it is more important to determine the presence or absence of microbial growth. Methods for this include dry weight measurement, volume measurement, turbidity measurement, and physiological index method. Dry weight measurement, volume measurement, and turbidity measurement methods have been widely used, but due to the disadvantage of requiring a long working time due to a large number of repetitions, the physiological index method has recently been used as a method for quickly and accurately diagnosing microbial growth. It is widely used. Among the physiological index methods, carbon dioxide is one of the main metabolites secreted by microorganisms as they grow, so the carbon dioxide content becomes an indicator that can reflect the growth state of microorganisms.

Therefore, the present invention not only facilitates the growth of the microorganisms by effectively neutralizing various antibiotics that may be present in the blood of sepsis patients who have been pre-treated with antibiotics (using cation exchange resin and adsorption resin), but also increases the amount of carbon dioxide secreted by the microorganisms. The purpose of this study is to verify the function of a colorimetric sensor that changes color and provide an anaerobic blood culture reagent containing it.

In the present invention, silicon was selected among compounds that have the property of being well permeable to carbon dioxide gas in order to use it as a raw material for a carbon dioxide colorimetric sensor, and it was determined which form of silicon can easily determine the pH change due to an increase in carbon dioxide. An experiment was conducted for this purpose. In the present invention, it was confirmed that a clear color change of the pH indicator occurred when room temperature curing silicone was used, and thus it was selected as a raw material for a carbon dioxide colorimetric sensor.

Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by an expert in the art. In addition, the experimental procedures specified in this specification are the same as those commonly performed in the technical field, unless specifically described.

Hereinafter, the present invention will be described in detail.

The present invention includes a medium containing a complex of a cation exchange resin (sodium vinylbenzensulfonate divinyl benzene polymer, CAS No. 63182-08-1) and an adsorption resin (divinylbenzene-ethylvinylbenzene-styrene copolymer, CAS No. 9052-95-3). , provides anaerobic blood culture reagents with antibiotic neutralizing ability.

The reagent is preferably used by mixing a cation exchange resin and an adsorption resin in a medium containing antibiotics. At this time, the cation exchange resin and the adsorption resin may be mixed at 2.5 to 3.5% (w/v) and 13 to 15% (w/v), respectively, based on the medium. Specifically, in mixing the cation exchange resin and the adsorption resin, the cation exchange resin is 1 to 5% (w/v), preferably 1.5 to 4.5% (w/v), 2.0 to 4.0% (w/v), based on the medium. w/v), more preferably 2.5-3.5% (w/v), 2.6-3.4% (w/v), 2.7-3.4% (w/v), 2.8-3.3% (w/v), 2.9 It may be ~3.2% (w/v), 3.0-3.1% (w/v), and the adsorption resin is 5-25% (w/v), preferably 10-20% (w/v) based on the medium. ), 11 to 19% (w/v), 12 to 18% (w/v), more preferably 13 to 17% (w/v), 14 to 16% (w/v), 14.1 to 15.9%. (w/v), 14.2~15.8%(w/v), 14.3~15.7%(w/v), 14.4~15.6%(w/v), 14.5~15.5%(w/v), 14.6~15.4% (w/v), 14.7-15.3% (w/v), 14.8-15.2% (w/v), 14.9-15.1% (w/v). The composite resin treated at this ratio can demonstrate excellent antibiotic neutralization ability.

Additionally, the reagent may further include a colorimetric sensor, and the colorimetric sensor may be a carbon dioxide detection colorimetric sensor.

The colorimetric sensor of the present invention includes silicone and a color indicator. The silicone may preferably be silicone that hardens at room temperature, and may be condensation type or addition type silicone, preferably addition type silicone.

On the other hand, addition-type silicone is one of the types of silicone and has good durability. It has excellent dimensional stability with a shrinkage rate of less than 0.2%, does not cause curing delay inside the silicone, and does not generate by-products such as alcohol or acetic acid during curing like condensation-type silicone. There is no odor when cured and there is no loss of mass during curing, so it has the advantage of almost no shrinkage. In general, most silicones that cure at room temperature are of the condensation type, but the present invention has a distinctive feature that is different from other technologies in that the addition type is used.

Meanwhile, in the present invention, the addition-type silicon may preferably have a tensile strength of 10 to 20 kgf/cm2 and an elongation of 150 to 300%, more preferably 14 to 18 kgf/cm2, 15 to 17 kgf/cm2, It may have a tensile strength of 15.5 to 16.5 kgf/cm2 and an elongation of 180 to 220%, 185 to 215%, 190 to 210%, 195 to 205%, and 197 to 203%. It is possible to manufacture a colorimetric sensor that shows a clear color change of the pH indicator by allowing carbon dioxide gas to pass through silicon under these conditions.

The silicone of the present invention includes 70 to 80% by weight of vinyl terminated polydimethylsiloxane, 15 to 25% by weight of silica gel, 1,3-diethenyl-1,1,3, Silicone (Agent A) containing 0.1% by weight or less of 3-tetramethyldisiloxane platinum complex (Platinum, 1,3-diethenyl-1,1,3,3-tetramethyldisiloxane complex) and vinyl terminated polydimethylsiloxane (Vinyl terminated polydimethylsiloxane) 70~80% by weight, Siloxanes, silicones, dimethyl, methyl hydrogen 15~25% by weight, Tetravinyltetramethylcycloterasiloxane 1~10 It may be a mixture of silicone (agent B) containing % by weight.

In addition, the silicone (agent A) may have a viscosity of 1 to 11 (Pa·s), and the silicone (agent B) may have a viscosity of 0.1 to 10 (Pa·s). Specifically, the silicone (agent A) may have a viscosity of 0.1 to 10 (Pa·s). ) has a viscosity of 1 to 20 (Pa·s), preferably 1 to 19 (Pa·s), 1 to 18 (Pa·s), 1 to 17 (Pa·s), 1 to 16 (Pa·s) ), 1 to 15 (Pa·s), 1 to 14 (Pa·s), 1 to 13 (Pa·s), 1 to 12 (Pa·s), 1 to 11 (Pa·s), more preferably is 2~11(Pa·s), 3~11(Pa·s), 4~11(Pa·s), 5~11(Pa·s), 6~11(Pa·s), 7~11( Pa·s), 8 to 11 (Pa·s), 9 to 11 (Pa·s), most preferably 10 (Pa·s), and the silicone (B agent) has a viscosity of 0.1 to 10 (Pa·s). Pa·s), preferably 0.1 to 9 (Pa·s), 0.1 to 8 (Pa·s), 0.1 to 7 (Pa·s), 0.1 to 6 (Pa·s), 0.1 to 5 (Pa·s) s), 0.1 to 4 (Pa·s), 0.1 to 3 (Pa·s), 0.1 to 2 (Pa·s), 0.1 to 1 (Pa·s), more preferably 0.1 to 0.9 (Pa·s) ), 0.2~0.9(Pa·s), 0.3~0.9(Pa·s), 0.4~0.9(Pa·s), 0.5~0.9(Pa·s), 0.6~0.9(Pa·s), 0.6~0.8 (Pa·s), most preferably 0.7 (Pa·s).

Additionally, the mixture may be a mixture of silicone (agent A) and silicone (agent B) at a weight ratio of 10:0.5 to 1.5, preferably 10:0.6 to 1.4, 10:0.7 to 1.3, or 10:0.8. It may be mixed at a weight ratio of ~1.2, 10:0.9~1.1, more preferably 10:1.

Meanwhile, in the reagent of the present invention, the color indicator is any one or more selected from bromothymol blue, phenolphthalein, thymol blue, and xylenol blue. It may contain, preferably, a mixture of bromothymol blue, phenolphthalein, thymol blue, and xylenol blue in a solution in which the indicator is dissolved. .

Meanwhile, the color indicator is preferably treated with a solution that can dissolve the indicator, preferably using a 0.2N NaOH solution containing 70 vol% glycerol and 60 vol% ethanol. At this time, the concentration of the mixing indicator may be 0.1 to 5.3 volume%, preferably 0.15 to 5.3 volume%, 0.2 to 5.3 volume%, 0.25 to 5.3 volume%, 0.3 to 5.3 volume%, more preferably 1 to 5.3 volume%. 5.3 vol%, 2-5.3 vol%, 3-5.3 vol%, 4-5.3 vol%, most preferably 4.2-5.3 vol%, but is not limited thereto. Through this, the presence or absence of microbial growth can be confirmed by clearly observing changes in the colorimetric sensor with the naked eye and in terms of reflectance values.

In addition, when using a mixture of the above four types of indicators, it is preferable to use 0.01 to 0.5 volume% of the color indicator, and most preferably 0.05 volume%. Through the mixing ratio and processing of these color indicators, the presence or absence of microbial growth can be confirmed by clearly observing changes in the colorimetric sensor both with the naked eye and in terms of reflectance values.

Meanwhile, in the reagent of the present invention, the anaerobic medium can be applied to any medium capable of growing sepsis-causing bacteria, such as pancreatic digest of casein and papaic digest of soybean. ), yeast extract, dextrose, sodium chloride, dibasic potassium phosphate, vitamin B ₁ (thiamine), vitamin B ₃ (nicotinic acid), vitamin Selected from B ₆ (pyridoxine), vitamin K ₁ , vitamin K ₃ (menadione), hemin, L-cysteine, sodium sulfite, and sodium thioglycolate A nutrient medium containing more than one of any one may be used. Preferably pancreatic digest of casein, papaic digest of soybean, yeast extract, dextrose, sodium chloride, Dibasic potassium phosphate, vitamin B ₁ (thiamine), vitamin B 3 (nicotinic acid), vitamin _{B 6 (} pyridoxine), vitamin K ₁ , vitamin K ₃ (menadione), hemin, L- It may be HN4 medium containing cysteine (L-cysteine), sodium sulfite, and sodium thioglycolate, but is not limited thereto. By using a medium manufactured with this configuration, anaerobic microorganisms can grow smoothly.

Meanwhile, in the reagent of the present invention, the medium is capable of neutralizing various antibiotics, and the antibiotics include, for example, beta-lactam series, glycopeptide series, and aminoglycoside. ) series, tetracycline series, cephalosporin series, it may be one or more selected from the group, ampicillin, vancomycin, gentamicin, tetracycline, cell It may be one or more selected from cefotaxime, but is not limited thereto.

Meanwhile, in the reagent of the present invention, the microorganism may be a sepsis-causing microorganism, and may be applied to any causative bacteria that causes sepsis, but is preferably an anaerobic microorganism, for example, Enterococcus faecalis , Enterococcus faecium, Escherichia coli , Klebsiella pneumoniae , Staphylococcus aureus , Staphylococcus epidermidis , Streptococcus anginosus, but is not limited thereto.

Meanwhile, in the reagent of the present invention, additional ingredients such as nutrients may be included for the growth of microorganisms, and are not limited to any medium as long as it is in accordance with media preparation techniques known in the art.

In addition, the present invention is an anaerobic polymer containing a complex of a cation exchange resin (sodium vinylbenzensulfonate divinyl benzene polymer, CAS No. 63182-08-1) and an adsorption resin (divinylbenzene-ethylvinylbenzene-styrene copolymer, CAS No. 9052-95-3). Provided is a reagent composition for diagnosing sepsis endowed with antibiotic neutralizing ability, including a medium and a colorimetric sensor.

In addition, the present invention includes the steps of 1) mixing blood collected from a patient to be diagnosed with the anaerobic blood culture reagent to obtain a mixed solution; 2) cultivating the mixed solution of step 1); and 3) a method of providing information for diagnosing sepsis, including the step of checking the color change after culturing, and determining that it is sepsis when the color of the colorimetric sensor changes from blue to yellow, green, or yellow. to provide.

The culture in step 2) may be cultured for 3 to 10 days, preferably for 4 to 9 days, 4 to 8 days, 4 to 7 days, or 4 to 6 days.

The color change in step 3) may be confirmed with the naked eye or by measuring a reflectance value, which may be measured with a photodiode with a wavelength of 610 nm, but is not limited thereto.

Additionally, the present invention provides a sepsis diagnostic kit including the anaerobic blood culture reagent.

Meanwhile, according to the following experiments in the present invention, the reagent of the present invention can promote the growth of microorganisms even when treated with antibiotics by mixing a cation exchange resin and an adsorption resin in a basic anaerobic medium (HN4), and can promote the growth of microorganisms using a carbon dioxide detection colorimetric sensor. It was confirmed that the carbon dioxide colorimetric sensor changes clearly with the naked eye to confirm the presence or absence of anaerobic microorganisms, and that the change in reflectance value shows high discrimination power. In particular, this is an excellent result that can be used as a replacement for existing products. It was confirmed that it represents.

Through this, the reagent of the present invention is manufactured by mixing a basic anaerobic medium (HN4) with a cation exchange resin and an adsorption resin, and includes a colorimetric sensor for detecting carbon dioxide to induce the growth of microorganisms by neutralizing several antibiotics in the blood culture of sepsis patients. Since it can clearly determine the presence or absence of sepsis-causing bacteria, it is expected to be applicable to various pharmaceutical industries or treatments.

Hereinafter, the contents of the present invention will be described in more detail through the following examples and examples. However, the scope of the present invention is not limited to the following examples, but also includes modifications of the technical idea equivalent thereto.

<Example 1> Selection of basic medium and determination of antibiotic susceptibility of test strains

In this example, basic media were selected for seven types of bacteria frequently isolated from sepsis patients, and their antibiotic susceptibilities were determined.

1-1. Selection of anaerobic basic medium

The strains used in this experiment were seven facultative anaerobic ATCC strains most frequently isolated from sepsis patients; Enterococcus faecalis (hereinafter ' E. faecalis ') ATCC 29212, Enterococcus faecium (hereinafter ' E. faecalis' ) ' E. faecium ') ATCC 700221, Escherichia coli, hereinafter ' E. coli ') ATCC 25922, Klebsiella pneumoniae, hereinafter ' K. pneumoniae ') ATCC 700603, Staphylococcus aureus (hereinafter referred to as Staphylococcus aureus) ' S. aureus ') ATCC 25923, Staphylococcus epidermidis (hereinafter referred to as Staphylococcus epidermidis) ' S. epidermidis ') ATCC 12228, Streptococcus anginosus (hereinafter ' S. anginosus ') ATCC12395.

To confirm the smooth growth of the seven ATCC strains presented above, single colonies of each strain cultured on a TSA (BD, 236950) plate at 37°C for 24 hours were cultured in 2 ml of a total of four media (Table 1). After inoculation, the cells were cultured in a shaking incubator (200 rpm, Daehan Science, IS-20R) at 37°C for 24 hours, then spread on a plate medium containing the same ingredients, and the number of grown bacteria was measured to confirm CFU. After uniformly correcting the confirmed CFU to 10 to 20 CFU, 10 to 20 CFU was inoculated into 5 ml of four different media and incubated for 12 hours, and the media were incubated under anaerobic conditions [(anaerobic jar, Kisan Bio, KS-SC3381) and (anaerobag, The growth potential was confirmed by counting CFU in [Kisan Bio, KS-B2002)]. The nitrogen source specified in Table 1 below consists of pancreatic digest of casein and yeast extract, the carbon source is dextrose, and the inorganic salts are sodium chloride and Dibasic. Potassium phosphate (dibasic potassium phosphate), growth factors are vitamin B ₁ (thiamine), vitamin B ₃ (nicotinic acid), vitamin B ₆ (pyridoxine), vitamin K ₁ , vitamin K ₃ (menadione), hemin, reducing agent It is composed of L-cysteine, sodium sulfite, and sodium thioglycolate.

Nutrient composition by type of medium used for selection of anaerobic basic medium nitrogen source carbon source Inorganic salts growth factor reducing agent Thioglycolate broth 2.0% 0.55% 0.5% - 0.1% HN1 2.0% 0.55% 0.5% 0.00123% 0.1% HN2 2.0% 0.55% 0.5% 0.00185% 0.1% HN3 2.0% 0.65% 0.5% 0.00155% 0.1% HN4 2.3% 0.65% 0.5% 0.00375% 0.11%

As a result, as shown in Figure 1, HN4 medium, which commonly showed the best growth ability for the seven ATCC strains, was selected as the anaerobic basic medium for this experiment.

1-2. Determination of antibiotic susceptibility of test strains

The seven ATCC strains used above were cultured in 2 ml of HN4 medium, an anaerobic medium, for 24 hours to determine CFU, and the final 10 ⁸ CFU were divided into 0.5, 1, 2.5, 5, 10, 20, 30, and After inoculating 5ml of HN4 medium containing concentrations of 40, 50, 75, and 100ug/ml and shaking culture for 3 days at 37°C, the minimum inhibitory concentration (MIC) of each antibiotic at which the inoculated strain did not grow was confirmed. .

Types of antibiotics used in experiments on antibiotic susceptibility and neutralizing ability of each strain antibiotic class antibiotic type β-lactam ampicillin (WAKO, 014-23302) glycopeptide vancomycin (Kisanbio, MB-V4882) aminoglycosides gentamicin (Kisanbio, MB-G4582) tetracycline tetracycline (Daejung, 8716-4150) cephalosporin cefotaxime (Kisanbio, MB-C4392)

As a result, as shown in Figure 2, the minimum inhibitory concentration of each antibiotic that prevents bacterial growth is 30ug/ml for ampicillin, 2.5ug/ml for vancomycin, and 75ug for gentamicin in the E. faecalis ATCC 29212 strain. /ul, tetracycline concentration of 25ug/ml, E. faecium ATCC 700221 strain, tetracycline concentration of 1ug/ml, E. coli ATCC 25922 strain, ampicillin 50ug/ml, gentamicin ( The concentration is 30ug/ml, tetracycline 2.5ug/ml, and cefotaxime 0.5ug/ml. For K. pneumoniae ATCC 700603 strain, gentamicin is 75ug/ml and tetracycline is 50ug. /ml concentration, in S. aureus ATCC 25923 strain, ampicillin 2.5ug/ml, vancomycin 2.5ug/ml, gentamicin 50ug/ml, tetracycline 2.5ug/ml, cells The concentration of cefotaxime is 2.5ug/ml, S. For the epidermidis ATCC 12228 strain, the concentration of vancomycin is 2.5ug/ml, gentamicin is 1ug/ml, and cefotaxime is 2.5ug/ml. For the S. anginosus ATCC 12395 strain, the concentration is ampicillin 10ug/ml, It was confirmed that the concentration of vancomycin was 2.5ug/ml, gentamicin was 50ug/ml, tetracycline was 10ug/ml, and cefotaxime was 2.5ug/ml, and among the seven ATCC bacteria, S. aureus was identified. It was confirmed that the ATCC 25923 strain had the lowest antibiotic susceptibility.

<Example 2> Verification of antibiotic neutralization ability against 7 strains using composite resin

In this example, the composite resin [based on the medium, 3.0% (w/v) of cation exchange resin (sodium vinylbenzensulfonate divinyl benzene polymer, CAS No. 63182-08-1) and adsorption resin (divinylbenzene-ethylvinylbenzene-styrene copolymer, CAS No. 9052-95-3) mixed with 15.0% (w/v)] was used to verify the neutralizing ability of several antibiotics against 7 ATCC strains.

2-1. E. faecalis Verification of antibiotic neutralization ability against ATCC 29212 strain

The cation exchange resin and adsorption resin were quantified and mixed in a 100 ml Erlenmeyer flask containing 10 ml of HN4 medium as shown in Table 1 above. Next, E. faecalis ATCC 29212 strain was 10 CFU and ampicillin was 30, 50, 75, 100 ug/ml. , vancomycin is 2.5, 5, 10, 25, 50, 75, 100ug/ml, gentamicin is 75, 100ug/ml, tetracycline is 25, 50, 75, 100ug/ml. Each was added individually and cultured with shaking at 37°C for 3 days, and then growth was confirmed using CFU of the inoculum in the flask. At this time, the HN4 medium was treated under anaerobic conditions, sealed, and cultured with shaking under the above conditions. A control flask was used under the same antibiotic conditions and culture conditions, but without the addition of cation exchange resin and adsorption resin.

As a result, as shown in Figure 3, in the HN4 medium treated with a quantitative cation exchange resin and adsorption value, ampicillin was 100ug/ml, vancomycin was 100ug/ml, gentamicin was 100ug/ml, Neutralizing ability was confirmed up to a concentration of 100ug/ml of tetracycline.

2-2. E. faecium Verification of antibiotic neutralization ability against ATCC 700221 strain

The cation exchange resin and adsorption resin were quantified in the same manner as above, mixed in a 100 ml Erlenmeyer flask containing 10 ml of HN4 medium, and sterilized. Then, 10 CFU for E. faecium ATCC 700221 strain, 1, 2.5, 5 for tetracycline, 10, 25, 50, 75, and 100 ug/ml were added individually and cultured with shaking at 37°C for 3 days, and then growth was confirmed using CFU of the inoculum in the flask. At this time, the HN4 medium was treated under anaerobic conditions, sealed, and cultured with shaking under the above conditions. A control flask was used under the same antibiotic conditions and culture conditions, but without the addition of cation exchange resin and adsorption resin.

As a result, as shown in Figure 4, the neutralizing ability of tetracycline up to a concentration of 100ug/ml was confirmed in HN4 medium treated with a cation exchange resin and a quantitative adsorption value.

2-3. E. coli Verification of antibiotic neutralization ability against ATCC 25922 strain

The cation exchange resin and adsorption resin were quantified in the same manner as above, mixed in a 100 ml Erlenmeyer flask containing 10 ml of HN4 medium, and sterilized. Then, 10 CFU of E. coli ATCC 25922 strain and 50, 75, 100 ug/ml of ampicillin were added. , gentamicin is 30, 40, 50, 75, 100ug/ml, tetracycline is 2.5, 5, 10, 25, 50, 75, 100ug/ml, and cefotaxime is 0.5, 0.75. , 1, and 2.5ug/ml were added individually and cultured with shaking at 37°C for 3 days, and then growth was confirmed using CFU of the inoculum in the flask. At this time, the HN4 medium was treated under anaerobic conditions, sealed, and cultured with shaking under the above conditions. A control flask was used under the same antibiotic conditions and culture conditions, but without the addition of cation exchange resin and adsorption resin.

As a result, as shown in Figure 5, in the HN4 medium with a quantitative complex treatment of cation exchange resin and adsorption value, ampicillin was 100ug/ml, gentamicin was 75ug/ml, and tetracycline was 100ug. /ml, cefotaxime was confirmed to have neutralizing ability up to a concentration of 1ug/ml.

2-4. K. pneumoniae Verification of antibiotic neutralization ability against ATCC 700603 strain

Cation exchange resin and adsorption resin were quantified in the same manner as above, mixed in a 100 ml Erlenmeyer flask containing 10 ml of HN4 medium, sterilized, and then K. pneumoniae ATCC 700603. 10 CFU of the strain, 75 and 100 ug/ml of gentamicin, and 50, 75, and 100 ug/ml of tetracycline were added individually, cultured with shaking at 37°C for 3 days, and then inoculated from the flask. Growth was confirmed with CFU. At this time, the HN4 medium was treated under anaerobic conditions, sealed, and cultured with shaking under the above conditions. A control flask was used under the same antibiotic conditions and culture conditions, but without the addition of cation exchange resin and adsorption resin.

As a result, as shown in Figure 6, neutralization ability was confirmed up to a concentration of 100ug/ml for gentamicin and 100ug/ml for tetracycline in HN4 medium with a quantitative complex treatment of cation exchange resin and adsorption value. .

2-5. S. aureus Verification of antibiotic neutralization ability against ATCC 25923 strain

Cation exchange resin and adsorption resin were quantified in the same manner as above, mixed in a 100ml Erlenmeyer flask containing 10ml of HN4 medium, sterilized, and then added to S. aureus ATCC 25923. The strain is 10CFU, ampicillin is 2.5, 5, 10, 25ug/ml, vancomycin is 2.5, 5, 10, 25, 50, 75, 100ug/ml, gentamicin is 50, 75, 100ug/ml, tetracycline at 2.5, 5, 10, 25, 50, 75, 100ug/ml, and cefotaxime at 2.5, 5, 10, 25, 50, 75, and 100ug/ml respectively. After adding and culturing with shaking at 37°C for 3 days, growth was confirmed using CFU of the inoculum in the flask. At this time, the HN4 medium was treated under anaerobic conditions, sealed, and cultured with shaking under the above conditions. A control flask was used under the same antibiotic conditions and culture conditions, but without the addition of cation exchange resin and adsorption resin.

As a result, as shown in Figure 7, in the HN4 medium treated with a quantitative cation exchange resin and adsorption value, ampicillin was 10ug/ml, vancomycin was 100ug/ml, and gentamicin was 75ug/ml. Neutralizing ability was confirmed up to a concentration of 75ug/ml for tetracycline and 50ug/ml for cefotaxime.

2-6. S. epidermidis Verification of antibiotic neutralization ability against ATCC 12228 strain

Cation exchange resin and adsorption resin were quantified in the same manner as above, mixed in a 100 ml Erlenmeyer flask containing 10 ml of HN4 medium, sterilized, and then added to S. epidermidis ATCC 12228. The strain is 10CFU, vancomycin is 2.5, 5, 10, 25, 50, 75ug/ml, gentamicin is 1, 2.5, 5, 10, 25, 50ug/ml, and cefotaxime is 2.5. , 5, 10, 25, and 50 ug/ml were added individually and cultured with shaking at 37°C for 3 days, and then growth was confirmed using CFU of the inoculum in the flask. At this time, the HN4 medium was treated under anaerobic conditions, sealed, and cultured with shaking under the above conditions. A control flask was used under the same antibiotic conditions and culture conditions, but without the addition of cation exchange resin and adsorption resin.

As a result, as shown in Figure 8, in the HN4 medium treated with a quantitative combination of cation exchange resin and adsorption value, vancomycin was 50ug/ml, gentamicin was 25ug/ml, and cefotaxime was 25ug. Neutralizing ability was confirmed up to a concentration of /ml.

2-7. S. anginosus Verification of antibiotic neutralization ability against ATCC 12395 strain

Cation exchange resin and adsorption resin were quantified in the same manner as above, mixed in a 100 ml Erlenmeyer flask containing 10 ml of HN4 medium, sterilized, and then added to S. anginosus ATCC 12395. The strain is 10CFU, ampicillin is 10, 25, 50, 75, 100ug/ml, vancomycin is 2.5, 5, 10, 25, 50, 75ug/ml, gentamicin is 50, 75, Add 100ug/ml, tetracycline at 10, 25, 50, 75, 100ug/ml, and cefotaxime at 2.5, 5, 10, 25, and 50ug/ml separately and incubate at 37°C for 3 days. After shaking culture, growth was confirmed using CFU of the inoculum in the flask. At this time, the HN4 medium was treated under anaerobic conditions, sealed, and cultured with shaking under the above conditions. A control flask was used under the same antibiotic conditions and culture conditions, but without the addition of cation exchange resin and adsorption resin.

As a result, as shown in Figure 9, in a medium treated with a cation exchange resin and a quantitative adsorption value, when antibiotics were treated alone, ampicillin was 75ug/ml, vancomycin was 50ug/ml, and gentamicin was 75ug/ml. Neutralizing ability was confirmed up to a concentration of 75ug/ml for gentamicin, 75ug/ml for tetracycline, and 25ug/ml for cefotaxime.

<Example 3> Selection of carbon dioxide colorimetric sensor raw materials and indicators

In this example, we attempted to select raw materials and indicators for a colorimetric sensor that changes color by detecting carbon dioxide secreted when microorganisms grow.

3-1. Selection of raw materials for carbon dioxide colorimetric sensor

The raw material for the carbon dioxide colorimetric sensor used in this example was determined to be silicon among compounds with the property of being well permeable to carbon dioxide gas, and two types of room temperature curable silicone were selected (Table 3).

Characteristics of carbon dioxide colorimetric sensor raw materials GS-H1
(Request for green silicone production) DY-H11
(Request for DY silicone production) A B A B color Transparency Transparency White Transparency Viscosity (Pa·s) 1.6 0.5 10 0.7 importance 1.044 0.9 1.10 0.98 mixing ratio Mix A:B 10:1 Mix A:B 10:1 Hardening room temperature room temperature Hardness 30 25 Elongation rate (%) 160 200 Tensile strength (kgf/㎠) 8.49 16.0 classification Additional type Additional type

GS-H1 silicone ingredient list Chemical name CAS number A B Content (%) Content (%) Vinyl terminated polydimethylsiloxane 68083-19-2 70~80 40~50 Silicon dioxide 112945-52-5 10~20 - Vinyl Q resin 68584-83-8 10~20 - Methylhydrosiloxane, Dimethylsiloxane copolymer, Trimethylsiloxane terminated 68037-59-2 - 50~60

DY-H11 silicone ingredient list Chemical name CAS number A B Content (%) Content (%) Vinyl terminated polydimethylsiloxane 68083-19-2 70~80 70~80 Silica gel 112926-00-8 15~25 - Platinum, 1,3-diethenyl-1,1,3,3-tetramethyldisiloxane complex 68478-92-2 0.1 or less - Siloxanes and silicones, dimethyl, methyl hydrogen 68037-59-2 - 15~25 Tetravinyltetramethylcycloterasiloxane 2554-06-05 - 1~10

In order to easily determine the pH change due to an increase in carbon dioxide, the indicators bromothymol blue, phenolphthalein, thymol blue, and xylene are used in each room temperature curable silicone. The xylenol blue indicator was mixed and dissolved in a 0.2N NaOH solution containing 70% glycerol and 60% ethanol, and then added to the silicon to a final concentration of 0.05%.

First, add 0.05% of the indicator mentioned above to 10 g of room temperature curing silicone agent A, mix well, add 1 g of agent B, mix, then dispense 2 g into 70 ml plastic bottles, defoame, and cure at room temperature for one day. After treatment with 30 ml of HN4 medium containing composite resin (mixture of cation exchange resin and adsorption resin) and anaerobic conditions, 10 CFU of S. aureus ATCC 25923 strain was inoculated and cultured with shaking for 30 days, and the color change of the carbon dioxide colorimetric sensor of the corresponding experimental group was visually observed. It was confirmed with the reflectance value measured by a photodiode (self-produced) with a wavelength of 610 nm. At this time, as a control group, the same culture conditions were used, but the S. aureus ATCC 25923 strain was not inoculated, and a plastic bottle containing only HN4 medium and composite resin was used.

As a result, as shown in Figure 10, in all experimental groups, the color of the carbon dioxide colorimetric sensor was blue at the start of culture, but changed to light green to yellow after 1 day of culture due to an increase in carbon dioxide secreted by the inoculated S. aureus ATCC 25923 bacteria. It was confirmed that the color of the colorimetric sensor changed. In particular, an indicator containing bromothymol blue, phenolphthalein, thymol blue, and xylenol blue mixed with DY-H11 silicone was used at 70% glycerol and 60% The colorimetric sensor color at the start of cultivation in the negative experimental group (HN4 medium and composite resin treatment) was the only experimental group dissolved in 0.2N NaOH solution containing ethanol and treated with final 0.05%, while maintaining the colorimetric sensor color during the 30-day incubation period. The reflectance value according to the color change also maintained a constant value from 1 day to 30 days after inoculation in the positive test group (inoculation with S. aureus ATCC 25923 strain), and a clear difference in discrimination between the negative test group and the positive test group was confirmed. . Accordingly, as DY-H11 silicon was confirmed to be the most stable silicon for this study than GS-H1 silicon, DY-H11 silicon was finally selected as the raw material for the carbon dioxide colorimetric sensor.

3-2. Selection of carbon dioxide colorimetric sensor indicator

When mixed with DY-H11 silicone, the room temperature curing silicone used above, the sensitivity of the colorimetric sensor according to the increase in carbon dioxide secreted by microorganisms as they grow, and when measured with a photodiode, are stable when stored for a long time and are easy to determine positive. To select an indicator, the four indicators above were mixed in a certain ratio and dissolved in a 0.2N NaOH solution containing 70% glycerol and 60% ethanol, and then the mixed indicator was allowed to have a concentration in the range of 0.1 to 5.3%. At this time, the 20 types of mixed indicators manufactured were each referred to as HXB1~20. The indicator was mixed with silicone in the same manner as above. That is, after dissolving HXB 1 to 20 in a solution containing 0.2N NaOH solution containing 70% glycerol and 60% ethanol, add the final 0.05% to 10 g of DY-H11A, mix well, and then add DY-H11. After adding 1g of B and mixing, dispensed and degassed 2g each into a 70ml plastic bottle, cured at room temperature for a day, then added 30ml of HN4 medium with composite resin (mixture of cation exchange resin and adsorption resin) and treated under anaerobic conditions. 10 CFU of the S. aureus ATCC 25923 strain was inoculated and cultured with shaking for 5 days. The color change of the test area was visually confirmed, and the reflectance value measured by a photodiode with a wavelength of 610 nm was measured every day.

As a result, as shown in Figure 11, in all experimental groups, the color of the carbon dioxide colorimetric sensor was blue and green on the first day of culture, but due to an increase in carbon dioxide secreted by the inoculated S. aureus ATCC 25923 bacteria from 1 day after culture, It was confirmed that the color of the colorimetric sensor changed to yellow, green, and yellow. In particular, in the experimental group treated with 0.05% of the HXB 20 indicator mixed with the colorimetric sensor, the colorimetric colorimetric color was clearly yellow to the naked eye compared to the positive group (inoculated with S. aureus ATCC 25923 strain) in the experimental group treated with the HXB 1 to 19 indicator. It was confirmed that not only the color of the sensor changed, but also the change in reflectance value showed the highest discriminatory power compared to the voice treatment group, and the type of indicator mixed with the silicone was finally selected as HXB 20.

<Example 4> Preparation of anaerobic microbial medium based on carbon dioxide colorimetric

A final 0.05% of the indicator HXB 20 was added to DY-H11A, the room temperature curing silicone selected according to Example 3, mixed, 0.1 times DY-H11B was added, mixed and degassed, and cured at room temperature for one day. Then, the composite resin (cation exchange resin) Add 30ml of HN3 medium, an anaerobic medium containing (mixture of adsorption resin), sterilize by adding 10% carbon dioxide and 70% nitrogen to the medium, then add 10CFU of S. aureus ATCC 25923 strain and sheep blood (Kisan Bio, MB-S1876) A mixture of 10ml, vancomycin 25ug/ml + cefotaxime 30ug/ml + ampicillin 7.5ug/ml + gentamicin 50ug/ml was added and cultured with shaking at 37°C for 5 days. At this time, as a comparison group, BIOMERIEUX's FN PLUS product, a ready-made product sold in the world market, was compared under the same conditions as above. After incubation, the color change of the corresponding experimental groups was confirmed by visual inspection and daily measurement of reflectance values measured by a photodiode with a wavelength of 610 nm.

As a result, as shown in Figure 12, the anaerobic blood culture reagent showed a microbial growth rate similar to that of BIOMERIEUX's FN PLUS product (when only S. aureus ATCC 25923 strain was inoculated), and when sheep blood and antibiotics were mixed, antibiotics were observed. Compared to BIOMERIEUX's FN PLUS, which has no neutralizing ability, antibiotic neutralization ability was confirmed only in this product, confirming that it has a superior antibiotic neutralization ability than BIOMERIEUX's FN PLUS product. In addition, it was confirmed that not only did the color of the colorimetric sensor change to a more visually clear yellow color, but the change in reflectance value also showed excellent overall discrimination. This clearly shows that the anaerobic blood culture reagent, which includes a composite resin mixed with a cation exchange resin and an adsorption resin and a carbon dioxide colorimetric sensor, exhibits superior efficacy compared to existing anaerobic blood culture bottle products.

Claims (26)

Anaerobic microbial growth medium containing a complex of 3.0% (w/v) of cation exchange resin and 15.0% (w/v) of adsorption resin based on the medium; and a colorimetric sensor containing silicone that cures at room temperature and a color indicator,
The cation exchange resin is sodium vinylbenzensulfonate divinyl benzene polymer (CAS No. 63182-08-1), and the adsorption resin is divinylbenzene-ethylvinylbenzene copolymer (divinylbenzene-ethylvinylbenzene- styrene copolymer, CAS No. 9052-95-3),
The silicone is vinyl terminated polydimethylsiloxane, silica gel, and 1,3-diethenyl-1,1,3,3-tetramethyldisiloxane platinum complex (Platinum, 1) , 3-diethenyl-1,1,3,3-tetramethyldisiloxane complex) containing silicone (Agent A), vinyl terminated polydimethylsiloxane, siloxane, silicone, dimethyl, methyl hydrogen (Siloxanes, silicones, dimethyl, methyl hydrogen), and silicone (B agent) containing tetravinyltetramethylcycloterasiloxane,
Anaerobic blood culture reagent with antibiotic neutralizing ability. delete delete The anaerobic blood culture reagent with antibiotic neutralizing ability according to claim 1, wherein the colorimetric sensor is a colorimetric sensor for detecting carbon dioxide. delete The anaerobic blood culture reagent imparted with antibiotic neutralizing ability according to claim 1, wherein the silicone is condensed type or addition type silicone. The anaerobic blood culture reagent imparted with antibiotic neutralizing ability according to claim 6, wherein the silicone is an addition-type silicone. The anaerobic blood culture reagent with antibiotic neutralizing ability according to claim 1, wherein the silicone has a tensile strength of 10 to 20 kgf/cm2 and an elongation of 150 to 300%. The method of claim 1, wherein the silicone is 75 to 80% by weight of vinyl terminated polydimethylsiloxane, 15 to 25% by weight of silica gel, and 1,3-diethenyl-1,1. , 3,3-tetramethyldisiloxane platinum complex (Platinum, 1,3-diethenyl-1,1,3,3-tetramethyldisiloxane complex) silicone (Agent A) containing 0.001 to 0.1% by weight, and vinyl terminated 70-80% by weight of vinyl terminated polydimethylsiloxane, 15-25% by weight of siloxanes, silicones, dimethyl, methyl hydrogen, Tetravinyltetramethylcycloterasiloxane ) Anaerobic blood culture reagent with antibiotic neutralizing ability, which is a mixture of 1 to 10% by weight of silicon (agent B). The antibiotic neutralizing ability according to claim 9, wherein the silicone (agent A) has a viscosity of 1 to 11 (Pa·s), and the silicone (agent B) has a viscosity of 0.1 to 10 (Pa·s). Anaerobic blood culture reagent. The anaerobic blood culture reagent with antibiotic neutralizing ability according to claim 9, wherein the mixture is a mixture of silicone (agent A) and silicone (agent B) at a weight ratio of 10:0.5 to 1.5. The method of claim 1, wherein the color indicator includes one or more selected from bromothymol blue, phenolphthalein, thymol blue, and xylenol blue. , Anaerobic blood culture reagent with antibiotic neutralizing ability. The method of claim 12, wherein the color indicator is a mixed indicator of bromothymol blue, phenolphthalein, thymol blue, and xylenol blue, and imparts antibiotic neutralizing ability. Anaerobic blood culture reagent. The anaerobic blood culture reagent with antibiotic neutralizing ability according to claim 13, wherein the mixing indicator has a concentration of 0.1 to 5.3% by volume after mixing. The anaerobic blood culture reagent with antibiotic neutralizing ability according to claim 13, wherein the color indicator is prepared by treating a 0.2N NaOH solution containing 70 vol% glycerol and 60 vol% ethanol. The anaerobic blood culture reagent with antibiotic neutralizing ability according to claim 13, wherein the sensor is prepared by processing 0.01 to 0.5% by volume of the color indicator. The method of claim 1, wherein the medium is pancreatic digest of casein, papaic digest of soybean, yeast extract, dextrose, sodium. Sodium chloride, dibasic potassium phosphate, vitamin B ₁ (thiamine), vitamin B 3 (nicotinic acid), vitamin B ₆ (pyridoxine) _, vitamin K ₁ , vitamin K ₃ (menadione), hemin An anaerobic blood culture reagent with antibiotic neutralizing ability, which is a medium containing one or more selected from hemin, L-cysteine, sodium sulfite, and sodium thioglycolate. . The method of claim 1, wherein the antibiotics are beta-lactam series, glycopeptide series, aminoglycoside series, tetracycline series, and cephalosporin series. It may be any one or more selected from among, and is one or more selected from ampicillin, vancomycin, gentamicin, tetracycline, and cefotaxime, and is given antibiotic neutralizing ability. Anaerobic blood culture reagent. The anaerobic blood culture reagent imparted with antibiotic neutralizing ability according to claim 1, wherein the microorganism is a sepsis-causing bacteria. The method of claim 1, wherein the anaerobic microorganisms are Enterococcus faecalis ATCC 29212, Enterococcus faecium ATCC 700221, Escherichia coli ATCC 25922, Klebsiella pneumoniae pneumoniae ) ATCC 700603, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, and Streptococcus anginosus ATCC12395. An anaerobic blood culture reagent with antibiotic neutralizing ability. The anaerobic blood culture reagent with antibiotic neutralizing ability according to claim 1, wherein the reagent facilitates the growth of anaerobic microorganisms and is used to diagnose the presence or absence of causative bacteria in sepsis patients. A reagent composition for diagnosing sepsis with antibiotic neutralizing ability, comprising the anaerobic blood culture reagent of any one of claims 1, 4, and 6 to 21. A sepsis diagnostic kit comprising the anaerobic blood culture reagent of any one of claims 1, 4, and 6 to 21. 1) mixing the blood collected from the patient with the anaerobic blood culture reagent of any one of items 1, 4, and 6 to 21 to obtain a mixed solution;
2) cultivating the mixed solution of step 1); and
3) It includes the step of checking color change after culturing,
A method of providing information for diagnosing sepsis, including the step of determining that it is sepsis when the color of the colorimetric sensor changes from blue to yellow, green, or yellow. The method of claim 24, wherein the culture in step 2) is cultured for 3 to 10 days. The method of claim 24, wherein the color change in step 3) is confirmed with the naked eye or by measuring reflectance values.