KR102405920B1 - Microbial growth medium with antibiotic neutralization capability - Google Patents

Abstract

The present invention provides a microbial growth medium that can facilitate the growth of microorganisms that cause sepsis by neutralizing various antibiotics that may exist in the blood during blood culture of sepsis patients, thereby providing antibiotics in the blood of pre-treated sepsis patients. By neutralizing various antibiotics that may exist, it facilitates the growth of microorganisms that cause sepsis, so it can be usefully used in diagnosing causative bacteria in sepsis patients.

Description

Microbial growth medium with antibiotic neutralization capability

The present invention relates to a microbial growth medium having antibiotic neutralizing ability, and more particularly, by neutralizing various antibiotics that may exist in the blood during blood culture of a sepsis patient, microbial growth that can facilitate the growth of microorganisms causing sepsis It's about badges.

Sepsis, which is caused by infection in the blood by microorganisms invading the human body, is a disease with a mortality rate of 40 to 70%, with 6 million deaths worldwide every year. (Seongnam Kim et al., 2000, Weekly Health and Illness, Vol. 13, No. 37: 2750-2760). Blood culture is one of the essential requirements for identifying the exact causative agent of sepsis for the use of strong and broad-spectrum antibiotics at the early stage of the disease, one of the principles of sepsis treatment (Woon-Sung Kim, Hyeon-Jeong Lee. 2013, J. Korean Med. Assoc. 56:819-826 ).

Sepsis has a variety of causative bacteria and clinical symptoms, so several examinations must be preceded. However, since symptoms appear and progress rapidly, treatment is prioritized according to the judgment of an expert. Use antibiotics. Due to such pre-treated antibiotics (antibacterial agents), it has been difficult to isolate pathogenic microorganisms from blood cultures of patients undergoing antimicrobial therapy (Darby J.M. 1997. Crit. Care Med. 25:989- 994).

In order to solve these problems, various methods have been tried to overcome them. Examples thereof include a method of diluting a blood sample in the culture medium, a method of adding an enzyme inactivating an antibiotic to the blood culture, a method of enhancing the recovery of bacteria using a lysis centrifugation method (Krogstad, D. J. et al. 1981. Antimicrob Agents Chemother. 20:272-274; Washington, J. A. III and Ilstrup, D. M. 1986. Rev. Infect. Dis. 8:792-802), there is a method using cation exchange and polymer adsorption resin (Lindsey, N. J., and Riely, P. E. 1981. J. Clin. Microbiol. 13:503-507; Hansen S. L. et al. 1983. Amer. J. Med. 31-36). In particular, it was found that the medium containing the cation exchange and adsorption resin was the most effective for neutralizing antibiotics among the several mentioned methods and shortened the detection time of positive cultured bacteria (Jorgensen, J. H. et al. 1997. J. Clin. Microbiol). 35:53-58;Lelievre, H. et al. 1997. Eur. J. Clin. Microbiol. Infect. Dis. 16:669-674; Vigan, E. F. et al. 2004. N. Microbiol. 248; Flayhart, D. et al. 2007. J. Clin. Microbiol. 45:816-821; Miller, N. S. et al. 2011. J. Clin. Microbiol. 49:1624-1627).

As such, rapid isolation and identification of microorganisms that cause bloodstream infection are essential factors for patient survival following effective antibacterial therapy, but the cation exchange resin and adsorption resin contained in blood culture, which must be carried out for this purpose, have not been clearly elucidated so far. is not in a state Accordingly, a new microbial medium containing a cation exchange resin and an adsorption resin that can neutralize various antibiotics, including ampicillin, a beta-lactam antibiotic, the first report of clinical efficacy against Gram-negative bacteria, was developed. It is necessary to manufacture.

U.S. Patent No. 5624814 (registration date: April 29, 1997) relates to a nutrient culture medium for separating antibiotics and other microbial growth inhibitors in body fluid samples, and provides osmotic protection to infecting microorganisms damaged by exposure to antibiotics. A growth medium and a culture medium having one or more resins capable of separating antibiotics are described.

The present invention is to select a cation exchange resin and an adsorption resin that can effectively neutralize various antibiotics that may exist in the blood of sepsis patients pretreated with antibiotics, verify their functions, and provide a microbial medium containing the same.

The present invention is prepared by mixing a medium containing antibiotics with a cation exchange resin HUCA01 (TRILITE MC-08, manufactured by Samyang trilite) and an adsorption resin HUAB01 (Amberlite XAD4, manufactured by Sigma). provide badges.

On the other hand, in the microbial growth medium of the present invention, the microorganism may preferably be a bacterium causing sepsis.

On the other hand, in the microbial growth medium of the present invention, the medium is preferably pancreatic digest of casein, papaic digest of soybean, dextrose, sodium It is preferable that the TSB medium containing chloride (sodium chloride), dibasic potassium phosphate (dibasic potassium phosphate).

On the other hand, in the microorganism growth medium of the present invention, the antibiotic is, for example, beta-lactam (β-lactam) series, glycopeptide (glycopeptide) series, aminoglycoside (aminoglycoside) series, tetracycline series (tetracycline) series , may be any one or more selected from the cephalosporin series.

The present invention provides a microbial growth medium that can facilitate the growth of microorganisms that cause sepsis by neutralizing several antibiotics, thereby neutralizing various antibiotics that may exist in the blood of sepsis patients pretreated with antibiotics to cause sepsis It can be usefully used in diagnosing the causative bacteria of sepsis patients by facilitating the growth of the microorganisms.

1 is a graph of the results obtained by calculating the log numerical value of the results of culturing 4 types of ATCC strains in 7 types of nutrient media as cfu.
2 is a graph showing the minimum growth concentration for 5 types of antibiotics according to 4 types of ATCC strains.
3 is a graph showing the neutralizing ability of each antibiotic alone against the E. coli ATCC 25922 strain using the composite resin.
4 is a graph showing the neutralizing ability of each mixed antibiotic for E. coli ATCC 25922 strain using a composite resin (A; Ampicillin, G; Gentamicin, T; Tetracycline, C; Cefotaxime).
5 is a graph showing the neutralizing ability of each antibiotic alone against the S. aureus ATCC 25923 strain using the composite resin.
6 is a graph showing the neutralizing ability of each mixed antibiotic for S. aureus ATCC 25923 strain using a composite resin (A; Ampicillin, V; Vancomycin, G; Gentamicin, T; Tetracycline, C; Cefotaxime).
7 is a graph showing the neutralizing ability of each single antibiotic and mixed antibiotic against K. pneumoniae ATCC 700603 strain using a composite resin (G; Gentamicin, T; tetracycline).
8 is a graph showing the neutralizing ability of each single antibiotic and mixed antibiotic against P. aeruginosa ATCC 27853 strain using a composite resin (G; Gentamicin, T; Tetracycline).
9 is a result of confirming the growth of the composite resin and each antibiotic in the medium containing the composite resin for each time period for the S. aureus ATCC 25923 strain.

Sepsis is caused by infection in the blood by microorganisms that have invaded the human body, and since the mortality rate is continuously increasing, interest in treating it is high. Since sepsis develops rapidly and progresses rapidly, treatment is preceded by the judgment of an expert. Due to such pre-treated antibiotics (antibacterial agents), it has been difficult to isolate microorganisms that cause sepsis from blood cultures of patients undergoing antibacterial therapy. Therefore, in the present invention, it is an object of the present invention to provide a novel microbial medium containing a cation exchange resin and an adsorption resin capable of neutralizing various antibiotics in a patient's blood.

The present invention is prepared by mixing a medium containing antibiotics with a cation exchange resin HUCA01 (TRILITE MC-08, manufactured by Samyang trilite) and an adsorption resin HUAB01 (Amberlite XAD4, manufactured by Sigma). provide badges. The cation exchange resin HUCA01 is composed of sodium vinylbenzensulfonate divinylbenzene polymer (CAS No. 63182-08-1), and the adsorption resin HUAB01 is Amberlite XAD4 adsorption resin (CAS No. 37380-42). -0). At this time, it is preferable to treat 1.5 to 4.5% of the cation exchange resin HUCA01 and 10 to 20% of the adsorption resin HUAB01, and more preferably 2.5 to 3.5% of the cation exchange resin HUCA01 and 13 to 17% of the adsorption resin HUAB01. it's good Through the composite resin treated in this ratio, the antibiotic neutralizing ability can be excellently exhibited.

On the other hand, in the microbial growth medium of the present invention, the microorganism may preferably be a bacterium causing sepsis. At this time, the sepsis causative agent is applied to any bacteria causing sepsis, for example, Escherichia coli , Staphylococcus aureus , Klebsiella pneumoniae , Pseudomonas ae It may be luginosa ( Pseudomonas aeruginosa ).

On the other hand, in the microbial growth medium of the present invention, the medium can be applied to any medium capable of growth of the bacteria causing sepsis, pancreatic digest of casein, papaic digest of soybein (papaic digest) of soybean), yeast extract, dextrose, sodium chloride, dibasic potassium phosphate (dibasic potassium phosphate) it is good to use a nutrient medium containing any one or more selected from . Preferably pancreatic digest of casein (pancreatic digest of casein), papaic digest of soybean (papaic digest of soybean), dextrose (dextrose), sodium chloride (sodium chloride), dibasic potassium phosphate (dibasic potassium phosphate) ) is preferably a TSB medium containing (dextrose) 0.15 ~ 0.35%, sodium chloride (sodium chloride) 0.4 ~ 0.6%, dibasic potassium phosphate (dibasic potassium phosphate) 0.15 ~ 0.35% containing TSB medium is preferred. By using the medium added in such a ratio, microbial growth can be performed smoothly.

On the other hand, in the microorganism growth medium of the present invention, the antibiotic is, for example, beta-lactam (β-lactam) series, glycopeptide (glycopeptide) series, aminoglycoside (aminoglycoside) series, tetracycline series (tetracycline) series , may be any one or more selected from the cephalosporin series, and any one selected from ampicillin, vancomycin, gentamicin, tetracycline, and cefotaxime It may be more than

On the other hand, in the microbial growth medium of the present invention, additional components such as nutrient components may be additionally included for the growth of microorganisms, and it is not limited as long as it follows a medium manufacturing technique known in the art.

On the other hand, according to the following experiment in the present invention, in the medium treated with the composite resin containing the cation exchange resin HUCA01 and the adsorption resin HUAB01 of the present invention, Escherichia coli , a kind of sepsis causative bacteria, Staphylococcus aureus ( Staphylococcus aureus ), Klebsiella pneumoniae , Pseudomonas aeruginosa for ampicillin, vancomycin, gentamicin, tetracycline cefotaxime), it was confirmed that the antibiotic neutralizing ability was excellent not only when one or more antibiotics selected from among them were treated alone, but also when treated in combination.

Through this, the microbial growth medium treated with the composite resin containing the cation exchange resin HUCA01 and the adsorption resin HUAB01 prepared in the present invention can be used to identify the causative bacteria in the blood culture of sepsis patients, which can be applied to various pharmaceutical industries or treatments. it is expected that it will be possible

Hereinafter, the content of the present invention will be described in more detail through the following Examples and Experimental Examples. However, the scope of the present invention is not limited only to the following examples, and includes modifications of technical ideas equivalent thereto.

[Example 1: Selection of basal medium and determination of antibiotic susceptibility of test strains]

In this example, the basal medium for 4 types of bacteria that are frequently isolated from sepsis patients was selected, and their antibiotic susceptibility was determined.

1) Selection of basic medium

The strains used in this experiment were the 4 strains of ATCC that have been isolated the most from sepsis patients; Escherichia coli (hereinafter referred to as ' E. coli ') ATCC 25922, Staphylococcus aureus ( hereinafter referred to as ' S. aureus ') ATCC 25923, Klebsiella pneumoniae , Hereinafter named ' K. pneumoniae ') ATCC 700603, Pseudomonas aeruginosa (hereinafter referred to as ' P. aeruginosa ') ATCC 27853.

In order to confirm the smooth growth of the four ATCC strains presented above, a total of 7 medium (Table 1) 2 ml of single colonies of each strain cultured at 37° C. for 24 hours on a TSA (BD, 236950) plate After inoculation at 37 ℃ in a shaker incubator (180 rpm, Daehan Science, IS-20R) for 24 hours incubation, smeared on a plate medium containing the same component, the number of grown bacteria was measured to determine cfu. Finally, after uniformly calibrating to 10-20 cfu from the confirmed cfu, 10-20 cfu was inoculated into 5 ml of 7 different media and cultured for 12 hours.

Nutrient composition by type of medium used for basic medium selection TSB H1 H2 H3 H4 H5 H6 Pancreatic digest of casein
(BD, 211705) 1.7% 1.5% 1.7% 1.7% 1.7% 1.7% 1.7% Papaic digest of soybean
(BD, 243620) 0.3% - 0.3% 0.3% 0.3% 0.3% 0.3% yeast extract
(BD, 212750) - 0.5% 0.3% 0.3% 0.3% 0.3% 0.15% Dextrose
(BD, 215530) 0.25% 0.2% 0.25% 0.25% 0.25% 0.25% 0.25% sodium chloride
(Daejung, 7548-4100) 0.5% - 0.5% 0.5% - - 0.25% Dibasic potassium phosphate
(Daejung, 6612-4400) 0.25% - 0.25% - 0.25% - 0.25%

As a result, as shown in FIG. 1, the E. coli ATCC 25922 strain showed the highest growth in TSB and H6 media, the S. aureus ATCC 25923 strain in TSB and H2 media, and the P. aeruginosa ATCC 27853 strain in TSB and H3 media. It was excellent, and K. pneumoniae ATCC 700603 strain was able to confirm the even growth in 6 types of medium other than H6 medium. Accordingly, TSB medium, which showed excellent growth in common for the four strains, was selected as the basic medium for this experiment.

2) Determination of antibiotic susceptibility of test strains

The 4 ATCC strains used above were cultured in 2 ml of TSB medium for 24 hours to confirm cfu, and the final 10 ⁸ cfu was 0.5, 1, 2.5, 5, 10, 20, 30, 40, The minimum inhibitory concentration (MIC; Minimum Inhibitory Concentration) of each antibiotic in which the inoculated strain does not grow was confirmed after inoculation in 5 ml of TSB medium containing 50, 75, and 100 ug/ml concentration and shaking culture at 37°C for 3 days. .

Types of antibiotics used for antibiotic susceptibility and antibiotic neutralization ability tests by strain antibiotic class type of antibiotic β-lactam ampicillin (WAKO, 014-23302) glycopeptide vancomycin (Kisanbio, MB-V4882) aminoglycosides gentamicin (Kisanbio, MB-G4582) tetracycline tetracycline (Daejung, 8716-4150) cephalosporins cefotaxime (Kisanbio, MB-C4392)

As a result, as shown in FIG. 2 , the minimum inhibitory concentration for each antibiotic in which bacteria cannot grow was 50 ug/ml of ampicillin, 30 ug/ml of gentamicin, and 30 ug/ml of tetracycline in the E. coli ATCC 25922 strain. ) 2.5 ug/ml, cefotaxime 0.5 ug/ml, in S. aureus ATCC 25923 strain, ampicillin 2.5 ug/ml, vancomycin 2.5 ug/ml, gentamicin 50 ug/ml, tetracycline 2.5 ug/ml, cefotaxime By confirming that the concentrations were 2.5 ug/ml, gentamicin 75 ug/ml and tetracycline 50 ug/ml in the K. pneumoniae ATCC 700603 strain, and gentamicin 25 ug/ml and tetracycline 50 ug/ml in the P. aeruginosa ATCC 27853 strain. Among the four strains, it was confirmed that the antibiotic sensitivity of S. aureus ATCC 25923 strain was the lowest.

[Example 2: Selection of composite resin and ratio to be used for antibiotic neutralization ability]

In this example, a composite resin including a cationic resin and an adsorption resin to be used for imparting antibiotic neutralizing ability and a ratio thereof were selected.

First, in order to select a resin to be used for imparting antibiotic neutralization ability, two types of cation exchange resins having a high binding force with a material having a positive charge and three types of adsorption resins having a high binding force with a nonionic substance (hydrophobic bond) were used (Table 3). ), the cation exchange resin HUCA01 in Table 3 is composed of sodium vinylbenzensulfonate divinylbenzene polymer (CAS No. 63182-08-1), and the adsorption resin HUAB01 is Amberlite XAD4 adsorption resin (CAS No. .37380-42-0).

Types and characteristics of cation exchange resins and adsorption resins used in antibiotic neutralization experiments cation exchange resin adsorption resin HUCA01
(TRILITE MC-08, manufactured by Samyang trilite) HUCA02
(LEWATIT S1567,
manufactured by Lanxess) HUAB01
(Amberlite XAD4, manufactured by Sigma) HUAB02
(TRILITE GSH-20, manufactured by Samyang trilite) HUAB03
(LEWATIT VPOC1064,
Lanxess
Produce) shape tan
spherical particles transparent dark
brown
spherical particles transparent white
spherical particles opaque white
spherical particles opaque white
spherical particles matrix Styrene-DVB Crosslinked polystyrene Styrene-DVB Styrene
-DVB Styrene exchange Sulfonic acid Sulfonic acid doesn't exist doesn't exist doesn't exist ionic Na ⁺ Na ⁺ neutrality neutrality neutrality moisture
content
(%) 43-49 44-50 54-68 58-68 54-63 density
(g/L) 845 840 650 650-750 600 Jin Bi-jung 1.28 1.28 1.015~1.025 1.0~1.1 1.02 uniform
Coefficient 1.1↓ 1.1↓ 1.6↓ 1.6↓ 1.1↓ Grain size (mm) 0.60±0.05 0.60±0.05 0.25 to 0.85 0.315~1.25 0.49±0.05 maximum temperature
(℃) 120 120 150 110 120 pH range 0-14 0-14 0-14 0-14 0-14 Ieast
resin layer
(mm) 800 800 760 300 800 linear speed
(m/h) 5 to 60 5 to 60 5-20 5-30 5-20

In a 100 ml Erlenmeyer flask, the above five resins were mixed with 10 ml of the TSB medium of Table 1 alone or in combination at a constant ratio of 0.9 to 15%, sterilized (autoclave), and then S. aureus ATCC 25923 strain 10 cfu and 10 ug/ml of ampicillin + 25 ug/ml of vancomycin + 10 ug/ml of tetracycline were added, and incubated with shaking at 37°C for 1 day, treated with cfu of the inoculum in the flask. Check whether neutralizing ability against antibiotics and the results are shown in Tables 4 to 9.

Antibiotic neutralization ability against S. aureus ATCC 25923 strain according to single use of cation exchange resin and adsorption resin (no antibiotic added) strain badge Antibiotic
(ug/ml) Resin (w/v) cfu
0 hour incubation 24 hour incubation S. aureus ATCC 25923 TSB, 10ml additive-free HUCA01,0.9% 10 10 ⁹ HUCA01,1.5% 10 10 ⁹ HUCA01,3.0% 10 10 ⁹ HUCA02,0.9% 10 10 ⁹ HUCA02,1.5% 10 10 ⁹ HUCA02,3.0% 10 10 ⁹ HUAB01,6.0% 10 10 ⁹ HUAB01,10.0% 10 10 ⁹ HUAB01,15.0% 10 10 ⁹ HUAB02,6.0% 10 10 ⁹ HUAB02,10.0% 10 10 ⁹ HUAB02,15.0% 10 10 ⁹ HUAB03,6.0% 10 10 ⁹ HUAB03,10.0% 10 10 ⁹ HUAB03,15.0% 10 10 ⁹

Antibiotic neutralization ability against S. aureus ATCC 25923 strain by combined use of cation exchange resin HUCA01 and 3 types of adsorption resin (no antibiotic added) strain badge Antibiotic
(ug/ml) Resin (w/v) cfu
0 hour incubation 24 hour incubation S. aureus ATCC 25923 TSB, 10ml additive-free HUCA01 (0.9%)
+HUAB01 (6.0%) 10 10 ⁹ HUCA01 (0.9%)
+HUAB01 (10.0%) 10 10 ⁹ HUCA01 (0.9%)
+HUAB01 (15.0%) 10 10 ⁹ HUCA01 (0.9%)
+HUAB02 (6.0%) 10 10 ⁹ HUCA01 (0.9%)
+HUAB02 (10.0%) 10 10 ⁹ HUCA01 (0.9%)
+HUAB02 (15.0%) 10 10 ⁹ HUCA01 (0.9%)
+HUAB03 (6.0%) 10 10 ⁹ HUCA01 (0.9%)
+HUAB03 (10.0%) 10 10 ⁹ HUCA01 (0.9%)
+HUAB03 (15.0%) 10 10 ⁹ HUCA01 (1.5%)
+HUAB01 (6.0%) 10 10 ⁹ HUCA01 (1.5%)
+HUAB01 (10.0%) 10 10 ⁹ HUCA01 (1.5%)
+HUAB01 (15.0%) 10 10 ⁹ HUCA01 (1.5%)
+HUAB02 (6.0%) 10 10 ⁹ HUCA01 (1.5%)
+HUAB02 (10.0%) 10 10 ⁹ HUCA01 (1.5%)
+HUAB02 (15.0%) 10 10 ⁹ HUCA01 (1.5%)
+HUAB03 (6.0%) 10 10 ⁹ HUCA01 (1.5%)
+HUAB03 (10.0%) 10 10 ⁹ HUCA01 (1.5%)
+HUAB03 (15.0%) 10 10 ⁹ HUCA01 (3.0%)
+HUAB01 (6.0%) 10 10 ⁹ HUCA01 (3.0%)
+HUAB01 (10.0%) 10 10 ⁹ HUCA01 (3.0%)
+HUAB01 (15.0%) 10 10 ⁹ HUCA01 (3.0%)
+HUAB02 (6.0%) 10 10 ⁹ HUCA01 (3.0%)
+HUAB02 (10.0%) 10 10 ⁹ HUCA01 (3.0%)
+HUAB02 (15.0%) 10 10 ⁹ HUCA01 (3.0%)
+HUAB03 (6.0%) 10 10 ⁹ HUCA01 (3.0%)
+HUAB03 (10.0%) 10 10 ⁹ HUCA01 (3.0%)
+HUAB03 (15.0%) 10 10 ⁹

Antibiotic neutralization ability against S. aureus ATCC 25923 strain by the combined use of cation exchange resin HUCA02 and 3 types of adsorption resin (no antibiotic added) strain badge Antibiotic
(ug/ml) Resin (w/v) cfu
0 hour incubation 24 hour incubation S. aureus ATCC 25923 TSB, 10ml additive-free HUCA02 (0.9%)
+HUAB01 (6.0%) 10 10 ⁹ HUCA02 (0.9%)
+HUAB01 (10.0%) 10 10 ⁹ HUCA02 (0.9%)
+HUAB01 (15.0%) 10 10 ⁹ HUCA02 (0.9%)
+HUAB02 (6.0%) 10 10 ⁹ HUCA02 (0.9%)
+HUAB02 (10.0%) 10 10 ⁹ HUCA02 (0.9%)
+HUAB02 (15.0%) 10 10 ⁹ HUCA02 (0.9%)
+HUAB03 (6.0%) 10 10 ⁹ HUCA02 (0.9%)
+HUAB03 (10.0%) 10 10 ⁹ HUCA02 (0.9%)
+HUAB03 (15.0%) 10 10 ⁹ HUCA02 (1.5%)
+HUAB01 (6.0%) 10 10 ⁹ HUCA02 (1.5%)
+HUAB01 (10.0%) 10 10 ⁹ HUCA02 (1.5%)
+HUAB01 (15.0%) 10 10 ⁹ HUCA02 (1.5%)
+HUAB02 (6.0%) 10 10 ⁹ HUCA02 (1.5%)
+HUAB02 (10.0%) 10 10 ⁹ HUCA02 (1.5%)
+HUAB02 (15.0%) 10 10 ⁹ HUCA02 (1.5%)
+HUAB03 (6.0%) 10 10 ⁹ HUCA02 (1.5%)
+HUAB03 (10.0%) 10 10 ⁹ HUCA02 (1.5%)
+HUAB03 (15.0%) 10 10 ⁹ HUCA02 (3.0%)
+HUAB01 (6.0%) 10 10 ⁹ HUCA02 (3.0%)
+HUAB01 (10.0%) 10 10 ⁹ HUCA02 (3.0%)
+HUAB01 (15.0%) 10 10 ⁹ HUCA02 (3.0%)
+HUAB02 (6.0%) 10 10 ⁹ HUCA02 (3.0%)
+HUAB02 (10.0%) 10 10 ⁹ HUCA02 (3.0%)
+HUAB02 (15.0%) 10 10 ⁹ HUCA02 (3.0%)
+HUAB03 (6.0%) 10 10 ⁹ HUCA02 (3.0%)
+HUAB03 (10.0%) 10 10 ⁹ HUCA02 (3.0%)
+HUAB03 (15.0%) 10 10 ⁹

Neutralizing ability of antibiotics against S. aureus ATCC 25923 strain according to single use of cation exchange resin and adsorption resin (antibiotic addition) strain badge Antibiotic
(ug/ml) Resin (w/v) cfu
0 hour incubation 24 hour incubation S. aureus ATCC 25923 TSB, 10ml ampicillin 10 ug/ml + vancomycin 25 ug/ml +
tetracycline
10 ug/ml HUCA01,0.9% 10 0 HUCA01,1.5% 10 0 HUCA01,3.0% 10 0 HUCA02,0.9% 10 0 HUCA02,1.5% 10 0 HUCA02,3.0% 10 0 HUAB01,6.0% 10 0 HUAB01,10.0% 10 0 HUAB01,15.0% 10 0 HUAB02,6.0% 10 0 HUAB02,10.0% 10 0 HUAB02,15.0% 10 0 HUAB03,6.0% 10 0 HUAB03,10.0% 10 0 HUAB03,15.0% 10 0

Antibiotic neutralization ability against S. aureus ATCC 25923 strain by combined use of cation exchange resin HUCA01 and 3 types of adsorption resin (antibiotic addition) strain badge Antibiotic
(ug/ml) Resin (w/v) cfu
0 hour incubation 24 hour incubation S. aureus ATCC 25923 TSB, 10ml ampicillin 10 ug/ml + vancomycin 25 ug/ml +
tetracycline
10 ug/ml HUCA01 (0.9%)
+HUAB01 (6.0%) 10 0 HUCA01 (0.9%)
+HUAB01 (10.0%) 10 0 HUCA01 (0.9%)
+HUAB01 (15.0%) 10 0 HUCA01 (0.9%)
+HUAB02 (6.0%) 10 0 HUCA01 (0.9%)
+HUAB02 (10.0%) 10 0 HUCA01 (0.9%)
+HUAB02 (15.0%) 10 0 HUCA01 (0.9%)
+HUAB03 (6.0%) 10 0 HUCA01 (0.9%)
+HUAB03 (10.0%) 10 0 HUCA01 (0.9%)
+HUAB03 (15.0%) 10 0 HUCA01 (1.5%)
+HUAB01 (6.0%) 10 0 HUCA01 (1.5%)
+HUAB01 (10.0%) 10 0 HUCA01 (1.5%)
+HUAB01 (15.0%) 10 0 HUCA01 (1.5%)
+HUAB02 (6.0%) 10 0 HUCA01 (1.5%)
+HUAB02 (10.0%) 10 0 HUCA01 (1.5%)
+HUAB02 (15.0%) 10 0 HUCA01 (1.5%)
+HUAB03 (6.0%) 10 0 HUCA01 (1.5%)
+HUAB03 (10.0%) 10 0 HUCA01 (1.5%)
+HUAB03 (15.0%) 10 0 HUCA01 (3.0%)
+HUAB01 (6.0%) 10 0 HUCA01 (3.0%)
+HUAB01 (10.0%) 10 0 HUCA01 (3.0%)
+HUAB01 (15.0%) 10 10 ⁹ HUCA01 (3.0%)
+HUAB02 (6.0%) 10 0 HUCA01 (3.0%)
+HUAB02 (10.0%) 10 0 HUCA01 (3.0%)
+HUAB02 (15.0%) 10 0 HUCA01 (3.0%)
+HUAB03 (6.0%) 10 0 HUCA01 (3.0%)
+HUAB03 (10.0%) 10 0 HUCA01 (3.0%)
+HUAB03 (15.0%) 10 0

Antibiotic neutralization ability against S. aureus ATCC 25923 strain by combined use of cation exchange resin HUCA02 and 3 types of adsorption resin (antibiotic addition) strain badge Antibiotic
(ug/ml) Resin (w/v) cfu
0 hour incubation 24 hour incubation S. aureus ATCC 25923 TSB, 10ml ampicillin 10 ug/ml + vancomycin 25 ug/ml +
tetracycline
10 ug/ml HUCA02 (0.9%)
+HUAB01 (6.0%) 10 0 HUCA02 (0.9%)
+HUAB01 (10.0%) 10 0 HUCA02 (0.9%)
+HUAB01 (15.0%) 10 0 HUCA02 (0.9%)
+HUAB02 (6.0%) 10 0 HUCA02 (0.9%)
+HUAB02 (10.0%) 10 0 HUCA02 (0.9%)
+HUAB02 (15.0%) 10 0 HUCA02 (0.9%)
+HUAB03 (6.0%) 10 0 HUCA02 (0.9%)
+HUAB03 (10.0%) 10 0 HUCA02 (0.9%)
+HUAB03 (15.0%) 10 0 HUCA02 (1.5%)
+HUAB01 (6.0%) 10 0 HUCA02 (1.5%)
+HUAB01 (10.0%) 10 0 HUCA02 (1.5%)
+HUAB01 (15.0%) 10 0 HUCA02 (1.5%)
+HUAB02 (6.0%) 10 0 HUCA02 (1.5%)
+HUAB02 (10.0%) 10 0 HUCA02 (1.5%)
+HUAB02 (15.0%) 10 0 HUCA02 (1.5%)
+HUAB03 (6.0%) 10 0 HUCA02 (1.5%)
+HUAB03 (10.0%) 10 0 HUCA02 (1.5%)
+HUAB03 (15.0%) 10 0 HUCA02 (3.0%)
+HUAB01 (6.0%) 10 0 HUCA02 (3.0%)
+HUAB01 (10.0%) 10 0 HUCA02 (3.0%)
+HUAB01 (15.0%) 10 0 HUCA02 (3.0%)
+HUAB02 (6.0%) 10 0 HUCA02 (3.0%)
+HUAB02 (10.0%) 10 0 HUCA02 (3.0%)
+HUAB02 (15.0%) 10 0 HUCA02 (3.0%)
+HUAB03 (6.0%) 10 0 HUCA02 (3.0%)
+HUAB03 (10.0%) 10 0 HUCA02 (3.0%)
+HUAB03 (15.0%) 10 0

[Experimental Example 1: Verification of antibiotic neutralizing ability for 4 strains of the composite resin prepared in Example 2]

In this experimental example, using the composite resin (cation exchange resin HUCA01 3.0% + adsorption resin HUAB01 15.0%) confirmed in Example 2, it was attempted to verify the neutralizing ability of various antibiotics against 4 strains.

One) E. coli Validation of antibiotic neutralization ability against ATCC 25922 strain

Quantitation with cation exchange resin HUCA01 3.0% + adsorption resin HUAB01 15.0%, mixed in a 100 ml Erlenmeyer flask containing 10 ml of the TSB medium of Table 1 above, and sterilized, E. coli ATCC 25922 strain 10 cfu, ampicillin 50 , 75, 100 ug/ml, gentamicin at 30, 40, 50 ug/ml, tetracyclin at 2.5, 5, 10, 25, 50, 75, 100 ug/ml, cefotaxime at 0.5, 0.75, and 1 ug/ml, respectively After adding alone and culturing with shaking at 37° C. for 1 day, growth of the inoculum in the flask was confirmed with cfu. In this case, as a control, the same antibiotic conditions and culture conditions were used, but flasks without addition of cation exchange resin and adsorption resin were used.

As a result, as shown in FIG. 3, in the medium in which the cation exchange resin and the adsorption value were quantitatively combined, when antibiotics were treated alone, ampicillin was 100 ug/ml, gentamicin was 40 ug/ml, and tetracycline was 100 ug/ml. , cefotaxime was confirmed to have neutralizing ability up to a concentration of 0.75 ug/ml.

Based on the neutralizing concentration of each single antibiotic identified above, the neutralizing ability was confirmed by mixing antibiotics in the same experimental method; A mixture of two antibiotics (ampicillin 100 ug/ml + gentamicin 30 ug/ml , ampicillin 100 ug/ml + gentamicin 40 ug/ml, ampicillin 100 ug/ml + tetracycline 100 ug/ml, ampicillin 100 ug/ml + cefotaxime 0.5 ug/ml, ampicillin 100 ug/ml + cefotaxime 0.75 ug/ml), a mixture of 3 antibiotics (ampicillin 100 ug/ml + gentamicin 30 ug/ml + tetracycline 100 ug/ml, ampicillin 100 ug/ml + gentamicin 30 ug /ml + cefotaxime 0.5 ug/ml), a mixture of 4 antibiotics (ampicillin 100 ug/ml + gentamicin 30 ug/ml + tetracycline 100 ug/ml + cefotaxime 0.5 ug/ml).

As a result, as shown in FIG. 4 (A; Ampicillin, G; Gentamicin, T; Tetracycline, C; Cefotaxime), in the medium in which the cation exchange resin and the adsorption value were quantitatively combined, two types of antibiotics were mixed. Neutralizing ability was confirmed at the concentrations of ampicillin 100 ug/ml + gentamicin 30 ug/ml, ampicillin 100 ug/ml + tetracycline 100 ug/ml, ampicillin 100 ug/ml + cefotaxime 0.5 ug/ml. The antibiotic neutralizing ability was confirmed at the concentration of ug/ml + gentamicin 30 ug/ml + tetracycline 100 ug/ml. This is because even if blood culture in which several antibiotics are simultaneously treated for E. coli ATCC 25922 strain by the cation exchange resin and adsorption resin contained in the medium is carried out, antibiotics can be simultaneously neutralized, so there is no problem in culturing the causative bacteria of sepsis is to present

2) S. aureus Validation of antibiotic neutralization ability against ATCC 25923 strain

As in the above experiment, quantified with cation exchange resin HUCA01 and adsorption resin HUAB01, mixed in a 100 ml Erlenmeyer flask containing 10 ml of TSB medium of Table 1, and sterilized, S. aureus ATCC 25923 strain 10 cfu, ampicillin 2.5 , 5, 10, 25 ug/ml, vancomycin 2.5, 5, 10, 25, 50, 75, 100 ug/ml, gentamicin 50, 75, 100 ug/ml, tetracyclin 2.5, 5, 10, 25, For 50, 75, 100 ug/ml, and cefotaxime, 2.5, 5, 10, 20, 30, 40 ug/ml were added individually and cultured with shaking at 37°C for 1 day, and then grown as cfu of the inoculum in the corresponding flask. The presence or absence was checked. In this case, as a control, the same antibiotic conditions and culture conditions were used, but flasks without addition of cation exchange resin and adsorption resin were used.

As a result, as shown in FIG. 5, in the medium in which the cation exchange resin and the adsorption value were quantitatively combined, when antibiotics were treated alone, ampicillin was 10 ug/ml, vancomycin was 100 ug/ml, and gentamicin was 75 ug/ml. , Neutralizing ability was confirmed up to a concentration of 75 ug/ml for tetracycline and 30 ug/ml for cefotaxime.

Neutralizing ability was confirmed by mixing antibiotics in the same experimental method based on the neutralizing concentration of each single antibiotic identified above; A mixture of two antibiotics (ampicillin 10 ug/ml + vancomycin 50 ug/ml, ampicillin 10 ug/ml + vancomycin 75 ug/ml, ampicillin 10 ug/ml + gentamicin 50 ug/ml, ampicillin 10 ug/ml + gentamicin 75 ug/ml, ampicillin 10 ug/ml + cefotaxime 30 ug/ml, ampicillin 10 ug/ml + tetracycline 25 ug/ml, ampicillin 10 ug/ml + tetracycline 50 ug/ml), a mixture of three antibiotics (ampicillin 10 ug /ml + vancomycin 50 ug/ml + gentamicin 50 ug/ml, ampicillin 10 ug/ml + vancomycin 50 ug/ml + gentamicin 75 ug/ml, ampicillin 10 ug/ml + vancomycin 50 ug/ml + cefotaxime 30 ug/ml , ampicillin 10 ug/ml + vancomycin 50 ug/ml + tetracycline 25 ug/ml, ampicillin 10 ug/ml + vancomycin 50 ug/ml + tetracycline 50 ug/ml), a mixture of 4 antibiotics (ampicillin 10 ug/ml + vancomycin 50 ug/ml + gentamicin 50 ug/ml + cefotaxime 30 ug/ml), a mixture of 5 antibiotics (ampicillin 10 ug/ml + vancomycin 50 ug/ml + gentamicin 50 ug/ml + cefotaxime 30 ug/ml + tetracycline 25 ug/ml) treatment results are shown in FIG. 6 .

As a result, as shown in FIG. 6 (A; Ampicillin, V; Vancomycin, G; Gentamicin, T; Tetracycline, C; Cefotaxime), in the medium in which the cation exchange resin and the adsorption value were quantitatively combined, two types of antibiotics were mixed treated: ampicillin 10 ug/ml + vancomycin 50 ug/ml, ampicillin 10 ug/ml + gentamicin 50 ug/ml, ampicillin 10 ug/ml + gentamicin 75 ug/ml, ampicillin 10 ug/ml + cefotaxime 30 ug/ ml, ampicillin 10 ug/ml + tetracycline 25 ug/ml, ampicillin 10 ug/ml + tetracycline 50 ug/ml, ampicillin 10 ug/ml + vancomycin 50 ug/ml + gentamicin 50 ug/ml ml, ampicillin 10 ug/ml + vancomycin 50 ug/ml + cefotaxime 30 ug/ml, ampicillin 10 ug/ml + vancomycin 50 ug/ml + tetracycline 25 ug/ml concentration, ampicillin 10 ug/ml in the mixed treatment group of 4 antibiotics ml + vancomycin 50 ug/ml + gentamicin 50 ug/ml + cefotaxime 30 ug/ml, ampicillin 10 ug/ml + vancomycin 50 ug/ml + gentamicin 50 ug/ml + cefotaxime 30 ug/ml When the concentration of ml + tetracycline 25 ug/ml, the antibiotic neutralizing ability was confirmed. This is because even if blood culture in which several antibiotics are simultaneously treated for S. aureus ATCC 25923 strain by the cation exchange resin and adsorption resin contained in the medium is carried out, antibiotics can be simultaneously neutralized, so there is no problem in culturing the causative bacteria of sepsis is to present

3) K. pneumoniae Validation of antibiotic neutralization ability against ATCC 700603 strain

As in the above experiment, quantified with cation exchange resin HUCA01 and adsorption resin HUAB01, mixed in a 100 ml Erlenmeyer flask containing 10 ml of the TSB medium of Table 1, and sterilized. Then, 10 cfu of K. pneumoniae ATCC 700603 strain, 75 gentamicin, 100 ug/ml and tetracycline were treated alone at concentrations of 50, 75, and 100 ug/ml, and gentamicin 100 ug/ml + tetracycline 100 ug/ml were mixed and added at a concentration of 100 ug/ml. Growth was confirmed by cfu of the inoculum.

As a result, as shown in FIG. 7 (G; Gentamicin, T; Tetracycline), in the medium in which the cation exchange resin and the adsorption value were quantitatively combined, when the antibiotic was treated alone, gentamicin was 100 ug/ml and tetracycline was 100 ug Neutralizing ability was confirmed up to the concentration of /ml, and the neutralizing ability was also confirmed at the concentration of 100 ug/ml of gentamicin + 100 ug/ml of tetracycline, which was mixed with the highest neutralizing concentration of each antibiotic. This is because even if blood culture in which several antibiotics are simultaneously treated for K. pneumoniae ATCC 700603 strain by the cation exchange resin and adsorption resin contained in the medium is carried out, antibiotics can be simultaneously neutralized, so there is no problem in culturing the causative bacteria of sepsis is to present

4) P. aeruginosa Validation of antibiotic neutralization ability against ATCC 27853 strain

As in the above experiment, quantified with cation exchange resin HUCA01 and adsorption resin HUAB01, mixed in a 100 ml Erlenmeyer flask containing 10 ml of the TSB medium of Table 1, and sterilized. Then, P. aeruginosa ATCC 27853 strain was 10 cfu, gentamicin was 25, 50 ug/ml, tetracycline was treated alone at concentrations of 50, 75, and 100 ug/ml, and gentamicin 25 ug/ml + tetracycline 50 ug/ml, gentamicin 25 ug/ml + tetracycline 75 ug/ml, gentamicin 25 ug/ml After mixing with tetracycline at a concentration of 100 ug/ml and culturing with shaking at 37°C for 1 day, growth of the inoculum in cfu was checked in the flask.

As a result, as shown in FIG. 8 (G; Gentamicin, T; Tetracycline), when antibiotics were treated alone, gentamicin was 25 ug/ml and tetracycline was 100 ug in the medium in which the cation exchange resin and the adsorption value were quantitatively combined. Neutralizing ability was confirmed up to the concentration of /ml, and the neutralizing ability was also confirmed at the concentration of 25 ug/ml of gentamicin + 50 ug/ml of tetracycline mixed with antibiotics. This is because the cation exchange resin and the adsorption resin contained in the medium enable simultaneous neutralization of antibiotics even if several types of antibiotics are simultaneously treated for P. aeruginosa ATCC 27853 strain, so there is no problem in culturing the bacteria causing sepsis is to present

5) Verification of binding force between composite resin and antibiotics

As in the above experiment, quantified with cation exchange resin HUCA01 and adsorption resin HUAB01, mixed in a 100 ml Erlenmeyer flask containing 10 ml of TSB medium of Table 1 above, sterilized, vancomycin 100 ug/ml, gentamicin 75 ug/ml, cefotaxime 30 After each treatment at a concentration of ug/ml, 100ul of the culture medium with shaking at 37°C for each time period (0, 1, 2, 3, 4 hours) was collected and absorbed in a sterile paper disc (Advantec, PD0815) 10 ⁸ cfu of S. aureus ATCC 25923 bacterial suspension was placed on a mixed TSA (BD, 236950) plate and incubated for 24 hours in a stationary incubator at 37 ° C. The clear zone appeared to confirm the presence or absence of binding between the composite resin and the antibiotic.

As a result, as shown in FIG. 9, in the medium in which the cation exchange resin and the adsorption value were quantitatively combined, vancomycin was 100 ug/ml, gentamicin was 75 ug/ml, and cefotaxime was treated at a concentration of 30 ug/ml at 37 ° C. It was confirmed that the treated resin and the antibiotic were 100% bound within 1 hour after incubation. This clearly shows that the quantitatively mixed composite resin has an excellent function in neutralizing antibiotics, and it was confirmed that the efficacy was expressed even when several antibiotics were simultaneously treated.

Claims (4)

3% (w/v) of cation exchange resin composed of sodium vinylbenzensulfonate divinyl benzene polymer (CAS No. 63182-08-1) and Amberlite XAD4 adsorption resin (CAS No. 37380-42-0) Sepsis causative bacteria growth medium with antibiotic neutralizing ability, characterized in that it was prepared by mixing 15% (w/v).
delete The method of claim 1,
The medium is
Contains pancreatic digest of casein, papaic digest of soybean, dextrose, sodium chloride, dibasic potassium phosphate Sepsis causative bacteria growth medium with antibiotic neutralizing ability, characterized in that it is a TSB medium.
The method of claim 1,
The antibiotic is
Beta-lactam (β-lactam) series, glycopeptide (glycopeptide) series, aminoglycoside (aminoglycoside) series, tetracycline (tetracycline) series, characterized in that at least one selected from the cephalosporin series A growth medium for bacteria causing sepsis with antibiotic neutralizing ability.

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