Field of the Invention

The present invention relates to an anthocyanin-based antibiotic resistance test, which is developed for detecting antibiotic resistant microorganisms and is based on color change.

Background of the Invention

Anthocyanins are colorful, water-soluble polyphenolic pigments which are found in various edible fruits, vegetables, flowers, and grains and are produced by plants as secondary metabolites. Anthocyanins are a subset of flavonoids, and their basic chemical structure is composed of 2-phenyl benzopyrylium or flavylium nucleus. They are formed by linking polyhydroxy and polymethoxy derivatives with one or more glucose, rhamnose, galactose or arabinose at the C3, C5, C7, C3’ and C5’ positions via glycoside bonds. Among the glycosylation positions, C3 is the most commonly found binding point in nature. Pelargonidin, cyanidin (Cn), delphinidin (Dp), peonidin (Pn), petunidin (Pt), malvidin (Mv), and apigenin are the primary anthocyanins found in nature, and are mostly found in plants. Brassica oleracea var. Capitata f. Rubra (red cabbage) is an edible vegetable source having a high yield in obtaining anthocyanin, which is widely used as natural colorant in foods. Anthocyanin molecules contained in red cabbage extract serve as a natural pH indicator and exhibit a wide spectrum of colors such as red, pink in acidic environments depending on the pH of the environment, purple in neutral environments, and blue, green, yellow, respectively, in alkaline environments depending on the increase in pH. Colistin (colistin sulfate) is an antibiotic drug discovered in 1947 and used as a treatment of last resort for multidrug -resistant Gram-negative infections, including pneumonia. It is included in World Health Organization’s List of Essential Medicines (medicines considered to be the most effective and safe to meet the most important needs in any health-care system). Phenotypic bacterial identification tests can be used in laboratories where there is a need to detect gram-negative bacteria resistant to the antibiotic colistin. In clinical laboratories, it is tried to detect microorganisms in samples taken from the patient. Before the physician starts treatment, it should be determined whether the detected bacteria are resistant to antibiotics. Although colistin-resistant microorganisms are frequently encountered in our country, the cost of the commercially available tests prevents their use, and tests performed with conventional methods that take a long time to obtain results are still carried out.

Carbapenems are antibiotics commonly used in severe bacterial infections and are used for multidrug-resistant bacterial infections. Carbapenems are members of the beta-lactam antibiotics class, which kill bacteria by binding to penicillin-binding proteins, thus inhibiting bacterial cell wall synthesis. However, concern has arisen in recent years over increasing rates of resistance to carbapenems. Before the treatment process, there is a need to detect gram-negative bacteria resistant to carbapenem group antibiotics, and phenotypic resistance tests developed for this purpose are used in the known state of the art. In clinical laboratories, attempts are made to detect microorganisms in samples taken from the patient. The applications in the art are based on the detection of carbapenem resistance in products in the form of a test for detecting carbapenem-resistant microorganisms by means of pH indicators present in the environment due to the presence of a free -COOH group in the antibiotic structure as a result of the degradation of carbapenem group antibiotics by cell lysis. In the current product existing in the technical field, the cells are lysed before the test is performed and therefore it contains extra tris buffer. It comprises the carbapenemase cofactor, which is present in the test solely for detecting the structural change of carbapenem. The antibiotic dose is in an amount as high as 2-3 mg/mL. Therefore, the said product contains components which will increase the cost. Since the mechanism of the said test is intended for detecting the carbapenemase activity, the test contains high doses of antibiotics. Furthermore, phenol red, bromothymol blue or bromocresol purple are used as pH indicators, which are obtained by chemical processes under laboratory conditions and may cause irritation when they come in contact with eyes and skin. They do not only have both corrosive and toxic properties but may also cause irritation in the respiratory system if inhaled. Therefore, they should not be inhaled in the form of vapors or gases. The organs of the respiratory system and kidneys are among the target organs. It is recommended to use protective materials to prevent contact with hands, face, and eyes.

EP3271474, known in the state of the art, discloses a test for determining susceptibility or resistance to polymyxins in Enterobactericicecie , a family of bacteria that includes pathogens such as Salmonella and Escherichia coli. Herein, the test result for the product is specified as 2-4 hours and phenol red is used as a pH indicator to measure the components of the test medium and the pH change.

Chinese patent application CN105861630A, known in the state of the art, relates to a method and a reagent kit for detecting the presence of carbapenemase-producing bacteria in a sample. The related document is the patent of the existing commercially available product. It is based on the detection of the resistance by means of pH indicators present in the environment due to the presence of a free - COOH group that occurs in the antibiotic structure as a result of the degradation of carbapenem group antibiotics by cell lysis. In this product, the cells are lysed before the test is performed and therefore it contains extra tris buffer. It comprises the carbapenemase cofactor, which is present in the test solely for detecting the structural change of carbapenem. The antibiotic dose is in an amount as high as 2- 3 mg/mL. Therefore, the product of the said document contains components that increase the cost, unlike the invention. Since the mechanism of the said test is intended for detecting the carbapenemase activity, the test contains high doses of antibiotics. Furthermore, phenol red, bromothymol blue or bromocresol purple are used as pH indicators, which are obtained by chemical processes under laboratory conditions and may cause irritation when they come in contact with eyes and skin. They do not only have both corrosive and toxic properties but may also cause irritation in the respiratory system if inhaled. Therefore, they should not be inhaled in the form of vapors or gases. The organs of the respiratory system and kidneys are among the target organs. It is recommended to use protective materials to prevent contact with hands, face, and eyes.

International patent application WO2015013177A2, known in the state of the art, discloses methods, reagents, kits, and systems for detecting drug-resistant bacteria, particularly carbapenemase and beta-lactamase producing bacteria. In the related patent application, the test which is prepared to analyze carbapenem resistance contains metal cation, protein solubilizing surfactant and membrane disintegrating surfactant, unlike the present invention. It is also stated that dyes such as resazurin, trypan blue, methylene blue can be used as dyes. All these dyes are the ones produced by chemical synthesis and as previously stated, they have disadvantages against the anthocyanin disclosed in our invention.

International patent application WO2017089823A1, known in the state of the art, discloses a method for detecting the presence of carbapenemase-producing bacteria in a sample. In the said document, emphasis is put on the enzymatic activity of the bacteria and not on their growth. Therefore, it contains 2-3 mg/mL antibiotic. In addition, toxicity indicators such as phenol red and bromothymol blue are used as pH indicators.

Summary of the Invention

The objective of the present invention is to realize a phenotypic identification test comprising anthocyanin as pH indicator. In the said invention, instead of indicators such as phenol red, bromothymol blue or bromocresol purple, which are used as pH indicators in applications in the art and have toxicity and irritation risks, biocompatible and cost-efficient anthocyanins which are obtained from natural red cabbage are used as pH indicators.

Another objective of the invention is to realize a phenotypic identification test which, unlike the known applications in the art, does not contain extra tris buffer as there is no need for lysis of the cells before the test is performed, and in which anthocyanin indicators naturally obtained from red cabbage are used as pH indicators, instead of materials obtained by chemical processes in the laboratory as pH indicators which cause irritation when they come in contact with the eyes and skin.

A further objective of the invention is to realize a phenotypic identification test, which allows to obtain results in a shorter period of time (1.5-2 h) compared to applications known in the art (2-4 h). It is highly important to detect resistant bacteria in terms of starting treatment with the right antibiotic. Therefore, it is necessary to make the correct resistance analysis within the shortest time possible. Thanks to the invention, the 2-4-hour time interval of fastest available test is reduced to the 1.5-2 -hour time interval. Thus, test results are obtained more rapidly, and treatment is started with the right antibiotic.

Detailed Description of the Invention

The invention is the use of an anthocyanin indicator obtained from red cabbage rich in anthocyanins for the detection of antibiotic resistant bacterial strains. The said antibiotics are colistin and carbapenem. Within the scope of the invention, anthocyanin is used as a pH indicator in detecting the said antibiotic-resistant bacterial strains in order to detect these antibiotic -resistant bacteria by color change. In the preferred embodiment of the invention, red cabbage is used for its rich anthocyanin content, and also, red beet (Beta vulgaris var. cruenta Alef), purple carrot (Daucus carota L.ssp. sativus var. atrorubens Alef), hibiscus (Hibiscus sabdariffa), and the red cabbage (Brassica oleracea L. ssp. capitata f. rubra) as the reference plant, which are also rich in anthocyanins, can be used.

In the invention, colistin or carbapenem can be used as antibiotics for detecting the resistant bacteria.

A method of anthocyanin-based phenotypic resistance test of the present invention, which is developed for detecting antibiotic-resistant bacterial strains, comprises the following steps:

- preparing red cabbage extract in order to use its rich anthocyanin content as a pH indicator,

- preparing test medium containing 10 g/L peptone, 1 g/L pulp extract, 75 g/L NaCl, and sterilizing in autoclave

- adjusting the pH of the red cabbage extract to 8, fdtering through a sterile membrane fdter with a pore size of 0.45 pm and adding to the test medium at a ratio of 1: 1,

- adding the antibiotic (colistin (polymyxin E) or carbapenem) to the test solution in order to detect the microorganisms,

- adding the bacteria solution to be analyzed into a blue test solution to which antibiotic (colistin (polymyxin E) or carbapenem) and anthocyanin obtained from red cabbage are added,

- observing for 1.5-2 hours for detecting the presence of antibiotic -resistant bacteria with the solution turning pink.

A method of anthocyanin-based phenotypic colistin resistance test of the present invention, which is developed for detecting colistin antibiotic-resistant bacterial strains, comprises the following steps:

- preparing red cabbage extract in order to use its rich anthocyanin content as a pH indicator, - preparing test medium containing 10 g/L peptone, 1 g/L pulp extract, 75 g/L NaCl, and sterilizing in autoclave

- adjusting the pH of the red cabbage extract to 8, fdtering through a sterile membrane fdter with a pore size of 0.45 pm and adding to the test medium at a ratio of 1: 1,

- adding colistin (polymyxin E) to the test solution in order to detect the microorganisms,

- adding the bacteria solution to be analyzed into a blue test solution to which colistin (polymyxin E) and anthocyanin obtained from red cabbage are added,

- observing for 1.5-2 hours for detecting the presence of colistin antibioticresistant bacteria with the solution turning pink.

A method of anthocyanin-based phenotypic carbapenem resistance test of the present invention, which is developed for detecting carbapenem antibiotic-resistant bacterial strains, comprises the following steps:

- preparing red cabbage extract in order to use its rich anthocyanin content as a pH indicator,

- preparing test medium containing 10 g/L peptone, 1 g/L pulp extract, 75 g/L NaCl, and sterilizing in autoclave

- adjusting the pH of the red cabbage extract to 8, fdtering through a sterile membrane fdter with a pore size of 0.45 pm and adding to the test medium at a ratio of 1: 1,

- adding carbapenem to the test solution in order to detect the microorganisms,

- adding the bacteria solution to be analyzed into a blue test solution to which carbapenem and anthocyanin obtained from red cabbage are added,

- observing for 1.5-2 hours for detecting the presence of carbapenem antibioticresistant bacteria with the solution turning pink. Within the scope of the invention, firstly, red cabbage extract, which is a plant rich in anthocyanin, is prepared in order to obtain an anthocyanin indicator, which is one of the key components. During the preparation of red cabbage extract, the following process steps are applied:

- separating, washing, and drying the red cabbage leaves,

- shredding the red cabbage leaves into small pieces in a food processor,

- placing the shredded plant parts in distilled water at 1 : 1 (w/v) (e.g., 100 grams of shredded plant parts to 100 ml of distilled water) in a beaker,

- extracting the anthocyanin contained in red cabbage leaves by boiling the resulting mixture on a heater for 30 minutes,

- filtering the obtained anthocyanin extract and keeping at -20 °C until the study time.

A detection kit comprises containers for detecting antibiotic -resistant bacterial strains, wherein the said containers comprise the following:

- a test medium containing antibiotic-resistant bacterial strains,

- antibiotic (colistin or carbapenem),

- red cabbage extract rich in anthocyanin as pH indicator.

A test medium containing antibiotic-resistant bacterial strains contains 8-12 g/L peptone, 900-1100 g/L pulp extract. In a preferred embodiment of the invention, the said test medium contains 10 g/L peptone, 1 g/L pulp extract, 75 g/L NaCl. The amount of antibiotic is preferably 2 yg/mL.

In an embodiment of the invention, a detection kit comprises containers for detecting colistin antibiotic-resistant bacterial strains, wherein the said containers comprise the following:

- a test medium containing colistin antibiotic-resistant bacterial strains, - colistin antibiotic,

- red cabbage extract rich in anthocyanin as pH indicator.

A test medium containing colistin antibiotic-resistant bacterial strains contains preferably 10 g/L peptone, 1 g/L pulp extract, 75 g/L NaCl. The amount of colistin antibiotic is preferably 2 pg/mL.

In an embodiment of the invention, a detection kit comprises containers for detecting carbapenem antibiotic-resistant bacterial strains, wherein the said containers comprise the following: a test medium containing carbapenem antibiotic-resistant bacterial strains, carbapenem antibiotic, red cabbage extract rich in anthocyanin as pH indicator.

A test medium containing carbapenem antibiotic-resistant bacterial strains contains preferably 10 g/L peptone, 1 g/L pulp extract, 75 g/L NaCl. The amount of carbapenem antibiotic is preferably 2 pg/mL.

The invention is novel in the field of phenotypic bacterial identification tests and relates to a phenotypic identification test, which is developed for detecting antibiotic resistant microorganisms and is based on color change as a result of the test.

In clinical laboratories, there is a need for phenotypic bacterial identification tests for detecting antibiotic-resistant gram-negative bacteria with the purpose of detecting microorganisms in samples taken from patients. Before the physician starts treatment, it should be determined whether the detected bacteria are resistant to antibiotics. At this stage, the test developed within the scope of the invention is applied and information is obtained on whether the bacteria are resistant to the colistin antibiotic. An important difference of the subject matter of the invention before the applications in the known state of the art is the use of anthocyanins obtained from commonly available red cabbage as pH indicators instead of indicators synthesized in laboratory conditions such as phenol red, bromothymol blue. Thus, within the scope of the invention, a biocompatible and cost-efficient test has been developed. In addition, the prepared test content and working mechanism shorten the test time and reduce the antibiotic dose used by 1 in 1000, making it rapid and much more cost-efficient. Since the basic mechanism of the invention is intended for the inhibition of bacterial growth, it contains 2 pg/mL of antibiotic, which indicates that we have reduced the required antibiotic dose by 1000 times.

In the invention of the present application, the basic working mechanism of the test is completely different from the applications in the known state of the art (from the approach of detection of resistance through pH indicators present in the environment due to the free -COOH group that occurs in the antibiotic structure as a result of the degradation of carbapenem group antibiotics by cell lysis) and is based on the change in pH value of the medium caused by acidic volatile components released into the environment due to the growth of resistant microorganisms in the presence of antibiotics.

In the state of the art, the test result for the product is 2-4 hours, and phenol red is used as a pH indicator to measure the components of the test medium and the pH change. These indicators are obtained by chemical processes under laboratory conditions and may cause irritation when they come in contact with eyes and skin. They do not only have both corrosive and toxic properties but may also cause irritation to the respiratory system if inhaled. Therefore, they should not be inhaled in the form of vapors or gases. The organs of the respiratory system and kidneys are among the target organs. It is recommended to use protective materials to prevent contact with hands, face, and eyes [1, 2], On the other hand, within the scope of the invention, the antibiotic susceptibility test contains anthocyanin indicators naturally obtained from red cabbage as pH indicators. Since anthocyanins, which are a plant-derived pH indicator, are obtained from a plant consumed in daily life, there are no risks such as toxicity and irritation compared to other indicators. Anthocyanins obtained from red cabbage are often used for coloring the food and fruit juices. In addition, regular consumption of food sources rich in anthocyanins is recommended due to their antioxidant effects [3] , Red cabbage, which is naturally grown and easily accessible in our country, is used as a plant source in antibiotic susceptibility test due to its high anthocyanin content.

Within the scope of the invention, during the use of the phenotypic resistance test to which red cabbage extract is added in order to detect the presence of resistant bacteria,

• the test solution turns from blue to pink after 2 hours depending on the growth of antibiotic-resistant bacteria,

• there is no color change in the presence of antibiotic sensitive Gram-negative bacteria,

• there is no color change in the presence of antibiotic sensitive Gram-positive bacteria.

Bacterial strains resistant to colistin or carbapenem antibiotics can be listed as follows: Acinetobacter baumannii, Aeromonas junii, Bacillus cereus, Bacteroides fragilis, Citrobacter amalonaticus, Citrobacter freundii, Citrobacter youngae, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Pandoraea pnomenusa, Proteus mirabilis, Proteus rettgeri, Proteus vulgaris, Providencia stuartii, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens, Shigella flewieri, Stenotrophomonas maltophilia, Ralstonia picketti, and Shewanella algae. These strains can be stated among the strains for which the phenotypic identification test of the invention can be used for detection purposes. An important difference of the subject matter of the invention from the applications in the known state of the art is that there is no need for the lysis of the cells before the test is performed and therefore it does not contain extra tris buffer. There is no need for the antibiotic cofactor in the test of the invention since it is not solely for detecting the structural change of the antibiotic.

Unlike all existing products and teachings in the state of the art, in the invention, biocompatible anthocyanin dye isolated from plants is used for the first time.

The anthocyanin levels (per 100 g of plant) of the plants that can be used as anthocyanin sources in the invention are as follows: Mulberry: 1.4-704 mg, Black elderberry: 17—463 mg, Blackcurrant: 25-305 mg. Sweet cherry: 7-- 143 mg, Blackberry: 10-139 mg, Lingonberry: 4-49 mg, Strawberry: 4-48 mg , Sour cherry: 3-44 mg, Red raspberry: 5-38 mg, Black grape: 3-39 mg, Plum: 5-34 mg, Lowbush blueberry: 11-26 mg, Black bean: 1-15 mg, Redcurrant: 2-11 mg, Red onion: 7 mg [4], Other anthocyanin-rich foods (purple corn, pomegranate, eggplant, black carrots, red cabbage, and purple cauliflower) contain 200-300 mg anthocyanin per 100 g of product [5] .

EXPERIMENTAL STUDIES FOR COLISTIN-RESISTANT BACTERIA

Preparation of the Test Medium

The test medium which is prepared to contain 10 g/L peptone, 1 g/L pulp extract, 75 g/L salt is sterilized in autoclave at 121°C for 15 min. The red cabbage extract is adjusted to pH 8, filtered through a sterile membrane filter with a pore size of 0.45 pm and added to the test medium at a ratio of 1 : 1. In the last step, antibiotic is added at 2 pg/mL. Colistin (polymyxin E) is added to the test medium for detecting colistin-resistant microorganisms. The prepared test medium is blue colored. Preparation of the Agar Test Medium

To prepare 1 L of solid medium, 10 g peptone, 1 g pulp extract, NaCl containing 75 g/L salt, 15 g agar are dissolved in 500 ml distilled water. It is sterilized in autoclave at 121°C for 15 min. It is removed from the autoclave at 70°C. For the red cabbage extract to be present in the agar at a ratio of 1 : 1, 500 ml of red cabbage extract is also filtered through a membrane filter with a pore size of 0.45 pm and heated to 70°C in a hot water bath. 2 pg/ml colistin is added. The extract and medium are brought together at the same temperature. It is poured into petri dishes before it cools. The prepared test medium is blue-purple colored.

Implementation of the Test

150 pL of anthocyanin test medium and 150 pL of bacterial suspension were added to each well. After 1.5-2 hours of incubation, there was no color change in the wells containing antibiotic-sensitive bacterial strains and in the control wells, and the blue color remained constant. A pink color was observed in the well containing antibiotic-resistant bacteria. Thus, it was clearly seen that a visible color change occurred. Bacteria are inoculated into the agar test medium with a sterile swab. If colistin-resistant bacteria are present after incubation, pink colored colonies are observed.

EXPERIMENT - 1

Preparation of the Test Medium

The test medium which is prepared to contain 10 g/L peptone, 1 g/L pulp extract, 75 g/L salt is sterilized in autoclave at 121°C for 15 min. The red cabbage extract is adjusted to pH 8, filtered through a sterile membrane filter with a pore size of 0.45 pm and added to the test medium at a ratio of 1 : 1. In the last step, antibiotic is added at 2 pg/mL. Colistin (polymyxin E) is added to the test medium for detecting colistin-resistant microorganisms. The prepared test medium is blue colored.

Implementation of the Test

150 pL of anthocyanin test medium and 150 pL of bacterial suspension were added to each well. After 1.5-2 hours of incubation, there was no color change in the wells containing antibiotic-sensitive bacterial strains and in the control wells, and the blue color remained constant. A pink color was observed in the well containing antibiotic-resistant bacteria. Thus, it was clearly seen that a visible color change occurred.

EXPERIMENT -2

Preparation of the Agar Test Medium

To prepare 1 L of solid medium, 10 g peptone, 1 g pulp extract, NaCl containing 75 g/L salt, 15 g agar are dissolved in 500 ml distilled water. It is sterilized in autoclave at 121°C for 15 min. It is removed from the autoclave at 70°C. For the red cabbage extract to be present in the agar at a ratio of 1 : 1, 500 ml of red cabbage extract is also filtered through a membrane filter with a pore size of 0.45 pm and heated to 70°C in a hot water bath. 2 pg/ml colistin is added. The extract and medium are brought together at the same temperature. It is poured into petri dishes before it cools. The prepared test medium is blue-purple colored.

Implementation of the Test

Bacteria are inoculated into the agar test medium with a sterile swab. If colistin- resistant bacteria are present after incubation, pink colored colonies are observed. EXPERIMENTAL STUDIES FOR CARBAPENEM-RESISTANT BACTERIA

Preparation of the Liquid Test Medium

The test medium which is prepared to contain 10 g/L peptone, 1 g/L pulp extract, 75 g/L salt is sterilized in autoclave at 121°C for 15 min. The red cabbage extract is adjusted to pH 8, fdtered through a sterile membrane filter with a pore size of 0.45 pm and added to the test medium at a ratio of 1 : 1. In the last step, antibiotic is added at 2 pg/mL. In the test, imipenem will be used as a model antibiotic for detecting carbapenem-resistant microorganisms. The test works with all carbapenem group antibiotics. The prepared test medium is blue colored.

Preparation of the Agar Test Medium

To prepare 1 L of solid medium, 10 g peptone, 1 g pulp extract, NaCl containing 75 g/L salt, 15 g agar are dissolved in 500 ml distilled water. It is sterilized in autoclave at 121°C for 15 min. It is removed from the autoclave at 70°C. For the red cabbage extract to be present in the agar at a ratio of 1 : 1, 500 ml of red cabbage extract is also filtered through a membrane filter with a pore size of 0.45 pm and heated to 70°C in a hot water bath. 2 pg/ml imipenem is added. The extract and medium are brought together at the same temperature. It is poured into petri dishes before it cools. The prepared test medium is blue-purple colored.

Implementation of the Test

150 pL of anthocyanin test medium and 150 pL of bacterial suspension were added to each well. After 1.5-2 hours of incubation, there was no color change in the wells containing antibiotic-sensitive bacterial strains and in the control wells, and the blue color remained constant. A pink color was observed in the well containing antibiotic-resistant bacteria. Thus, it was clearly seen that a visible color change occurred. Bacteria are inoculated into the agar test medium with a sterile swab. If carbapenem-resistant bacteria are present after incubation, pink colored colonies are observed.

EXPERIMENT - 1

Preparation of the Liquid Test Medium

The test medium which is prepared to contain 10 g/L peptone, 1 g/L pulp extract, 75 g/L salt is sterilized in autoclave at 121°C for 15 min. The red cabbage extract is adjusted to pH 8, filtered through a sterile membrane filter with a pore size of 0.45 pm and added to the test medium at a ratio of 1 : 1. In the last step, antibiotic is added at 2 pg/mL. In the test, imipenem will be used as an antibiotic for detecting carbapenem-resistant microorganisms. The prepared test medium is blue colored.

Implementation of the Test

150 pL of anthocyanin test medium and 150 pL of bacterial suspension were added to each well. After 1.5-2 hours of incubation, there was no color change in the wells containing antibiotic-sensitive bacterial strains and in the control wells, and the blue color remained constant. A pink color was observed in the well containing antibiotic-resistant bacteria. Thus, it was clearly seen that a visible color change occurred.

EXPERIMENT - 2

Preparation of the Agar Test Medium

To prepare 1 L of solid medium, 10 g peptone, 1 g pulp extract, NaCl containing 75 g/L salt, 15 g agar are dissolved in 500 ml distilled water. It is sterilized in autoclave at 121°C for 15 min. It is removed from the autoclave at 70°C. For the red cabbage extract to be present in the agar at a ratio of 1 : 1, 500 ml of red cabbage extract is also filtered through a membrane filter with a pore size of 0.45 pm and heated to 70°C in a hot water bath. 2 pg/ml imipenem is added. The extract and medium are brought together at the same temperature. It is poured into petri dishes before it cools. The prepared test medium is blue-purple colored.

Implementation of the Test

Bacteria are inoculated into the agar test medium with a sterile swab. If carbapenem-resistant bacteria are present after incubation, pink colored colonies are observed.

REFERENCES

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Cytotoxicity of phenol red in toxicity assays for carbon nanoparticles. International journal of molecular sciences, 13(10), 12336- 12348.

[2]. Phenol Red, sc -203752, Material Safety Data Sheet, Santa Cruz

Biotechnology, 2010

[3]. Ahmadiani, N., Robbins, R. J., Collins, T. M., & Giusti, M. M. (2014).

Anthocyanins contents, profdes, and color characteristics of red cabbage extracts from different cultivars and maturity stages. Journal of agricultural and food chemistry, 62(30), 7524-7531.

[4]. Manolescu, B. N., Oprea, E., Mititelu, M., Ruta, L. L., & Farcasanu, I. C.

(2019). Dietary anthocyanins and stroke: A review of pharmacokinetic and pharmacodynamic studies. Nutrients, 11(7), 1479.

[5]. Mattioli, R., Francioso, A., Mosca, L., & Silva, P. (2020). Anthocyanins: A comprehensive review of their chemical properties and health effects on cardiovascular and neurodegenerative diseases. Molecules, 25(17), 3809.