DESCRIPTION

Technical Field Related To Invention

The invention relates to a rapid, economical and reliable Methicillin Resistant Staphylococcus aureus (MRSA) diagnostic test with natural content, which contains less chemicals, by using the color changing property with pH change anthocyanin in Brassica oleracea var. capitata f. rubra (red cabbage) and by meeting the NaCl need from Salicomia europaea (sea beans) extract or directly using NaCl.

Known State of the Invention (Prior Art)

Antibiotic resistance has become one of the most important health problems of our day and it threatens our future due to increasing use of antibiotics. One of the most important bacteria resistant to antibiotics is Methicillin Resistant Staphylococcus aureus (MRSA). Today, most of the infections that occur in hospitals are caused by MRSA, and rapid diagnosis and treatment of MRSA is vital.

In addition, because of the resistance of MRSA to beta-lactam group antibiotics, broad- spectrum antibiotics that are used for prevention before diagnosis or during the time for waiting for the results do not have an effect on MRSA and therefore this leads to waste of drugs.

In the prior art, a rich medium using Tryptic soy broth, which is the most sensitive (97.3%) among the culture methods, has been found. Besides this, it has been observed that chromogenic agar has 88.9% sensitivity and blood agar has 77.2% sensitivity rate. The suitability of the enrichment method is debatable since it requires an additional 24-hour incubation period although it is sensitive. Results can be obtained in 24 hours by seeding the samples into chromogenic agar without enrichment.

In molecular studies with PCR, results can be obtained within 3 hours. In terms of specificity, it can give positive faulty results, although rare. It may be appropriate to use in cases where results need to be taken urgently, but the high cost and need for qualified personnel limits its wide use. The methods to be commonly used can be enrichment method using tryptic soy broth or direct cultivation methods on chromogenic agar in emergency situations.

In another study, three different PCR methods were tested for the diagnosis of MRSA and S. aureus in samples taken from the upper and lower respiratory tract. The 3 PCR tests evaluated in this study are reasonably reliable rapid tests for detecting S. aureus and MRSA directly from clinical specimens suspected of a staphylococcal infection, however they have been found more appropriate to be performed in addition to bacterial culture today.

Nowadays chromogenic agar, rich culture and molecular methods are used for the diagnosis of MRSA. It is possible to obtain the results with high accuracy within 3 hours with the molecular method, but the widespread use of these methods is limited since the device and consumables used in these methods are expensive and also it requires trained personnel.

For these reasons, there is a need for an inexpensive, natural, reliable and rapid diagnostic test for the diagnosis of MRSA.

Summary and Aims of the Invention

The present invention relates to a diagnostic test (kit) using the color changing property with pH change of anthocyanin in red cabbage and by meeting the NaCl need from sea beans extract or directly using NaCl, which meets the above-mentioned requirements, eliminates the disadvantages and provides some additional advantages.

The separation of MRSA was practically and rapidly carried out in the liquid medium to be prepared by taking into consideration the resistance of methicillin with completely natural, common, accessible and very low cost red cabbage and sea beans.

Also, one of its important features is that plants which grow in Turkey and are low cost, such as red cabbage and sea beans have been used. Thus, it is possible to prepare a cost effective and very practical test. Since this method has more natural content, it is an environmentally friendly MRSA diagnostic method.

In the invention, extracts obtained from plants are used, 6 hours of incubation is sufficient, the cost is low and it does not require equipment-device compared to other molecular diagnosis methods.

Methicillin Resistant S. aureus (MRSA) diagnostic kit can be obtained with the application of the diagnostic test of the invention.

The summarized advantages of the inventive MRSA diagnostic test are as follows

^■ Rapid Result

^■ Reliable Result

^■ Originating from Edible Plant

^■ Low Environmental Pollution/Less Chemical Waste Innovative

^■ Low Cost ^■ Minimum Need for Equipment-Device-Personnel

^■ Easily Applicable to Industry

^■ Domestic and National Production

^■ High domestic consumption and export potential

Definitions of Drawings Illustrating the Invention

Figure 1: The process steps of the diagnostic test method of MRS A using red cabbage and sea bean extract are shown schematically.

Definitions of Elements/Pieces/Parts Forming the Invention

1. Preparation of Red Cabbage Extract

2. Preparation of Sea Beans Extract

3. Preparation of Bacterial Strains

4. Preparation of Liquid and/or Solid Media

5. Inhibition of Staphylococci other than MRSA by Adding 4 pg/ml Cefoxitin to the Medium Consisting of Red Cabbage Extract and Sea Beans Extract

6. 6 Hours of Incubation of Red Cabbage Extract and Medium Containing Sea Beans Extract with Methicillin Resistant S. Aureus (MRSA)

Detailed Description of the Invention

In the diagnostic test of Methicillin Resistant Staphylococcus aureus (MRSA); Brassica oleracea var. capitata f. rubra (red cabbage) extract is used as a pH indicator and sea beans extract or directly NaCl are used to meet the need for salt.

The diagnostic test of Methicillin Resistant Staphylococcus aureus (MRSA) comprises Brassica oleracea var. capitata f. rubra L. (red cabbage) extract as a pH indicator and Salicornia europaea (sea beans) extract for meeting the need for salt.

1. Preparation of the Extracts

1.1. Preparation of Red Cabbage Extract

Method 1: Red cabbage leaves are cut into small pieces by being passed through a food processor. 100 grams of material is weighed and taken into a beaker with 100 ml of distilled water. The mixture is extracted in a microwave oven (900 W, microwave power) for 2 minutes. The obtained anthocyanin extract is filtered and kept at -20°C until the running time.

Method 2: Red cabbage is cut into small pieces by being passed through a food processor and extracted with alcohol of 70% for 4 hours. The obtained extract is filtered. The solvent is evaporated with the evaporator, the extract is kept at -20°C until the experiment time.

Both methods were applied and positive results were obtained.

1.2. Preparation of Sea Beans Extract

The parts of the sea beans plant that are above-ground are divided into small pieces and taken into a beaker. They are subjected to mobile maceration at room temperature for 8 hours by adding distilled water. At the end of the period, the extract is filtered and an amount of water is evaporated with the evaporator. In order for the extract to dry completely, it is frozen at -80°C and lyophilized until dried.

1.2.1 Salt Detection from Sea Beans with Mohr Method

In order to calculate the sea beans extract to be used in the medium, firstly, the amount of salt it contains must be found. Mohr Method is used to determine the amount of salt in any product by titrating with AgN0 .

Titrant: 0.1 N Silver nitrate (AgN0 ) solution Indicator: 5% of potassium chromate (K ₂ Cr0 ₄ ) solution

Sample: 0.5 g of sea beans extract was dissolved in 125 ml of distilled water (stock solution)

The amount of salt obtained from sea beans was calculated.

The Formula of Mohr Method (Vl-v _blank ) * N * mEq * F * 100 / g _sampie Vl=titrant used in titration (ml)

V _biank = The amount of titrant used in blank test (ml)

N= Titrant normality (0.1 AgN0 ₃ ) mEq= 0.0585 for NaCl

F= 1 for 0.1 N AgN0 ₃ gsampie= The amount of used sample (g)

Blank Titration

The mixture was titrated with 50ml of distilled water + 1 ml of indicator titrant. Color change was observed after 8 drops in total Vbi _ank = 0.4ml

1. Titration

25 ml of the stock solution were taken; the solution was titrated by adding 1ml of indicator and 25ml of distilled water.

Total used titrant = V1 = 11 ml (1 lml-0.4ml)*0.1*0.0585* 1* 100/0. lg =61.58%

2. Titration 59.84% 3. Titration 63.15%

In other words, the average salt ratio was found to be 61.5% (w/w).

1.2.2. The Amount of Salt of Sea Beans

Mannitol-only agar medium was used as a reference in the prepared medium. The salt ratio of mannitol-only medium is 75 (g/1). In this way, microorganisms other than staphylococci will have been inhibited. The salt concentration of Salicorniaeuropaea (sea beans) was determined by Mohr Method and dilution was carried out accordingly. While preparing liquid or solid media, the same amount of extract is used each time based on the salt content of 61.5%. There is no need to calculate the amount of salt before each test.

The diagnostic test of Methicillin Resistant Staphylococcus aureus (MRSA) contains sea beans extract such that it contains 75 g/L salt. The said salt can be obtained from sea bean extract or salt can be used directly.

2. Bacterial Strains

In the invention, 98 S. aureus strains isolated from wound samples of hospitalized patients were used in the clinical microbiology laboratory. The colony morphology, Gram staining, catalase test, mannitol test, DNase and coagulase tests were applied for identification of bacteria. In order to determine methicillin resistance, cefoxitin disk diffusion testing, automated systems (Phoenix 100, Becton Dickinson, USA) were used and multiplex PCR was used for determination of mecA gene and production in chromogenic CHROM agar MRSA (CHROM agar Microbiology, Salubris, Turkey) media.

Cefoxitin disk diffusion test (30 pg, Oxoid, England) was studied by Kirby-Bauer disk diffusion method. The presence of mecA gene, which is regarded as the gold standard in the detection of methicillin resistance in strains, was studied using double primer gel-based multiplex PCR (Seeplex, Seegene Inc, Korea) method in accordance with the manufacturer's recommendations. All strains were inoculated on chromogenic CHROM agar MRSA medium and plates were incubated in an aerobic environment at 35°C. The results were evaluated at the end of twenty-four and forty-eight hours in accordance with the manufacturer's recommendations. The strains forming pink-red colonies in twenty-four hours were defined as methicillin resistant S.aureus (MRSA). The incubation period of plates with no growth in twenty-four hours was extended to forty-eight hours, and formation of colorless colonies or no growth in plates after 48 hours was evaluated as methicillin sensitive S.aureus (MSSA). ATCC 43300 strains for MRSA and ATCC 25923 strains for MSSA were used as standard strains. The sensitivity, specificity, positive predictive value (PPV) and negative predictive values (NPV) of the methods used in the study were calculated in detecting methicillin resistance. Red cabbage extract causes color change in the range of red-pink-purple-green-yellow at pH 1-14. This color change is observed as pink-red in acidic pH ranges (pH 1-4), purple color in neutral pH value (pH 7), yellow and green tones in basic pH range (pH 8-12).

3. Preparation of Liquid Medium

Per 1 liter, 10 g of peptone, 1 g of meat extract, sea bean extract including 75 g/L of salt or NaCl and red cabbage extract prepared according to method 1 at a ratio of 1:1 was added to the prepared medium. Peptone, meat extract, sea bean extract are added to 500 ml of distilled water and then sterilized in autoclave at 121°C for 15 minutes. 500 ml of red cabbage extract is added to the prepared medium by filtering through a membrane filter with a pore size of 0.45 pm. 4 pg/ml of cefoxitin is added. The prepared liquid medium is purple in color.

4. Preparation of Solid Medium

For the preparation of 1 L of solid medium, 10 g peptone, 1 g meat extract, sea bean extract containing 75 g/L salt or NaCl, and 15 g of agar are dissolved in 500 ml of distilled water. The mixture is sterilized in an autoclave at 121°C for 15 minutes. It is taken out of the autoclave at 70°C. In order to have the red cabbage extract in the agar at a ratio of 1:1, 500 ml of red cabbage extract is filtered through a membrane filter with a pore size of 0.45 pm and it is heated up to 70°C in a hot water bath. 4 pg/ml of cefoxitin is added. The extract and the medium are combined at the same temperature. The mixture is poured into petri dishes before it cools. The prepared solid medium is blue-purple in color.

5. Test Application on Liquid and Solid Media

In order to perform the test in a standardized way, 1 ml of liquid medium is added to 0.5 McFarland 1 ml of bacteria solution. Visible pink color formation was observed in solutions containing MRSA in 6 hours. If measured spectrophotometrically, samples containing MRSA give a positive result in 3 hours depending on the increase in absorbance.

Bacteria are planted on solid media with sterile swab. If MRSA is found as a result of incubation, pink colonies are seen.

6. Standardization Studies of the Medium

In order to determine the optimal concentrations of red cabbage extract and sea bean in the medium and the incubation times with bacteria, 4 pg/ml cefoxitin was added to this medium to inhibit staphylococci other than MRSA. Non-inhibited MRSA's change the purple color of anthocyanins into pink-red by decreasing the pH of the environment. For this purpose, the effect of the concentration of red cabbage extract, incubation time and temperature on the color change to be observed first was investigated, respectively.

6.1. Anthocyanin Liquid Media Experiment 1

1 ml of anthocyanin medium and 1 ml of bacterial suspension were added to each well by using bacterial suspensions prepared with Nutrient Broth (NB). As a result, 10.66 mg/ml of red cabbage and 75 mg/ml of NaCl were obtained in each well. After 6 hours of incubation, the wells containing MRS A turned pink, while wells with MSS A (Methicillin sensitive S. aureus) and control remained purple.

6.2. Anthocyanin Liquid Media Experiment 2

E.coli was also used in the prepared medium besides MRSA and MSSA. After 6 hours of incubation, MRS A color became pink while no change was observed in E.coli and MSSA color compared to the control. In addition, no color change was observed in Gram negative bacteria such as E.coli. It has been observed that MRSA can be easily separated from other bacteria due to high salt concentration and anthocyanin.

6.3. Anthocyanin Liquid Media Experiment 3

The experiment was repeated with antibiotic medium in order to eliminate bacteria sensitive to cefoxitin in anthocyanin liquid medium. 0.5 McFarland bacterial suspensions were plated into a 24- well plate. More types of bacteria (5 types of gram negative: E. coli, P. aeuriginosa, K. pneumoniae, S. typhi, B. Subtilis, 6 types of gram positive: MRSA, MSSA, E.faecium, S. pyogenes, S. pneumoniae, E. faecalis ) and fungus ( Candida albicans ) were used.

According to the result obtained at the end of 6 hours, no change was observed in the wells except for those containing MRSA and they were purple in color in the same manner as the control, while pink color was observed in the well containing MRSA.

6.4. Anthocyanin Liquid Media Experiment 4

Liquid media tests were tested to contain MRSA with an increase of 10 times between 1-100.000 colonies, also at concentrations other than 0.5 McFarland. Clear pink color formation was observed even in a single colony.

6.5. Anthocyanin Liquid Media Experiment 5

A test has also been carried out for MRSE (Methicillin resistant S. epidermidis ), which is one of the causes of serious nosocomial infections other than MRSA. The plasma was added to the prepared liquid medium and tested in order to distinguish it from MRSE since both of them have methicillin resistance. As a result of the incubation period, while pink color formation was observed in the samples with MRSE, both pink color change and clot formation were observed in the solutions containing MRSA. Thus, both the presence of MRSE and the presence of MRSA were detected.

6.6. Anthocyanin Solid Media Experiment

While MSSA and E.coli did not grow in solid media plated with MRSA, MSSA and E.coli, MRSA grew therein and a pink color formation was observed in the solid medium.