WO2025022409A1

WO2025022409A1 - A kit for identifying bacteria and determining antibiotic sensitivity

Info

Publication number: WO2025022409A1
Application number: PCT/IN2024/050303
Authority: WO
Inventors: Roshan Ratnakar Naik; Lakshmi Swaroopa Naik
Original assignee: Individual
Current assignee: Individual
Priority date: 2023-07-21
Filing date: 2024-03-23
Publication date: 2025-01-30
Anticipated expiration: 2026-01-21

Abstract

The present invention relates to a kit for identifying bacteria and determining antibiotic sensitivity. The kit of the present invention includes the following components: a differential media formulation; distilled water; a color developer; a set of antibiotics; a control test; a set of Eppendorf tubes and a dropper. The differential media formulation includes peptone- 03gm by weight%, meat Extract-02gm by weight% and a nitrate source-0.6gm by weight%. the kit of the present invention enables bacterial identification and determining antibiotic susceptibility in lesser time than that of the conventional tests thereby reducing the time required in the treatment of urinary tract infection and thus reducing the incidences of antibiotic resistance.

Description

“A KIT FOR IDENTIFYING BACTERIA AND DETERMINING

ANTIBIOTIC SENSITIVITY”

FIELD OF THE INVENTION:

The present invention relates to a kit for identifying bacteria and determining antibiotic sensitivity, and more particularly to a kit for identifying bacteria and determining antibiotic sensitivity based on a differential media.

BACKGROUND OF THE INVENTION:

Urinary tract infections (UTIs) account for 150 million cases annually worldwide with a prevalence rate of 33.54% in India. The most common UTI pathogens are Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis and Staphylococcus saprophyticus and all are nitrite positive. Amongst these, 90% of all UTI infections are caused by E. coli.

Prescription without diagnosis is common during UTI treatment as reliable diagnosis takes 2-3 days and not getting the right treatment contributes towards AMR and recurrent UTIs in 20-30% women. Antimicrobial resistance (AMR) continues to be a global public health challenge due to improper and excessive use of antibiotics in human health, animal welfare leading to severe antimicrobial resistant infections, disease complications and mortality. The excessive usage of antibiotics is also leading to water and soil pollution, affecting aquatic organisms leading to AMR and there is possibility of resistance transfer into food chain and human body through seafood consumption. The current diagnosis of urinary tract infections (UTIs) involves a 2-minute dipstick analysis which is a qualitative test for the presence of nitrite in urine and gives a pink or red color precipitate suggesting UTI infection. The test has a sensitivity of 25-75% and specificity of 94-100% but exact identification of microorganisms and the right antibiotics is not possible. Patients often try home remedies or take over the counter medication given by pharmacists or doctor prescription based on symptoms but not based on urine culture/microbiology analysis. Use of wrong antibiotics contributes to antibiotic resistance, recurrent UTIs and worsening of symptoms. When symptoms persist beyond 1-2 days then the doctors recommend urine culture that usually involves a series of different media compositions to isolate single colonies using differential media, to identify bacteria using Indole, Methyl Red, Voges Proskauer and Citrate (IMViC), Triple Sugar Iron (TSI) test or similar tests and antibiotic susceptibility tests, leading to a wait of nearly 72 hours to identify bacteria and the correct antibiotics.

Reliable diagnosis of urinary tract infection takes 2-3 days. When outpatients visit a doctor, antibiotics are prescribed based on trial and error coupled with physician experience in treating similar cases. If antibiotics don’t work, patients revisit the doctor, leading to different antibiotic prescription or they order urine culture tests depending on the severity of the case. For inpatients, usually urine culture is common practice but choosing the right antibiotic is still trial and error due to urgency of treatment.

The gold standard urine culture method requires substantial laboratory setup and trained personnel to run the analysis. This is not available in remote areas, or the primary health center must send the samples (e.g., urine) to the laboratory for further analysis leading to treatment delay and severity of conditions.

Various technologies are being developed to reduce the time required for the detection of Urinary Tract Infection. The Indian Patent Application 201831007555 discloses a diagnosis kit for detection of Urinary Tract Infection (UTI) through agglutination method. However, there is no teaching for determining the right antibiotic to be used.

Thus, there is a need for an improved kit for identifying bacteria and determining antibiotic sensitivity in less time and thus reducing the problem of antibiotic resistance.

SUMMARY OF THE INVENTION:

The present invention discloses a kit for identification of bacteria and determining antibiotic sensitivity, comprising: a differential media formulation; distilled water; a color developer; a set of antibiotics; a control test; a set of Eppendorf tubes and a dropper. The differential media formulation includes Peptone- 03 gm by weight%; Meat Extract- 02 gm by weight%; and A nitrate source- 0. 6gm by weight%. The nitrate source is sodium nitrate or potassium nitrate. The differential media formulation is dispersed in 10 ml of water wherein the water is clean tap water or distilled water. The color developer in the kit is Griess reagent. The set of antibiotics in the kit are selected from Nitrofurantoin (NFN), Trimethoprim (TMP), Sulfamethoxazole (SMZ) Vancomycin (VAN), Piperacillin (PIP), Cefoperazone (CFP) and Meropenem (MEM). The present invention discloses a method for identifying the bacteria having the following steps: dissolving the media formulation in 10 ml of water using the dropper; distributing 1ml of dissolved media into individual Eppendorf tubes and inoculate with 2 drops of urine sample; incubating the tubes for 6-8 hours at 37°C; and adding 2 drops of color developer into the Eppendorf tubes and observing the tubes for color change at end of 1 minute.

The present invention discloses a method for determination of antibiotic sensitivity having the following steps: dissolving the media formulation in 10 ml of water using the dropper; distributing 1ml of dissolved media into individual Eppendorf tubes and inoculate with 2 drops of urine sample; adding 100 ul of antibiotics into individual tubes along with a negative control tube (no antibiotic) and incubating for 6-8 hours at 37°C; and adding 2 drops of color developer into the Eppendorf tubes and observing the tubes for color change at end of 1 minute. In another embodiment,

In another embodiment for determination of antibiotic sensitivity, the predefined amount of dissolved media added into individual Eppendorf tubes is 40- 100 ul. The predefined amount of urine sample from suspected UTI patient is 10- 50 ul. The antibiotics are selected from vancomycin (VAN), piperacillin (PIP), cefoperazone (CFP), meropenem (MEM). The amount of antibiotics used is 10-20 ul. The tubes are incubated for predefined period of 1 hr at predefined temperature of 37°C. The predefined amount of color developer is 20 ul. The results are recorded using a camera or smartphone and analysed using a suitable software. BRIEF DESCRIPTION OF DRAWINGS:

FIG. 1 depicts the methodology of conventional urine culture and testing of antibiotic sensitivity.

FIG. 2a and b show graphs indicating the growth curve of gram positive and gramnegative bacteria in media composition of varying concentrations.

FIG. 3a depicts the test results of bacterial identification using the media composition of the present invention.

FIG. 3b depicts the test results of antibiotic susceptibility using the kit of the present invention.

FIG. 4 depicts the test results of antibiotic susceptibility using an alternative embodiment of the kit of the present invention.

DESCRIPTION OF THE INVENTION:

References in the specification to "one embodiment" or "an embodiment" means that a particular feature, structure, characteristic, or function described in connection with the embodiment is included in at least one embodiment of the invention. The appearances of the phrase “in one embodiment” in various places in the specification are not necessarily all referring to the same embodiment.

References in the specification to “preferred embodiment” means that a particular feature, structure, characteristic, or function described in detail thereby omitting known constructions and functions for clear description of the present invention. The foregoing description of specific embodiments of the present invention has been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed and obviously many modifications and variations are possible in light of the above teaching.

The inventors of the present invention have developed a kit for the rapid detection of bacteria followed by testing of antibiotic susceptibility for the same . The kit ensures reduced time in identifying the bacteria and identifying the suitable antibiotic for the treatment of urinary tract infection.

In one aspect the present invention relates to kit for the quantitative or qualitative susceptibility testing for the most clinically significant urinary tract infection causing microorganisms.

The kit of the present invention includes the following components:

1. a differential media formulation;

2. water;

3. a color developer;

4. a set of antibiotics;

5. a control test;

6. a set of Eppendorf tubes; and

7. a dropper. In another aspect the present invention, the differential media formulation includes: i) peptone- 03 gm by weight%; ii) meat extract- 02 gm by weight%; and iii) a nitrate source- 0. 6gm by weight%;

In this preferred embodiment, the above components of the media formulation are added to 100 ml of clean tap water or distilled water. The nitrate source is sodium nitrate or potassium nitrate.

Further, the color developer in the kit is Griess reagent. The set of antibiotics included in the kit are Nitrofurantoin (NFN), Trimethoprim (TMP) and Sulfamethoxazole (SMZ).

Now a preferred method for identifying the bacteria is discussed as follows:

1. dissolving the media formulation in a predefined amount of water using the dropper;

2. distributing a predefined amount of dissolved media into individual Eppendorf tubes and inoculate with predefined amount of urine sample;

3. incubating the tubes for predefined period at a predefined temperature; and

4. adding predefined amount of color developer by gently squeezing the bottle into the Eppendorf tubes and observe for color change at end of 1 minute.

In this embodiment the predefined amount of water is 10 ml wherein the water is clean tap water or distilled water. The predefined amount of dissolved media added into individual Eppendorf tubes is 1 ml. The predefined amount of urine sample from suspected UTI patient is 2 drops (approx. 50-100 ul) The tubes are incubated for predefined period of 6 hrs at a predefined temperature of 37°C. The predefined amount of color developer is 2 drops.

A yellow, yellowish orange, pink to red color precipitate indicates presence of S. aureus, MRSA, E. coli and P. aeruginosa respectively that helps in bacterial identification.

Further, a preferred method for determination of antibiotic sensitivity is discussed as follows:

3. adding predefined amount of antibiotics into individual tubes along with a negative control tube (no antibiotic) and incubating for predefined period at a predefined temperature; and

In this embodiment the predefined amount of water is 10 ml wherein the water is clean tap water or distilled water. The predefined amount of dissolved media added into individual Eppendorf tubes is 1 ml. The predefined amount of urine sample from suspected UTI patient is 2 drops (approx. 50-100 ul) The reference antibiotics are Nitrofurantoin (NFN), Trimethoprim (TMP) and Sulfamethoxazole (SMZ) wherein the predefined amount of antibiotic is 100 ul and concentration is 1 ug/ml. The tubes are incubated for predefined period of 6 hrs at a predefined temperature of 37°C. Incubation beyond 6-8 hours is not recommended as it would not yield correct results. This is due to non-linear relationship between bacterial growth-related product formed and developed color beyond 8 hrs leading to color saturation at the upper red end The predefined amount of color developer is 2 drops.

For antibiotic sensitivity, lower the color change (yellow) indicates that the bacteria are antibiotic sensitive while higher the color change (dark red) indicates that the bacteria are antibiotic resistant. The color change is compared against a visual color guide or by measuring the optical density at 600 nm in a visible spectrophotometer.

In another embodiment for determination of antibiotic sensitivity, the predefined amount of dissolved media added into individual Eppendorf tubes is 40- 100 ul. The predefined amount of urine sample from suspected UTI patient is 10- 50 ul. The antibiotics are selected from vancomycin (VAN), piperacillin (PIP), cefoperazone (CFP), meropenem (MEM). The amount of antibiotics used is 10-20 ul. The tubes are incubated for predefined period of 1 hr at predefined temperature of 37°C. The predefined amount of color developer is 20 ul.

The results are recorded using a camera or smartphone and analysed using a suitable software. These and other embodiments will be apparent to those of skill in the art and others in view of the following detailed description of some embodiments. It should be understood, however, that this summary and the detailed description illustrate only some examples of various embodiments and are not intended to be limiting to the invention as claimed. The following examples illustrate the invention but are not limiting thereof.

EXAMPLES:

Example 1: Selection of concentration for media composition of present invention

The following concentrations of the media composition were prepared.

Nitrate broth (IX): peptone (0.5 gm), HM (or meat) extract (0.33 gm) and sodium nitrate (or potassium nitrate) (0.1 gm) in 100 ml distilled water or clean tap water.

Nitrate broth (3X): peptone (1.5 gm), HM (or meat) extract (1 gm) and sodium nitrate (0.3 gm) in 100 ml distilled water or clean tap water.

Nitrate broth (6X): peptone (03 gm), HM (or meat) extract (02 gm) and sodium nitrate (0.6 gm) in 100 ml distilled water or clean tap water.

Nitrate broth (9X): peptone (4.5 gm), HM (or meat) extract (2.7 gm) and sodium nitrate (0.9 gm) in 100 ml distilled water or clean tap water.

Bacteria were inoculated in the above media formulations and growth was observed. Results: FIG. 2a and b shows the growth curve of a representative gram-negative bacteria (E. coli) and a gram-positive bacteria (S. aureus) in various multiples of nitrate broth (lx, 3x, 6x, 9x). For both bacteria, there was an initial lag phase of 2 hrs. followed by exponential phase which peaks at 5 hr. followed by gradual decline in growth phase as measured by optical density at 600 nm in a spectrophotometer. The graphs indicate that the 6X media is optimal for bacterial growth.

Example 2: Components of the Diagnostic Kit

Media Formulation: 10 units

Color Developer: 10 ml

Water: 15 ml

Dropper: 1 No

Eppendorf tubes: 10 No

Negative test: 1

Nitrofurantoin (NFTN): 1

Trimethoprim (TMP): 1

Sulfamethoxazole (SMZ): 1

Example 3: Identification of Bacteria

1. Open the bottle containing the media formulation and dissolve it in 10 ml of water using the provided dropper.

2. Distribute 1 ml dissolved media into individual Eppendorf tubes (10 No. provided) and inoculate with 2 drops (approx. 50-100 ul) of urine sample. 3. Incubate tubes for 6 hr at 37°C.

4. Add 2 drops of color developer by gently squeezing the bottle into the Eppendorf tubes and observe for color change at end of 1 minute.

5. A yellowish orange, pink to red color precipitate indicates presence of MRS A, E. coli and P. aeruginosa respectively, that helps in bacterial identification as shown in FIG. 3a.

Example 4: Determination of Antibiotic Susceptibility

2. Distribute 1 ml dissolved media into individual Eppendorf tubes (10 No. provided) and inoculate with 2 drops (approx. 50-100 ul) of urine sample.

3. Similarly, add 100 ul antibiotics of interest (NFN/TMP/SMZ, etc.) into individual tubes along with a negative control tube (no antibiotic) and incubate for 6 hr at 37°C.

5. For antibiotic sensitivity test, a suspected UTI urine sample tested with nitrofurantoin (NFN) vs Trimethoprim/Sulfamethoxazole (TMP/SMZ), exhibiting sensitivity towards the latter. Here, lower the color change (yellow) indicates antibiotic sensitive while higher the color change (dark red) indicates antibiotic resistant as shown in FIG. 3b. 6. The color change is compared against a visual color guide or by measuring the optical density at 600 nm in a visible spectrophotometer.

Example 5: Determination of Antibiotic Susceptibility

2. Distribute 40 ul dissolved media into individual Eppendorf tubes (10 No. provided) and inoculate with 40 ul of urine sample.

3. Similarly, add antibiotics of interest (vancomycin (VAN), piperacillin (PIP), cefoperazone (CFP), meropenem (MEM)) into individual tubes along with a negative control tube (no antibiotic) and incubate for 1 hr at 37°C.

4. Add 20 ul of color developer by gently squeezing the bottle into the Eppendorf tubes and observe for color change at end of 1 minute.

5. Results were recorded using a camera or smartphone and analysed using suitable software as shown in FIG. 4.

In the context of the present invention, the kit of the present invention enables bacterial identification and determining antibiotic susceptibility in lesser time than that of the conventional tests thereby reducing the time required in the treatment of urinary tract infection and thus reducing the incidences of antibiotic resistance. The use of Eppendorf tubes reduces headspace leading to less oxygen availability for microbes in nutrient broth and urine. Thus, the reaction process is completed in less time unlike 24 hrs in glass tubes. The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, to thereby enable others, skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated.

It is understood that various omission and substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but such are intended to cover the application or implementation without departing from the scope of the present invention.

Claims

CLAIMS:

1. A kit for identification of bacteria and determining antibiotic sensitivity, comprising: a) a differential media formulation; b) distilled water; c) a color developer; d) a set of antibiotics; e) a control test; f) a set of Eppendorf tubes; and g) a dropper.

2. A kit as claimed in claim 1, wherein the differential media formulation includes: a) peptone- 03 gm by weight%; b) meat extract- 02 gm by weight%; and c) a nitrate source- 0. 6 gm by weight%.

3. The differential media formulation as claimed in Claim 2, wherein the nitrate source is sodium nitrate or potassium nitrate.

4. A kit as claimed in claim 1, wherein the differential media formulation is dispersed in 10 ml of water wherein the water is clean tap water or distilled water.

5. A kit as claimed in claim 1, wherein the color developer in the kit is Griess reagent.

6. A kit as claimed in claim 1, wherein the set of antibiotics in the kit are selected from Nitrofurantoin (NFN), Trimethoprim (TMP), Sulfamethoxazole (SMZ) Vancomycin (VAN), Piperacillin (PIP), Cefoperazone (CFP) and Meropenem (MEM).

7. A method for using the kit as claimed in Claim 1 for identifying the bacteria having the following steps: a) dissolving the media formulation in 10 ml of water using the dropper; b) distributing 1ml of dissolved media into individual Eppendorf tubes and inoculate with 2 drops of urine sample; c) incubating the tubes for 6-8 hours at 37°C; and d) adding 2 drops of color developer into the Eppendorf tubes and observing the tubes for color change at end of 1 minute.

8. A method for using the kit as claimed in Claim 1 for determination of antibiotic sensitivity having the following steps: a) dissolving the media formulation in 10 ml of water using the dropper; b) distributing 1ml of dissolved media into individual Eppendorf tubes and inoculate with 2 drops of urine sample; c) adding 100 ul of antibiotics into individual tubes along with a negative control tube (no antibiotic) and incubating for 6-8 hours at 37°C; and d) adding 2 drops of color developer into the Eppendorf tubes and observing the tubes for color change at end of 1 minute.

9. A method for using the kit as claimed in Claim 1 for determination of antibiotic sensitivity having the following steps: a) dissolving the media formulation in 10 ml of water using the dropper; b) distributing 40-100 ul of dissolved media into individual Eppendorf tubes and inoculate with 10-50 ul of urine sample; c) adding 10-20 antibiotics into individual tubes along with a negative control tube (no antibiotic) and incubating for 1 hour at 37°C; and d) adding 20 ul drops of color developer into the Eppendorf tubes and observing the tubes for color change at end of 1 minute.