A DISINFECTANT FORMULATION FOR NOSOCOMIAL INFECTIONS AND METHOD OF SYNTHESIS THEREOF

Abstract

A synergistic disinfectant formulation, for nosocomial infections, and method of synthesis thereof, are disclosed. Said synergistic formulation broadly comprises: L-aqueous phase of wheat straw biomass; sodium lauryl sulphate; ethanol; and ethylenediaminetetraacetic acid. The disclosed disinfectant formulation offers at least the following synergistic advantages and effects: improved efficacy (or an efficacy that is comparable to that of commercially available disinfectants); effective against biofilms; reduced risk of complications; and/or lower incidence of side effects or allergies.

Specification

Description:TITLE OF THE INVENTION: A DISINFECTANT FORMULATION FOR NOSOCOMIAL INFECTIONS AND METHOD OF SYNTHESIS THEREOF
FIELD OF THE INVENTION
The present disclosure is generally related to nosocomial infections. Particularly, the present disclosure is related to formulations for nosocomial infections. More particularly, the present disclosure is related to: a disinfectant formulation for nosocomial infections; and method of synthesis thereof.
BACKGROUND OF THE INVENTION
Nosocomial infections (also referred to as health-care associated infections (HAIs)), are majorly acquired during the process of receiving health care by nosocomial (also referred as "ESKAPE") pathogens. They contribute to significant morbidity, mortality, and financial burden on patients, families, and healthcare systems. The emergence of multi-drug-resistant organisms is another complication seen with HAIs.
Existing medications for nosocomial infections involve use of hypochlorites, alcohols, hydrogen peroxide, quaternary ammonium compounds, and phenolic compounds. However, the medications are not effective against spore formers. Other alternatives such as UV irradiation, surface modification, and self-disinfecting surfaces suffer from various drawbacks, but not limited to: high production costs; limited effectiveness due to increased antimicrobial resistance; and/or potential health hazards (due to presence of harmful chemicals).
There is, therefore, a need in the art, for: a disinfectant formulation for nosocomial infections; and method of synthesis thereof, which overcome the aforementioned drawbacks and shortcomings.
SUMMARY OF THE INVENTION
A synergistic disinfectant formulation, for nosocomial infections, and method of synthesis thereof, are disclosed. Said synergistic disinfectant formulation broadly comprises: L-aqueous phase of wheat straw biomass; sodium lauryl sulphate; ethanol; and ethylenediaminetetraacetic acid. Said L-aqueous phase of wheat straw biomass is in a ratio of: about 1:5 with said sodium lauryl sulphate and said ethanol; about 1:1 with said ethylenediaminetetraacetic acid.
The disclosed disinfectant formulation offers at least the following synergistic advantages and effects: improved efficacy (or an efficacy that is comparable to that of commercially available disinfectants); effective against biofilms; reduced risk of complications; and/or lower incidence of side effects or allergies.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates results of minimum inhibitory concentration analyses, in accordance with an embodiment of the present disclosure;
Figure 2A, Figure 2B, Figure 2C, and Figure 2D illustrate results of minimum biofilm eradication concentration analyses, in accordance with an embodiment of the present disclosure; and
Figure 3A, Figure 3B, Figure 3C, and Figure 3D illustrate results of phenol coefficient analyses for phenol, commercial formulation, synthesised formulation, and L-aqueous phase of wheat biomass, respectively, in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
Throughout this specification, the use of the words "comprise" and "include", and variations, such as "comprises", "comprising", "includes", and "including", may imply the inclusion of an element (or elements) not specifically recited. Further, the disclosed embodiments may be embodied, in various other forms, as well.
Throughout this specification, the use of the word "synthesis", and its variations, is to be construed as: "produce; manufacture; and/or the like".
Throughout this specification, the use of the phrase "synthesised formulation" is to be construed as: "synthesised disinfectant formulation for nosocomial infections".
Throughout this specification, the use of the phrase "aqueous formulation" is to be construed as: "synthesised formulation".
Throughout this specification, the use of the phrase "formulated disinfectant" is to be construed as: "synthesised formulation".
Throughout this specification, the use of the phrases "commercial formulation" and "commercial product" is to be construed as: "commercially available formulation that comprises: about 2.4584% w/w of benzalkonium chloride solution, lauryl alcohol ethoxylate, sodium bicarbonate, cocamidopropyl betaine, perfume, tetra sodium EDTA, and denatonium benzoate".
Throughout this specification, the use of the acronym "L-AQ" is to be construed as: "Lyophilised aqueous phase of wheat straw biomass".
Throughout this specification, the use of the acronym "EDTA" is to be construed as: "Ethylenediaminetetraacetic acid".
Throughout this specification, the use of the acronym "SCD" is to be construed as: "Soybean Casein Digest media".
Throughout this specification, the use of the acronym "YPD" is to be construed as: "Yeast extract Potato Dextrose media".
Throughout this specification, the use of the acronym "RPMI" is to be construed as: "Roswell Park Memorial Institute media".
Throughout this specification, the use of the acronym "PBS" is to be construed as: "Phosphate Buffered Saline media".
Throughout this specification, the use of the acronym "MIC" is to be construed as: "Minimum Inhibitory Concentration".
Throughout this specification, the use of the acronym "MBEC" is to be construed as: "Minimum Biofilm Eradication Concentration".
Throughout this specification, the use of the acronym "MRSA" is to be construed as: "Methicillin Resistant Staphylococcus aureus".
Throughout this specification, the use of the acronym "CFU" is to be construed as: "Colony Forming Unit".
Throughout this specification, the use of the acronym "CDC is to be construed as: "Centre for Disease Control".
Throughout this specification, the use of the acronym "ASTM" is to be construed as: "American Society for Testing and Materials".
Throughout this specification, the use of the word "cultured strains" is to be construed as: "pure culture of MRSA cultured in SCD media; pure culture of Acinetobacter baumannii cultured in SCD media; and pure culture of Candida auris cultured in YPD media".
Throughout this specification, the use of the word "mixed species" is to be construed as: "mixed culture comprising strains of MRSA, strains of Acinetobacter baumannii, and strains of Candida auris cultured in RPMI media".
Throughout this specification, the use of the acronym "M+C" is to be construed as: "media and cells alone, which serve as a control".
Throughout this specification, the disclosure of a range is to be construed as being inclusive of: the lower limit of the range; and the upper limit of the range.
Throughout this specification, the words "the" and "said" are used interchangeably.
Throughout this specification, the words "sulfate" and "sulphate" are used interchangeably.
Also, it is to be noted that embodiments may be described as a method. Although the operations, in a method, are described as a sequential process, many of the operations may be performed in parallel, concurrently, or simultaneously. In addition, the order of the operations may be re-arranged. A method may be terminated, when its operations are completed, but may also have additional steps.
A synergistic formulation for disinfecting nosocomial infections (also referred as "synthesised disinfectant formulation", "synthesised formulation", and/or "formulation") is disclosed. The disclosed formulation was synthesised and tested as follows:
Said synergistic formulation broadly comprises: L-aqueous phase of wheat straw biomass (about 1%); sodium lauryl sulphate (about 5% to about 15%); ethanol (about 5% to about 35%); and ethylenediaminetetraacetic acid (about 1% to about 5%). Further, a person skilled in the art will appreciate the fact that the synthesised formulation may comprise distilled water as per requirements.
The L-aqueous phase of wheat straw biomass is in a ratio of: about 1:5 with said sodium lauryl sulphate; and said ethanol; and about 1:1 with said ethylenediaminetetraacetic acid.
The disclosed formulation was synthesised and tested as follows:
Said wheat straw biomass was procured from local farms of Rajasthan and Tamil Nadu. About 30 g of biomass was dried at about 60 ºC overnight, followed by size reduction to about 1 mm to obtain a milled biomass.
Synthesis of L-Aqueous Phase of Wheat Biomass
The milled biomass was subjected to pyrolysis to obtain a bio-oil aqueous phase. Pyrolysis was carried out in a rotating bed reactor with a heating zone of about 0.5 m and an outer diameter of about 74 mm, with a nitrogen flowrate of about 1 L/min. The temperature was set to about 550 ºC with a heating rate of about 25 ºC/min. A chiller unit was connected to supply cold water at about 8 ºC to condense aerosols generated during the reaction.
The pyrolysis was followed by gravity separation to obtain 2 separate phases (an aqueous phase and a bio-oil phase). The aqueous phase was lyophilised to obtain a lyophilised aqueous (L-AQ) phase of the wheat biomass.
Synthesis of the Formulations
As shown below, the disinfectant formulations were synthesised, by adding different concentrations of the L-AQ, sodium lauryl sulphate, ethanol, and EDTA one after the other, followed by simple mixing to attain a homogeneous solution.
For example: about 5% SLS was prepared by mixing about 5 grams of the SLS in about 100 mL of water; about 1% EDTA was prepared by mixing about 1 gram of the EDTA in about 100 mL of water; about 5% ethanol was prepared by mixing about 5 mL of the ethanol in about 95 mL of water; and about 1% L-AQ was prepared by mixing about 1 mL of the L-AQ in about 99 mL of water.
Components Combination 1 Combination 2 Combination 3
Sodium Lauryl Sulfate 5% 10% 15%
Ethanol 5% 20% 35%
EDTA 1% 3% 5%
L-AQ 1% 1% 1%
Water Makeup to 100% Makeup to 100% Makeup to 100%
pH 8 8 8
It was observed that the combination 1 was homogenous and stable, whereas in the combinations 2 and 3, the SLS was saturated and started precipitating out. Therefore, the combination 1 was used for further analyses.
Microbial Culture Conditions
Clinical isolates of Acinetobacter baumannii (AB2) (GenBank Accession No. MF443128), methicillin resistant Staphylococcus aureus (GSA)-44 GenBank Accession No. JN315148); and Candida auris (GenBank Accession ID MK108049) strains were used. At a cell density of about 107 CFU/mL, the MRSA, A. baumannii, and C. auris strains were added in a ratio of about 1:1:1.
 
Minimum Inhibitory Concentration (MIC) Analyses
MIC assays were performed to determine the lowest concentration of the L-AQ, which prevents the growth of the pathogens.
As illustrated, in Figure 1, about 100 µL of fresh media (SCD, YPD, and RPMI), about 50 µL of cultured strains, and about 50 µL of the L-AQ of varying dilutions from about 0.1% to about 1% (v/v) were added in a 96-well plate, and incubated for about 24 hours at about 37ºC.
About 30 µL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) dye was added to the plate and kept at dark conditions for about 30 minutes for metabolic reactions to take place.
As illustrated, in Figure 1, purple colour formation showed metabolically active cells, whereas no colour change indicated dead cells. The L-AQ showed a MIC of about 0.7%, about 0.8%, about 0.5%, and about 1% for A. baumannii, MRSA, C. auris, and mixed species, respectively.
Minimum Biofilm Eradication Concentration (MBEC) Analyses
About 100 µL of media (SCD, YPD, and RPMI) was added with about 50 µL of the cultured strains, in a 96-well plate. The plate was incubated for about 24 hours at about 37ºC. The next day, planktonic cells were removed without disturbing biofilms.
Fresh media was supplemented at various concentrations (from about 0.1% to about 1 % (v/v)) of the L-AQ and the plate was further incubated at about 37ºC for about 24 hours.
After incubation, spent media was removed and the wells were washed with distilled water to remove the planktonic cells. The biofilms were stained with about 0.4% crystal violet (CV) for about 30 minutes. The CV was removed and excess stains were washed with water.
The wells were then solubilised with about 70% ethanol and absorbance was recorded using an ELISA plate reader at about 595 nm. The wells with only cells served as control. Biofilm eradication was then calculated using the equation below (OD stands for Optical Density),
Percentage biofilm eradication (%)=(Control OD-Sample OD)/(Control OD)*100
As illustrated, in Figure 2A, Figure 2B, and Figure 2C, about 0.1% of the L-AQ eradicated about 80% of A. baumannii, about 65% of MRSA, and about 65% of C. auris biofilms. As illustrated, in Figure 2D, about 0.4% of the L-AQ eradicated about 60% of the mixed species biofilms.
Phenol Coefficient Analyses
Efficacy of the synthesised formulation was analysed by Rideal Walker phenol coefficient test. About 5% (w/v) of phenol, about 5% (w/v) of a commercial formulation, about 5% (w/v) of the synthesised formulation, and about 5% (w/v) of the L-AQ were diluted in water.
About 100 µL of the cultured strains were added to: about 1 mL of a diluted phenol solution, about 1 mL of the diluted commercial formulation, about 1 mL of the diluted synthesised formulation, about 1 mL of the diluted L-AQ for a contact time of about 10s, about 5 min, about 10 min, and about 15 min.
The plates were incubated for about 48 hours at about 37ºC. Following incubation, cell growth was observed and recorded. The phenol coefficient was calculated by the below formula,
Phenol coefficient = (Test dilution)/(Phenol dilution )
The results are illustrated, in Figure 3A, Figure 3B, Figure 3C, Figure 3D, and the below tables. "+" designation represents growth and "-" designation represents no growth.
 
MRSA

A. baumannii

C. auris

 
Mixed Species

A person skilled in the art will appreciate the fact that, though the commercial formulation yields results across a broader range of dilutions, the commercial formulation suffers from various drawbacks, which have been outlined in the "Background of the Invention" subhead.
As illustrated, in Figure 4D and the table below, the phenol coefficient of the L-AQ was less than 1. Upon addition of excipients (sodium lauryl sulphate, ethanol, and ethylenediaminetetraacetic acid), the phenol coefficient was observed to be greater than 1, indicating a synergistic effect of the L-AQ with the excipients.
Phenol Coefficient Value MRSA A. baumannii C. auris Mixed Species.
Formulated Disinfectant 16 8 2 2
L-AQ 0.25 0.25 0.25 0.25
Biofilm Disrupting Efficacy Analyses
Biofilm disrupting efficacy of the synthesised formulation was analysed by CDC biofilm reactor. Testing material used was stainless steel coupons used in hospital contact surfaces. The disinfectant efficacy against the biofilms was tested as per ASTM E2871 (ASTM International, (2018)).
Overnight cultures of about 107 strains of MRSA, A. baumannii, and C. auris were inoculated in respective autoclaved media and incubated at about 37 ºC under dynamic shaking conditions at about 150 rpm for about 24 hours to form biofilms on the stainless steel coupons.
Said steel coupons were taken out the next day, followed by rinsing in distilled water to remove loosely adhering planktonic cells and immersing in about 4 mL of the synthesised formulation for a contact time of about 10 seconds.
Then, the steel coupons were immersed in PBS (about 1X) and subjected to vortexing for about 30 seconds, followed by sonication at about 50 kHZ and a final vortex for about 30 seconds to disintegrate the biofilms attached to the coupons.
The steel coupons without treatment with disinfectant served as a control. The biofilm cells were serially diluted and plated to enumerate the CFU present.
Log CFU/mL = (No.of colonies)/(Volume plated)*dilution factor
Log reduction =(Control CFU-Treated CFU)/(Control CFU)*100
As illustrated in the below table, it was observed that there were no cells present, when disinfected with the synthesised formulation, which clearly indicated that the synthesised formulation was not only effective on the planktonic cells, but also effective on the biofilm cells.
S. No. Test Pathogen Control (x 106 CFUs/mL) Treated- Commercial (CFU/mL) Log reduction (%) Treated- Formulation (CFU/mL) Log Reduction (%)
1. MRSA 20±2 Nil 100 Nil 100
2. A. baumannii 14±2 Nil 100 Nil 100
3. C. auris 8±1 Nil 100 Nil 100
4. Mixed sp. 6±2 (SCD)
1.06±1(YPD) Nil 100 Nil 100
From the above table, it is clear that, though the synthesised formulation does not comprise any hazardous chemicals, its efficacy is comparable to that of commercially available disinfectants.
The disclosed disinfectant formulation offers at least the following synergistic advantages and effects: improved efficacy (or an efficacy that is comparable to that of commercially available disinfectants); effective against biofilms; reduced risk of complications; and/or lower incidence of side effects or allergies.
It will be apparent to a person skilled in the art that the above description is for illustrative purposes only and should not be considered as limiting. Various modifications, additions, alterations, and improvements, without deviating from the spirit and the scope of the disclosure, may be made, by a person skilled in the art. Such modifications, additions, alterations, and improvements, should be construed as being within the scope of this disclosure.
, Claims:1. A synergistic disinfectant formulation, for nosocomial infections, comprising: L-aqueous phase of wheat straw biomass; sodium lauryl sulphate; ethanol; and ethylenediaminetetraacetic acid, with:
said L-aqueous phase of wheat straw biomass being in a ratio of 1:5 with said sodium lauryl sulphate;
said L-aqueous phase of wheat straw biomass being in a ratio of 1:5 with said ethanol; and
said L-aqueous phase of wheat straw biomass being in a ratio of 1:1 with said ethylenediaminetetraacetic acid.
2. The synergistic formulation, for nosocomial infections, as claimed in claim 1, wherein:
concentration of said L-aqueous phase of wheat straw biomass is 1%;
concentration of said sodium lauryl sulfate is 5%;
concentration of said ethanol is 5%; and
concentration of said ethylenediaminetetraacetic acid is 1%.

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