FIELD OF INVENTION

The invention relates to field of biotechnology particularly to an antibiofouling composition
and the method of dissolution of microorganismal biofilms.
BACKGROUND OF THE INVENTION
Biofilm can be broadly defined as aggregates or colonies of microorganism in which the
microbial cells are embedded in a self-produced matrix, the matrix can is termed as
extracellular polymeric substances (ECS). The aggregates or the colonies in a biofilm either
are adherent to each other and also to the surface on which the biofilm is formed. The ECS
matrix has a complex composition which is composed of a number of substances produced by
the biofilm-forming microorganism itself such as exopolysaccharides (EPS), extracellular
DNA (e-DNA), extracellular RNA (e-RNA), proteins and amyloidogenic proteins. The
exopolysaccharides (EPS) self-organization determines the inter-molecular interactions and the
mechanical properties of the ECS. The main component of the matrix is water which is up to
97% and contains the structural and functional components of the matrix: soluble, gel-forming
polysaccharides, proteins and eDNA. The matrix also contains the insoluble components such
as amyloids, cellulose, fimbriae, pili and flagella, all of which form a part of the ECS matrix.
The matrix also contains pores and channels between microcolonies that form voids in the
matrix. Furthermore, cationic exopolysaccharide has been shown to crosslinks eDNA to
provide structural integrity to the matrix.
Biofilm of microorganisms display a totally different property than the planktonic bacteria (free
bacteria), this is primarily due to the fact that the microorganism in a biofilm live in totally
different microenvironment as compared to planktonic bacteria. The microenvironment of
biofilms is composed of heterogenous microorganismal population as biofilms may consists of
many species of microorganism. Biofilms display a heterogeneous physiological activity which
leads to steep gradients of electron acceptors and donors, pH value and redox conditions. Even
in mono species biofilms heterogeneity is observed due to phenotypic variation that arises from
fluctuating gene expression over time in individual cells and differential gene expression
between different cells. Therefore, localized physiological activity of microbial cells in a
biofilm which are immobilized in the matrix and are also spatially separated, contributes to the
formation of gradients and other spatial heterogeneities, which results in multilayered biofilms
such as microbial mats or flocs. Biofilms therefore display unique properties such as
desiccation tolerance, antibiotic resistance. Antibiotic resistance can be due to the complex
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process such as quenching the activity of antimicrobial substances that diffuse through the
biofilm in a form of inhibition known as diffusion–reaction inhibition or resistance of cells in
the biofilm to antimicrobials can be enhanced by uptake of resistance genes by horizontal gene
transfer. As a result the microorganism within biofilms have shown 10–1000 times more
antibiotics resistance than the planktonic cells.
Biofilms can be regarded as a most widely distributed and successful mode of survival of the
microorganisms forming the biofilm. Owing the versatility of biofilms, they derive a number
of important processes in the nature such as biogeochemical cycling processes of most elements
in water, soil, sediments and subsurface environments.
Also biofilms are known to be associated with persistent infections in plants and animals,
including humans. Approximately 80% of chronic and recurrent microbial infections in the
human body are due to bacterial biofilm. Biofilms are known to form on biotic surfaces such
as dental plaque, periodontitis, cystic fibrosis lung infections, chronic wounds, soft tissue
fillers, otitis media, chronic osteomyelitis, chronic rhinosinusitis, psoriasis, endocarditis,
urinary tract infections, human gastrointestinal tract and can contribute to the process of
infection. For example it has been reported that in ulcerative colitis and other inflammatory
bowel diseases, biofilms have been described covering the entire mucosa and entering the
crypts, mainly comprising of Bacteroides fragilis. On the other hand, in healthy subjects these
biofilm-like structures were not found, or if detected, was up to 100-fold lower amounts of
cells. In addition to this, growth of the biofilm on medical devices including implants,
catheters, artificial heart valves, teeth, contact lenses can pose a considerable challenge for the
management of the conditions which requires replacements of these medical implants. The
biofilms growing on a medical devices such as implants can easily spread in the body through
urinary tract and the blood stream. This leaves the doctors with few alternatives such as
replacing implants, which adds to the cost and also to inconvenience of the patients. It can also
lead to developing of chronic infection and risk a patient’s life.
Recently, Bacterial membrane vesicles are widely accepted as bacterial secretion system known
as membrane vesicles (MVs). It is produced by both gram positive & gram negative bacteria.
These MVs carries various cargo molecules like lipopolysaccharides, Peptidoglycan ,
periplasmic & cytoplasmic proteins, toxins and nucleic acids. Bacterial MVs are proven to be
safe & effective & also serving as promising agent for development of new therapies. MVs
protects the cargo inside from degradation due to action of nucleases and proteases. Number of
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qualities are associated with bacterial MVs which can be used therapeutically. However certain
limitation with the usage as vehicles in delivering of drugs, is that MVs can be stored in liquid
form at 4? for about year and at 37? not more than 3 months
Secretion of vesicles from gram positive bacteria is MVs and from gram negative bacteria is
OMVs commonly known as EVs (extracellular vesicles) defines as spherical, membranous
vesicle generated from a microbial cell surface with size ranging from 20-500 nm in diameter.
MVs release from gram positive bacteria is a quite complex process due to presence of thick
peptidoglycan layer having pore size of 2nm restricts the release of MVs 20-400nm in size.
Release of MVs of gram positive bacteria is only possible through the disruption of crosslink
of NAM & NAGpeptidoglycan through the presence of degrading enzyme & surfactant
proteins. It involves diversified events depending on cell lytic enzyme viz peptidoglycan
degradation followed by cytoplasmic membrane bleb protrusion via endolysin & peptidoglycan
remodelling via hydrolysing enzyme autolysins. In Bacillus, phage encoded endolysin
promotes pore formation in peptidoglycan layer and facilitates the release of MVs.
Dorward & Garon, 1990 provided 1
st evidence for release of MVs from gram positive bacteria
of size ranging from 10-500nm comprising simple architecture containing cell membrane &
cytoplasm. MVs of gram positive bacteria contain fatty acids, phospholipids, cytoplasmic
proteins, membrane associated virulence proteins, lipoteichoic acid, peptidoglycan, ex-DNA
and ex-RNA.
As outlined above existence of biofilms by disease causing microorganism is well-recognized
now and therefore efforts are being made at possible ways to contain it. A few of the prior art
have reported methods/ antibiofouling agents for dissolution of biofilms as under;
US9314479B2 published on 19
th April 2016 discloses a method of inhibiting biofilm
formation, comprising administering to a subject a polyamino acid selected from the group
consisting of polyaspartic acid and polyglutamic acid, wherein the polyamino acid is
administered directly to or proximal to the site of biofilm formation. The polyamino acid
disclosed in the invention is between 50 and 300 amino acids in length. The invention also
disclose protease inhibitor and anti-DNA compound as a part of method for inhibition of
biofilm formation. The invention is particularly directed towards cystic fibrosis and persistent
neutrophil accumulation in cystic fibrosis and aims to inhibit the adherence of, or response to
chemoattractants by neutrophils, or inhibits cytokine, chemokine or chemoattractant that
attracts or enhances neutrophil activity.
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Similarly, US9809477 published on 13th August, 2015 discloses a method for preventing
biofilm formation on a surface, comprising subjecting the surface to one or more bacterial
strains. The bacterial strains used in the application is Bacillus blend. The invention also
discloses the use of enzymes such as alpha-amylases, cellulases, lipases, mannanases, oxidases,
pectate lyases, peroxidases, and proteases, or a mixture thereof in the antifouling composition.
Further the application discloses the use of the method mostly on abiotic surfaces.
EP2533801 published on 19th December, 2012 discloses pharmaceutical or anti-biofouling
composition for disrupting a biofilm or preventing biofilm formation comprising an isolated
microbial deoxyribonuclease polypeptide and an excipient, wherein the microbial
deoxyribonuclease is a class Bacillus bacterial deoxyribonuclease. The patent therefore teaches
use of DNAase for disruption of the biofilms. The invention is based on the findings that DNA
and RNA a structural components of biofilm and therefore the inventors employed DNAase for
disruption of biofilms. The DNAase used in the invention is derived from Bacillus and is a
bacterial deoxyribonuclease. The composition can be used on dental surfaces as in a
mouthwash, dental paste, liquid dentifrice, mouthwash, gingival massage ointment etc in
humans and in wide range of human diseases. The composition can be used on medical
implants/catheters.
The prior art therefore discloses some of the antibiofouling compounds, however there is still a
need to develop effective antibiofouling composition that can be scaled up to the industrial level
with ease at an effective cost and have better and more effective mechanism for dissolution of
the biofilms.
OBJECT OF THE INVENTION
The object of the present invention is to develop anti-filming agent for dissolution of
microorganismal biofilm.
Another object of the present invention is to develop composition for dissolution of
microorganismal biofilm that is easy to use, economical and effective in dissolution of biofilms
effectively from biotic surfaces.
A still further object of the present invention is to develop a method of dissolution/elimination
of microorganismal biofilm by administering a suitable dose of anti-filming composition antifilming agent
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SUMMARY OF THE INVENTION
This summary is provided to introduce a selection of concepts in a simplified format that is
further described in the detailed description of the invention. This summary is neither intended
to identify key or essential inventive concepts of the invention and nor is it intended for
determining the scope of the invention.
The present invention discloses an anti-filming agent for dissolution of microorganismal
biofilm, wherein the anti-filming agent is extracellular-ribonucleic acid (ex-RNA).
The present invention discloses an anti-filming composition for dissolution of microorganismal
biofilm comprising; extracellular-ribonucleic acid (ex-RNA) is encapsulated in a extra cellular
vesicles and is dispensed in suitable pharmaceutical excipients.
The present invention discloses a method of dissolution/elimination of microorganismal
biofilm by administering a suitable dose of anti-filming composition containing anti-filming
agent, wherein the anti-filming agent is extracellular-ribonucleic acid (ex-RNA).
BRIEF DESCRIPTION OF FIGURES:
These and other features, aspects, and advantages of the present invention will become better
understood when the following detailed description is read with reference to the accompanying
figures in which like characters represent like parts throughout the figures, wherein:
Figure 1A depicts optical density obtained by reading 96-well microtiter plates of control
versus Bacillus sp., Streptococcus pneumoniae and Pseudomonas aeruginosa bacterial strains
showing growth of the bacteria in microtiter plate;
Figure 1B depicts the OD values in a bar graph of the microtiter plate;
Figure 2 depicts biofilm formation on a petri dish;
Figure 3 depicts biofilm of Pseudomonas aeruginosa treated with (A) Distilled water (negative
control); (B) Vancomycin (Positive control), (C) Combination 2- Rnase (ng/ml)+
antibiofouling M9 composition, (D) Combination 3- DNase (ng/ml)+ Rnase (ng/ml)+
antibiofouling M9 composition (E) Composition M9 - antibiofouling composition of the
present invention (extracellular ribonucleic acid (exRNA) encapsulated in extracellular
vesicles 6µg/ml; phosphate buffer saline in the range of 0.5%; sodium chloride 0.9%; glycerin
in the range of 4%.; and double distilled water 20%) of the present invention, as is clear from
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the figure, a zone of inhibition of 2.5 cm is clearly visible with the Composition M9 -
antibiofouling M9 composition of the present invention, whereas no inhibition is seen in any
of the plates from A-E.
Figure 4 depicts biofilm of Streptococcus pneumoniae treated with (A) Distilled water
(negative control); (B) Vancomycin (Positive control), (C) Combination 2- Rnase (ng/ml)+
antibiofouling M9 composition, (D) Combination 3- DNase (ng/ml)+ antibiofouling M9
composition+ Rnase (ng/ml) (E) Composition M9 - antibiofouling composition of the present
invention (extracellular ribonucleic acid (exRNA) encapsulated in extracellular vesicles
6µg/ml; phosphate buffer saline in the range of 0.5%; sodium chloride 0.9%; glycerin in the
range of 4%.; and double distilled water 20%) of the present invention, as is clear from the
figure, a zone of inhibition of 3 cm is clearly visible with the Composition M9 - antibiofouling
composition of the present invention, whereas no inhibition is seen in any of the plates from
A-E;
Figure 5 depicts biofilm of Staphylococcus aureus treated with (A) Distilled water (negative
control), (B) Combination 2- Rnase (ng/ml)+ antibiofouling M9 composition (C) Composition
M9 - antibiofouling composition of the present invention (extracellular ribonucleic acid
(exRNA) encapsulated in extracellular vesicles 6µg/ml; phosphate buffer saline in the range of
0.5%; sodium chloride 0.9%; glycerin in the range of 4%.; and double distilled water 20%),
(D) Vancomycin (Positive control), as is clear from the figure, a zone of inhibition of 3 cm is
clearly visible with the Composition M9 - antibiofouling composition of the present invention
whereas no inhibition is seen in any of the plates from A-D;
Figure 6 depicts the biofilm formation by Mutant P. aeruginosa?relA/?spoT and the
inhibition of the biofilm by the composition of the present invention, Figure 6(a) depicts no
inhibition zone in distilled water (negative control), vancomycin (positive control) and Figure
6 (b) shows composition zone of inhibition of 3 cm of the present invention;
Figure 7 depicts Rel A gene degradation by positive control vancomycin (A); distilled water
(B); M9 Composition (C) and C-DNA sample of RelA without M9 Composition/ vancomycin
(D); and
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Figure 8 depicts size distribution by intensity reports wherein the extra cellular vesicles is
revealed to be an anionic molecule with a zeta potential (-15) to (-20) mV and 100-1000nm.
Further, skilled artisans will appreciate that elements in the figures are illustrated for simplicity
and may not have been necessarily been drawn to scale. For example, the flow charts illustrate
the method in terms of the most prominent steps involved to help to improve understanding of
aspects of the present invention. Furthermore, in terms of the construction of the device, one
or more components of the device may have been represented in the figures by conventional
symbols, and the figures may show only those specific details that are pertinent to
understanding the embodiments of the present invention so as not to obscure the figures with
details that will be readily apparent to those of ordinary skill in the art having benefit of the
description herein.
DETAILED DESCRIPTION:
For the purpose of promoting an understanding of the principles of the invention, reference will
now be made to the embodiment illustrated in the figures and specific language will be used to
describe the same. It will nevertheless be understood that no limitation of the scope of the
invention is thereby intended, such alterations and further modifications in the illustrated
system, and such further applications of the principles of the invention as illustrated therein
being contemplated as would normally occur to one skilled in the art to which the invention
relates. It will be understood by those skilled in the art that the foregoing general description
and the following detailed description are exemplary and explanatory of the invention and are
not intended to be restrictive thereof.
Reference throughout this specification to “an aspect”, “another aspect” or similar language
means that a particular feature, structure, or characteristic described in connection with the
embodiment is included in at least one embodiment of the present invention.
Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar
language throughout this specification may, but do not necessarily, all refer to the same
embodiment The terms "comprises", "comprising", or any other variations thereof, are intended
to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps
does not include only those steps but may include other steps not expressly listed or inherent
to such process or method.
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Similarly, one or more devices or sub-systems or elements or structures or components
proceeded by "comprises... a" does not, without more constraints, preclude the existence of
other devices or other sub-systems or other elements or other structures or other components
or additional devices or additional sub-systems or additional elements or additional structures
or additional components. Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary skill in the art to
which this invention belongs. The system, methods, and examples provided herein are
illustrative only and not intended to be limiting.
Extracellular-ribonucleic acid (ex-RNA) may be defined as regulatory RNAs of about 40–100
nucleotides in length, derived from bacteria and binds to target mRNAs or proteins;
Extra cellular vesicles may be defined as extracellular vesicles and the multivesicular body
(MVB), with the plasma membrane;
Encapsulation may be defined as coating or engulfing the extracellular-ribonucleic acid (exRNA) molecule in extra cellular vesicles;
Pharmaceutical excipients may be defined as inert diluents, such as calcium carbonate, sodium
carbonate, lactose, sucrose, glucose, mannitol, cellulose, starch, calcium phosphate, sodium
phosphate, kaolin and the like. Binding agents, buffering agents, and/or lubricating agents may
also be used. Tablets and pills can additionally be prepared with enteric coatings. The
composition may optionally contain sweetening, flavoring, coloring, perfuming, and
preserving agents in order to provide a more palatable preparation.
Biofilms are known to show marked recalcitrance to the known antibiotics and can be cause of
a number of difficult to treat bacterial infections. The present invention discloses an antifilming agent for dissolution of microorganismal biofilm, wherein the anti-filming agent is
extracellular-ribonucleic acid (ex-RNA). RNA-based therapeutics can be an RNA molecules
or analogs directly used as therapeutic drugs or a RNA-targeted small-molecule medications.
The RNA-based therapeutics may be RNA aptamers (e.g., pegaptanib) which are RNA
oligonucleotides that bind to a specific target with high affinity and specificity. Similarly,
RNA-based therapeutics also include ASOs or antisense, microRNA (miRNA), short or small
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hairpin RNAs (shRNAs), guideRNA (gRNA) and small interferingRNA (siRNAs).
Ribozymes are a specific group of RNA molecules that cleaves target RNAs in specific
sequences via hammerhead or hairpin structures.
The RNAs are prone to catabolism by serum RNases and are required to pass the cellular
membrane barriers to access intracellular targets. RNA aptamers may directly bind to
extracellular or may target cell surface, or intracellular proteins. Similarly, ASOs, siRNAs, and
miRNA mimics may be delivered into cells to target intracellular mRNAs or functional noncoding (ncRNAs) through complementary base pairings, leading to gene silencing or control
of gene expression for the treatment of diseases.
RNA drugs may be “actively delivered” to targeted cells or tissues through
encapsulation/formulation with specific materials or carried by viral vectors, plasmid DNA
(pDNAs) or intact cells. If the RNA molecule is to be carried by a viral vector it may necessitate
use of a DNA/gene materials or engineered cells, whereas encapsulation/formulation with
specific materials keeps the RNA substances as the active ingredients inside a carrier vehicle
such as extra cellular vesicles. A nanoparticle encapsulation of RNA is a commonly used
method for RNA drug to be delivered at the therapeutic site. The encapsulation of RNA in a
nanoparticle physically protects RNA from degradation by RNase and facilitate cellular uptake
and endosomal escape. Cationic polymers are used to electrostatically condense the negatively
charged RNA into nanoparticles and can be used for delivery such as poly-L-lysine,
polyamidoamine, polyethyleneimine, and chitosan.
Lipids and lipid-like materials may be used as nanoparticle-based delivery vehicles for RNA.
The cationic lipids are often used to electrostatically bind the nucleic acid. The lipids that are
positively charged only at acidic pH (ionizable lipids) are used as a RNA drug delivery
mechanism. The ionizable lipids enhance efficacy through helping with endosomal escape and
reduces toxicity. Lipids are also capable through electrostatic interactions with RNA and
hydrophobic interactions self-assemble into well-ordered nanoparticle structures. These
structures are known as lipoplexes. Similarly, lipid nanoparticles (LNPs) are achieved by
adding hydrophobic moieties, such as cholesterol and PEG-lipid for enhancing nanoparticle
stability. Conjugation of a bioactive ligand such as N-acetylgalactosamine ,to the RNA allow
it to enter the cell of interest.
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Both prokaryotic and eukaryotic cells secrete exosomes. In prokaryotes, both gram positive
and gram negative bacteria secrete exosomes. In addition, extra cellular vesicles are nanosized
vesicles secreted by a variety of cells, such as immune system. Extra cellular vesicles are
known to participate in long-distance intercellular communications facilitating transfer of
proteins, functional mRNAs and microRNAs. Extra cellular vesicles possess an intrinsic ability
to cross biological barriers. The extra cellular vesicles therefore can be effectively used for
delivering the RNA drugs to target cell. In one embodiment the present invention discloses an
anti-filming composition for dissolution of microorganismal biofilm comprising; extracellularribonucleic acid (ex-RNA); extra cellular vesicles; and other excipients.
The present invention discloses a method of dissolution/elimination of microorganismal
biofilm by administering a suitable dose of anti-filming composition, wherein the anti-filming
agent is extra cellular-ribonucleic acid (ex-RNA).
The present invention discloses a method of dissolution/elimination of microorganismal
biofilm as claimed in claim 1, wherein the anti-filming composition comprises; extracellular
ribonucleic acid (exRNA) and is in the range of 4-12µg/ml; extracellular vesicle is bound by a
single membrane phospholipid layer comprising glycopeptides; lipopeptide; polysaccharides;
short chain fatty acids including propionic acid and small peptides, encapsulating extracellular
ribonucleic acid (exRNA); phosphate buffer saline in the range of 0.4-1%; sodium chloride
0.9%; glycerin in the range of 1-5%.; and double distilled water.
The present invention discloses a method of dissolution/elimination of microorganismal
biofilm, wherein the anti-filming composition comprises extracellular ribonucleic acid
(exRNA); phosphate buffer saline in the range of 0.4-1%; sodium chloride 0.9%; glycerin in
the range of 1-5%.; and double distilled water 10-20%, wherein the extracellular ribonucleic
acid (exRNA) is encapsulated in extracellular vesicle (EV), wherein the extracellular
ribonucleic acid (exRNA) and extracellular vesicle (EV) are derived from extra polymeric
substances (EPS) from gram positive bacteria, wherein extra polymeric substances (EPS) is
derived is from gram positive non-pathogenic bacteria.
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In one embodiment the anti-filming agent may be encapsulated in a multivesicular body such
as extra cellular vesicles.
In the present invention, the mechanism of action of the ani-filming agent may be due to the
inhibition of the m-RNA. The anti-filming agent being encapsulated in a extra cellular vesicles.
The fusion of extra cellular vesicles with the plasma membrane of the bacterial cells facilitates
the release of extra cellular vesicles into the extracellular space. The extracellular- RNA is then
released into the extracellular space of the bacterial cell. The anti-filming agent\ extracellularribonucleic acid (ex-RNA) then acts by inhibiting the messenger-ribonucleic acid (m-RNA) of
the bacteria either by antisense technique or by ribozymal cleavage, this in turn results in killing
of bacteria entrapped in the microorganismal biofilm. This mechanism of action eventually
leads to dissolution of the microorganismal biofilm.
In one embodiment the extracellular vesicle is bound by a single membrane phospholipid layer
comprising glycopeptides; lipopeptide; polysaccharides; short chain fatty acids including
propionic acid and small peptides and extracellular vesicle encapsulates extracellular
ribonucleic acid (exRNA) and is in the range of 4-12µg/ml.
In one embodiment, the extracellular ribonucleic acid (exRNA) inhibits the messengerribonucleic acid (m-RNA) by antisense/ribozymal cleavage.
In still another embodiment, the extracellular ribonucleic acid (exRNA) is selected from a
group comprising micro Ribonucleic acid (miRNA), small interfering Ribonucleic acid
(siRNA), guide Ribonucleic acid (gRNA) cRNA (circular Ribonucleic acid), piwi Ribonucleic
acid (piRNA).
The extra cellular vesicles which encapsulates the anti-filming agent is composed of single
membrane phospholipid layer; lipopeptide; small peptides; and polysaccharides. Further in still
another embodiment the extra cellular vesicles of the present invention is anionic molecule
with a zeta potential (-15) to (-20)mV and 100-1000 nm in size.
Alternatively, the compositions can be administered by oral ingestion. Compositions intended
for oral use can be prepared in solid or liquid forms, according to any method known to the art
for the manufacture of pharmaceutical compositions.
The excipients used in the composition may be admixed with non-toxic pharmaceutically
acceptable excipients. These include, for example, inert diluents, such as calcium carbonate,
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sodium carbonate, lactose, sucrose, glucose, mannitol, cellulose, starch, calcium phosphate,
sodium phosphate, kaolin and the like. Binding agents, buffering agents, and/or lubricating
agents may also be used. Tablets and pills can additionally be prepared with enteric coatings.
The composition may optionally contain sweetening, flavoring, coloring, perfuming, and
preserving agents in order to provide a more palatable preparation.
The composition of the present invention may be administered parenterally (e.g., by
intramuscular, intraperitoneal, intravenous, intraocular, intravitreal, subconjunctival, ,
subcutaneous injection or implant or may be given systemically.
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and
granules In another embodiment the anti-filming composition is formulated as a powder, a
liquid, a solution, a cream, a gel or a paste.
The anti-filming composition of the present invention may further comprise antifungal agent,
antibacterial compound, antiviral compound, antiparasitic compound.
In still another embodiment the present application discloses a method of
dissolution/elimination of microorganismal biofilm by administering a suitable dose of antifilming composition anti-filming agent, wherein the anti-filming agent is extracellularribonucleic acid (ex-RNA).
The microorganismal biofilm may form on a biotic surfaces. The biotic surfaces may be
selected from a group comprising lung tissue, teeth surfaces; skin surface, tonsils, hair scalp;
biofilms formed in chronic infections such as dental plaque, periodontitis, cystic fibrosis lung
infections, chronic wounds, soft tissue fillers, otitis media, chronic osteomyelitis, chronic
rhinosinusitis, psoriasis, endocarditis, urinary tract infections, human gastrointestinal tract.
The antibacterial compound may be selected from aminoglycosides, carbapenems,
cephalosporins, fluoroquinolones, glycopeptides and lipoglycopeptides (such as vancomycin),
macrolides (such as erythromycin and azithromycin), monobactams, oxazolidinones,
penicillin, polypeptides, rifamycin, sulphonamides, streptogramins (such as quinupristin and
dalfopristin), tetracyclines, carbapenems, cephalosporins, monobactams, penicillins,
chloramphenicol, clindamycin, daptomycin, fosfomycin, lefamulin, metronidazole, mupirocin
, nitrofurantoin, and tigecycline.
The antifungal compound may be selected from polyene antifungal drugs such as amphotericin,
nystatin, pimaricin, azole antifungal drugs- fluconazole, itraconazole, and ketoconazole
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allylamine and morpholine antifungal drugs- allylamines (naftifine, terbinafine), antimetabolite
antifungal drugs-5-Fluorocytosine.
The antiviral compounds may be selected from Acyclovir, Adefovir, Amantadine, Ampligen,
Amprenavir, Atazanavir, Atripla, Baloxavir Marboxil, Biktarvy, Bulevirtide, Cidofovir,
Cobicistat, Combivir, Daclatasvir (Daklinza), Darunavir, Delavirdine, Descovy,
Didanosine, Docosanol, Dolutegravir, Doravirine, Edoxudine, Efavirenz, Elvitegravir,
Emtricitabine, Enfuvirtide, Entecavir, Etravirine, Famciclovir, Fomivirsen, Fosamprenavir,
Foscarnet, Ganciclovir, Ibacitabine, Ibalizumab, Idoxuridine, Imiquimod, Imunovir, Indinavir,
Lamivudine, Letermovir, Loviride, Maraviroc, Methisazone, Moroxydine, Nelfinavir,
Nevirapine, Nexavir, Nitazoxanide, Norvir, Penciclovir, Peramivir, Penciclovir, Peramivir,
Pleconaril, Podophyllotoxin, Raltegravir, Remdesivir, Ribavirin, Rilpivirine, Rimantadine,
Ritonavir, Saquinavir, Simeprevir, Sofosbuvir, Stavudine, Taribavirin, Telaprevir,
Telbivudine, Tenofovir alafenamide, Tenofovir disoproxil, Tipranavir, Trifluridine, Trizivir,
Tromantadine, Truvada, Umifenovir, Valaciclovir, Valganciclovir, Vicriviroc, Vidarabine,
Zalcitabine, Zanamivir, Zidovudine.
The antiparasitic agent can be selected from Antiprotozoal Agents, Antimalarials, Antibabesial
agents, Antiamoebic agents, Antigiardial agents, Trypanocidal agents, Antileishmanial agents,
Anticestodal drugs, Antinematodal drugs, Antinematodal drugs, Ectoparasiticides,
Antiscabietic agents, Pediculicides.
EXAMPLE
The following examples are for illustration purposes and are not to be construed as limiting the
invention disclosed in this document to only the embodiments disclosed in these examples.
Example 1
Biofilm formation
Bacterial strains (Bacillus M9, Streptococcus pneumoniae and Pseudomonas aeruginosa) were
grown in Luria broth at 370C for 48-96 hours in a shaking incubator. This was followed by
inoculation of each bacterium in a 3-to-5-ml culture and grown to stationary phase. The cultures
were diluted 1:100 in the desired media and 100 µl of each diluted culture pipetted into each
of four wells in a fresh microtiter 96-well plate and incubated overnight. After incubation, the
contents of the wells are decanted into a discard container. Each well is washed three times
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with 200ul of sterile phosphate buffered Saline (PBS; pH 7.2) using micropipette to remove all
nonadherent cells while simultaneously providing preservation of the biofilm integrity. After
washing, the remaining attached bacteria should be heat fixed by exposing them to hot air at
600C for 60 min. The fixation is carried out by Bouin’s reagent. Following fixation plates are
treated with methanol for 20 min. This is followed emptying of microtiter plates by simple
flicking, and the microplates are left to air dry overnight in an inverted position at room
temperature.
The modified Christensen’s method includes resolubilization of the dye and measures the
biofilm formed both on the bottom and walls of the well for staining. The adherent biofilm
layer formed in each microtiter plate well is stained with 150 mL of crystal violet used for
Gram staining (2% Hucker crystal violet) for 15 min at room temperature. To demonstrate the
presence of slimy material, Alcian blue, which selectively stains acid mucopolysaccharides is
used. The plates are read with microplate reader spectrophotometer. Figure 1A show the optical
density obtained by reading 96-well microtiterplates of control versus Bacillus M9,
Streptococcus pneumoniae and Pseudomonas aeruginosa bacterial strains, Figure 1B depicts
the OD values in a bar graph,
Example 2
Composition of the present invention
Gram-positive non-pathogenic bacteria was mutated in lab through homologous recombination
method using a suitable bacteriophage strain, mutated bacteria was grown on a suitable culture
media for 22-24 hrs. at 37oC. After formation of the full grown biofilm on the medium. The
biofilm was scrapped from the surface and suspended in the conversion buffer to extract extra
polymeric substances (EPS) from the biofilm. The conversion buffer used for suspension of
extra polymeric substances (EPS) were carried out in 6 different solution- double distilled
water; 10% ethanol (10 ml ethanol in double distilled water); 5% Dimethyl sulfoxide (DMSO)
(5ml DMSO in double distilled water); 0.9% Sodium chloride (NaCl) 0.9gms NaCl dissolved
in 90ml double distilled water, making up the volume to 100ml; Phosphate buffer saline (PBS),
0.819 gms PBS dissolved in 90ml double distilled water, making up the volume to 100ml,
adjusting the pH to 7.4.
The homogenization of the mixture was carried out at room temperature (250C) for 1 minute
in a homogenizer (NAMCO, Asia 1936). The suspension obtained after the homogenization is
16
then centrifuged, (REMI- R8C) at 4500rpm for 5 mins. The supernatant obtained in then
filtered twice with membrane filter pore size, 0.45 µm followed by membrane filter pore
size, 0.22µm.
The anti-biofouling agent extracellular-ribonucleic acid by mixing extracellular ribonucleic acid
(exRNA) is in the range of 4-12µg/ml encapsulated in extracellular vesicle is bound by a single
membrane phospholipid layer comprising glycopeptides; lipopeptide; polysaccharides; short
chain fatty acids including propionic acid and small peptides, encapsulating extracellular
ribonucleic acid (exRNA); phosphate buffer saline in the range of 0.4-1%; sodium chloride
0.9%; glycerin in the range of 1-5% dissolved in double distilled water. This composition was
used to carry out the further experiments.
Example 3
Biofilm dissolution experiments
The gram-negative bacterial pathogen Pseudomonas aeruginosa is a prominent clinical
concern. The culture was prepared by streaking a loopful form a fresh culture of Pseudomonas
aeruginosa into 25ml of King’s Medium on a Petri dish were incubated at 370C for 24 hours
and allowed development of biofilm.
Streptococcus pneumoniae (pneumococcus) is a major gram-positive human pathogen and
currently the leading cause of community-acquired pneumonia, meningitis, and bloodstream
infections in the elderly, the young, and patients with immunosuppressive illness and chronic
diseases. Streptococcus pneumonia was grown on Nutrient Agar Medium (NAM) containing
Peptone; sodium chloride; meat extract; yeast extract; agar with pH was maintained at 7.4 (at
250C). Streptococcus pneumonia was cultured at 30-37°C for 24 hours. After 24 h we see
fully grown biofilm on the plate.
Staphylococcus aureus it’s grown on soya agar medium and cultured 30-37°C for 24 hours.
After 24 hours, fully grown biofilm on the plate was observed. .
In the test plates, wholes were punched in the petri dish and the following combination were
added:
17
Table 1: Different combination testing against the bacterial pathogens
Combination Pseudomonas
aeruginosa
Streptococcus
pneumonia
Staphylococcus
aureus
Antibiofouling
composition M9 -
extracellular
ribonucleic acid
(exRNA)
encapsulated in
extracellular vesicles
6µg/ml; phosphate
buffer saline in the
range of 0.5%;
sodium chloride
0.9%; glycerin in the
range of 4%.; and
double distilled
water 20%.
+++ +++ +++
Combination 2-
Rnase (ng/ml)+
antibiofouling M9
composition
_ _ _
Combination 3-
DNase (ng/ml)+
antibiofouling M9
composition
_ _ _
Combination 4-
DNase (ng/ml)+
Rnase (ng/ml)+
antibiofouling M9
composition
_ _ _
Vancomycin
(ng/ml)- positive
control
_ _ _
D.W (negative
control) (1ml)
_ _ _
+++ = significant inhibition observed
- = No inhibition
18
Figure 3, 4, 5 clearly indicates the composition of the present invention clearly show a zone of
inhibition varying from 2.5 cm to 3 cm, clearly showing evidence of the composition of the
present invention inhibiting the microbial film formed by the bacterial pathogens including but
not limited to Pseudomonas aeruginosa, Streptococcus pneumonia, Staphylococcus aureus.
The zone of inhibition was detected with the composition of the present invention against all
three bacterial pathogens. The absence of zone of inhibition with enzyme RNase and DNase
show that the antibiofouling agent is an ribonucleic acid molecule. Moreover, no zone of
inhibition was observed with negative control i.e. distilled water and the antibiotic -
vancomycin.
Vancomycin is a glycopeptide antibiotic used to treat severe but susceptible bacterial infections
such as MRSA (methicillin-resistant Staphylococcus aureus) infections. Clearly, the
experiment shows that vancomycin is not able to inhibit the resistant biofilms formed by the
pathogenic bacteria pseudomonal aeruginosa, Streptococcus pneumonia, Staphylococcus
aureus.
Example 4
Detection of the Extra cellular vesicle layer
Extra cellular vesicle layer was lysed with the help of a lysis buffer to dissolve the extra cellular
vesicle layer in the antibiofouling composition of the present invention (Sample A), and in a
separate control no lysis buffer was added to antibiofouling composition (Sample B). The
sample A & B were passed by binding column. and reading (260/280 ratio) was taken in
microtiter spectrophotometer. Nucleic acids have absorbance maxima at 260 nm. The ratio of
this absorbance maximum to the absorbance at 280 nm has been used as a measure of purity in
both DNA and RNA extractions. A 260/280 ratio of ~1.8 is generally accepted as “pure” for
DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. In Sample A, the (260/280) ratio
of the reading obtained was 2.122 and the concentration of RNA was detected to be of
12.669ng/µl. Whereas, in Sample B the (260/280), the ratio of the reading obtained was 1.417
(same as distilled water) and its concentration was 2.695ng/µl. The results clearly indicates that
the antibiofouling agent is encapsulated in a extra cellular vesicle which acts carrier for the
antibiofouling agent.
19
Example 5
RelA and SpoT gene analysis
Microorganisms continuously monitor their surroundings and adaptively respond to
environmental cues. One way to cope with various stress-related situations is through the
activation of the stringent stress response pathway. The bacterial stringent response is a crucial
survival response to various environmental stress conditions including biofilm formation
(Strugeon et al., 2016).
In Pseudomonas aeruginosa this pathway is controlled and coordinated by the activity of the
RelA and SpoT enzymes that metabolize the small nucleotide secondary messenger molecule
(p)ppGpp. Intracellular ppGpp concentrations are crucial in mediating adaptive responses and
virulence. Targeting this cellular stress response has recently been the focus of an alternative
approach to fight antibiotic resistant bacteria. As a consequence, cells rapidly trigger a cellular
reprogramming response that causes bacteria to decelerate energy-consuming processes (such
as macromolecular synthesis and growth) and redirect resources toward energy generation,
stress coping and expression of biosynthetic genes. Activation of the stringent stress response
pathway occurs through expression of the genes responsible for the synthesis of the small
signalling nucleotide guanosine-Penta phosphate (pppGpp) that is quickly hydrolysed to the
active form guanosine-tetraphosphate (ppGpp). In Gram-negative bacteria, two highly
conserved homologous proteins, RelA and SpoT, regulate the intracellular concentrations of
ppGpp (Pletzer et al., 2016).
RelA is a mono-functional synthase that binds to the ribosome and upon entry of an uncharged
tRNA molecule that blocks the ribosome, converts GTP and ATP to pppGpp and hence ppGpp.
Conversely, SpoT is a bi-functional enzyme that responds to many other types of stresses such
as carbon, phosphorus, fatty acid or iron starvation, and triggers the synthesis of ppGpp.
Additionally, SpoT possesses another functional domain that allows it to also hydrolyze
ppGpp. The second messenger ppGpp functions as a pleiotropic regulator to coordinate the
stress response. Rapid accumulation of intracellular ppGpp triggers a switch from cell growth
to survival mode through several mechanisms including binding and altering of the specificity
of RNA polymerase, interaction with proteins involved in translation, replication and RNA
turnover, crosstalk with other second messenger molecules such as c-di-GMP, and by
regulation of cellular processes such as quorum sensing
20
The composition of the present invention have potent broad-spectrum activities including
antimicrobial, antibiofilm and immunomodulatory properties. The distinct mechanism of
action against biofilms has been linked to a disruption of the stringent stress response since
anti-biofouling agent by binding to and triggering the degradation of ppGpp. Disabling the
stringent response therefore prevents intracellular accumulation of ppGpp
Mutant P. aeruginosa?relA/?spoT were streaked on plates containing King’s agar. Each plate
was streaked and incubated at 30-37°C for 24 hours. After 24 h we see fully grown biofilm on
the plate. Holes were punched on to the plates containing biofilm of P. aeruginosa?relA/?spoT
and the samples (a) distilled water (negative control) (b) vancomycin (positive control) (c)
composition of the present invention.
The results (Figure 6 (a) shows no inhibition zone in distilled water (negative control),
vancomycin (positive control) and Figure 6 (b) shows composition zone of inhibition of 3 cm
of the present invention.
Example 6
Degradation of Small signalling nucleotide guanosine-Penta phosphate (pppGpp)- by the
composition of the present invention.
The fully sequenced and widely reported laboratory strain P. aeruginosa. Overnight cultures
were prepared from -?80 °C frozen culture stocks in a nutrient-rich LB broth at 37 °C under
shaking conditions at 180 rpm. This culture was subsequently used to inoculate proteosepeptone-glucose-ammonium-salts (PPGAS) medium (Zhang and Miller 1992). The minimum
inhibitory concentration (MIC) of the test inhibitors against P. aeruginosa determined using
the resazurin six plate assay (Elshikh et al. 2016).
The cell pellets were collected from different growth phase cultures by spinning them at
13,000×g for 2–3 min at room temperature and the RNA extracted using JetGene RNA
Purification Kit (Thermo Fisher Scientific). The cells were lysed with occasional vortexing in
a buffer solution with 1× TE buffer, 15 mg/ml lysozyme and 20 mg/ml proteinase K (Promega).
The samples were then transferred to a 2-ml Lysing Matrix A tube (MP Biomedicals) with ßmercaptoethanol containing RLT buffer (provided in the kit) for enhanced lysis. The contents
in the lysing matrix tubes were then homogenised using the FastPrep™ FP 200 cell disrupter
at speed 5.5 for 30 s. A double DNA-digestion treatment was done to ensure that the RNA was
21
free of any genomic DNA (gDNA) contamination. The RNA isolated was quantified using the
Nanodrop spectrophotometer with A260/A280 ratio of 1.8–2.1 being considered as pure. The
integrity of the samples was checked by agarose gel electrophoresis for presence of two sharp
distinct bands representing 23S and 16S rRNA. The integrity was further verified by analysing
the samples in an Agilent 2100 Bioanalyzer where RNA Integrity Number (RIN) values greater
than 8 were observed for all samples. The RIN is based on a numbering system from 1 to 10
with 1 being the most degraded and 10 being the most intact. The RNA samples were aliquoted
and stored at -?80°C.
Reverse transcription quantitative polymerase chain reaction
First-strand cDNA was synthesised using Superscript™ Reverse Transcriptase II (Invitrogen).
Each reaction mix contained DNase-treated RNA (500 ng), 20–250 ng random primers
(Promega), 10 mM dNTPs and RNase free water to make to the reaction volume 15.6µl. The
reactions were heated at 65 °C for 5 min before adding 5× strand buffer, 0.1 M DTT and RNase
inhibitor (RNAse out™ Invitrogen) in final concentrations of 1×, 10 µM and 40 units,
respectively. The reactions were incubated at 25 °C for 2 min before adding Superscript™ II
Reverse Transcriptase (200 units final concentration) (Invitrogen). The RT reactions were
carried out at a series of temperature starting with 25°C for 10 min, 42°C for 50 min and 70°C
for 15 min. The first-strand cDNA synthesis was performed for all the biological triplicates
from each time point. A negative reaction without reverse transcriptase was included in every
run. All cDNA samples were stored at -?20 °C prior to use.
The cDNA synthesised was then used as a template for real-time PCR amplification using the
ROCHE LightCycler LC480 system with a SYBR-Green probe. Since PCR efficiency is highly
dependent on primer specificity, therefore a qPCR calibration curve was generated from each
primer set using PAO1 gDNA. Only those primers which gave a calibration curve with a slope
value between -?3.1 and -?3.6 that translated into amplification efficiencies of 90–110% were
used for PCR quantification. The binding specificity of these primers were also validated postamplification by generating a melt curve for each primer set with the presence of a single sharp
peak eliminating the chances of any non-specific binding.
The qPCR 10 µl reaction mix each contained 2× SYBR Green master mix (1×), forward and
reverse primers (1 µM), cDNA template and molecular grade water. Negative controls in form
of –RT (no reverse transcriptase) and no template control NTC (no DNA template added) were
included to rule out any contamination during the preparation process. A positive control in the
22
form of gDNA was also included. The cut-off values for residual gDNA amplification and
NTC were set at greater than 35 and 40 cycles, respectively. The cycling parameters were as
follows: initial denaturation at 95 °C for 5 min, 40–50 cycles of denaturation at 95°C for 10s,
annealing at 59 °C for 10s, extension at 72 °C for 10s.
Reference gene validation
A total of one gene (Rel-A(r) Rel-A(f)) were analysed. The BestKeeper is a free excel-based
tool that correlated the coefficient of the candidate gene with a Best Keeper Index to generate
the most stable gene. The genes Rel-A(R) and Rel-A(F) were identified as most stable for use.
Relative gene expression data analysis
System (LC480 software, version 2)-generated analysis was performed on the real-time PCR
data. The threshold values (Cq) from each of the qPCR run were extracted from the LC480
system using the second derivative maximum method (Rasmussen 2001). Data analysis was
performed by taking the arithmetic mean of the Cq values of the technical replicates and
transferring it into log values to generate the relative quantities (RQ). The RQ values of the
target genes were then divided by geometric mean of reference gene RQs (Rel-A*(R)Rel-(F))
to give normalised relative quantity value (NRQ). The output was the calibration normalised
ratio (CNRQ) which was used in extrapolating information on the expression profile of the
target genes.
The results clearly show that the composition of the present invention have potent broadspectrum activities including antibiofilm. The above example show that the distinct mechanism
of action against biofilms has been linked to a disruption of the stringent stress response
composition of the present invention acts by binding to and triggering the degradation of
ppGpp. M9- composition have potent broad-spectrum activities including antibiofilm. The
distinct mechanism of action against biofilms of the M9 Composition has been linked to a
disruption of the stringent stress response since anti-biofilm M9.drug act by binding to and
triggering the degradation of ppGpp. Disabling the stringent response therefore prevents
intracellular accumulation of ppGpp, The M9 composition anionic molecule targets
the Pseudomonas aeruginosa stringent response in vitro P. aeruginosa?relA/?spoT. Figure 7,
shows that rel A gene totally degraded by M9 composition (C) when compare to +ve
23
(Vancomycin) (A) and –ve (distilled water) controls (B), while as no degradation is shown in
sample containing no M9 Composition (D).
Therefore, it disables the stringent response by prevention of intracellular accumulation of
ppGpp, Thus, the composition of the present invention targets the Pseudomonas
aeruginosa stringent response in P. aeruginosa?relA/?spoT. The rel A gene is completely
degraded by composition of the present invention as compared to +ve(Vancomycin) and –ve
(distilled water) controls.
Example 7
Size distribution of the Extra cellular vesicles
The extra cellular vesicles is an anionic molecule with a zeta potential (-15) to (-20) mV and
100-1000nm in size as elucidated by the size distribution by intensity reports (Figure 8).
While specific language has been used to describe the disclosure, any limitations arising on
account of the same are not intended. As would be apparent to a person in the art, various
working modifications may be made to the method in order to implement the inventive concept
as taught herein. The figures and the foregoing description give examples of embodiments.
Those skilled in the art will appreciate that one or more of the described elements may well be
combined into a single functional element. Alternatively, certain elements may be split into
multiple functional elements. Elements from one embodiment may be added to another
embodiment. For example, orders of processes described herein may be changed and are not
limited to the manner described herein. Moreover, the actions of any flow diagram need not be
implemented in the order shown; nor do all of the acts necessarily need to be performed. Also,
those acts that are not dependent on other acts may be performed in parallel with the other acts.
The scope of embodiments is by no means limited by these specific examples. Numerous
variations, whether explicitly given in the specification or not, such as differences in structure,
dimension, and use of material, are possible. The scope of embodiments is at least as broad as
given by the following claims

We Claim,

1. A method of dissolution/elimination of microorganismal biofilm by administering a
suitable dose of anti-filming composition, wherein the anti-filming agent is extra
cellular-ribonucleic acid (ex-RNA).
2. The method of dissolution/elimination of microorganismal biofilm as claimed in claim
1, wherein the anti-filming composition comprises;
a. extracellular ribonucleic acid (exRNA) and is in the range of 4-12µg/ml.
b. extracellular vesicle is bound by a single membrane phospholipid layer
comprising glycopeptides; lipopeptide; polysaccharides; short chain fatty acids
including propionic acid and small peptides, encapsulating extracellular
ribonucleic acid (exRNA);
c. phosphate buffer saline in the range of 0.4-1%;
d. sodium chloride 0.9%;
e. glycerin in the range of 1-5%.; and
f. double distilled water.
3. The method of dissolution/elimination of microorganismal biofilm as claimed in claim
1, wherein the wherein the extracellular ribonucleic acid (exRNA) and extracellular
vesicle (EV) are derived from extra polymeric substances (EPS) from gram positive
bacteria.
4. The method of dissolution/elimination of microorganismal biofilm as claimed in claim
1, wherein extra polymeric substances (EPS) is derived is from gram positive nonpathogenic bacteria.
5. The method of dissolution/elimination of microorganismal biofilm as claimed in claim
2, wherein the extracellular vesicle is anionic molecule with a zeta potential (-1) to (-
30)mV and 20-500 nm in size.
6. The method of dissolution/elimination of microorganismal biofilm as claimed in claim
1, wherein the extracellular ribonucleic acid (exRNA) inhibits the messengerribonucleic acid (m-RNA) by antisense/ribozymal cleavage.
25
7. The method of dissolution/elimination of microorganismal biofilm as claimed in claim
1, wherein the extracellular ribonucleic acid (exRNA) is selected from a group
comprising micro Ribonucleic acid (miRNA), small interfering Ribonucleic acid
(siRNA), guide Ribonucleic acid (gRNA) cRNA (circular Ribonucleic acid), piwi
Ribonucleic acid (piRNA).
8. The method of dissolution/elimination of microorganismal biofilm as claimed in claim
1, wherein the microorganismal biofilm is formed on a biotic surfaces.
9. The method of dissolution/elimination of microorganismal biofilm as claimed in claim
1, wherein composition is formulated as a powder, a liquid, a solution, a cream, a gel
or a paste.
10. The method of dissolution/elimination of microorganismal biofilm as claimed in claim
1, wherein composition further comprises of antifungal agent, antibacterial compound,
antiviral compound, antiparasitic compound.
11. The method of dissolution/elimination of microorganismal biofilm as claimed in claim
1, wherein the wherein the biotic surfaces is selected from a group comprising lung
tissue, teeth surfaces; skin surface, tonsils, hair scalp; biofilms formed in chronic
infections such as dental plaque, periodontitis, cystic fibrosis lung infections, chronic
wounds, soft tissue fillers, otitis media, chronic osteomyelitis, chronic rhinosinusitis,
psoriasis, endocarditis, urinary tract infections, human gastrointestinal tract.
12. The method of dissolution/elimination of microorganismal biofilm as claimed in claim
1, wherein the method further comprises of an antifungal agent, antibacterial
compound, antiviral compound, antiparasitic compound. ,