Int.J.Curr.Microbiol.App.Sci (2015) Special Issue-2: 302-316
ISSN: 2319-7706 Special Issue-2 (2015) pp. 302-316
http://www.ijcmas.com
Original Research Article
Plasmid Curing Activity by Seed Extracts of Cuminum cyminum, Coriandrum
sativum and Myristica fragrans Houtt. and Fruit Peel Extracts of Orange,
Banana and Pineapple Against Gram Negative Bacteria
Yogini P. Soman, Jyoti A. Mohite, Swati M. Thakre,
Sayali R. Raokhande and Shilpa S. Mujumdar*
Department of Microbiology, Modern College of Arts, Science and Commerce,
Shivajinagar, Pune 411005, India
*Corresponding author
ABSTRACT
Keywords
Antibiotic
resistance,
Cuminum
cyminum,
Coriandrum
sativum and
Myristica
fragrans Houtt.,
Fruit peels,
Plasmid curing
In this study, previously cultured isolates of Acinetobacter spp., Pseudomonas spp.,
E.coli, Klebsiella pneumonia and Proteus spp. were used. Methanolic extracts of
seeds of Cuminum cyminum, Coriandrum sativuma and Myristica fragransHoutt.
as well as fruit peel extracts of orange, banana and pineapple were prepared to
study plasmid curing property of all extracts against Acinetobacter spp.,
Pseudomonas spp., E.coli, Klebsiella pneumoniae and Proteus spp. Plasmid DNA
were isolated by Kado and Liu (1981) method from respective isolates. Three
plasmid DNA bands and one plasmid DNA band were observed under UV
transilluminator by agarose gel electrophoresis from isolates of Acinetobacter
spp.,Pseudomonas spp., Klebsiella pneumoniae, E.coli and Proteus spp.
respectively. The susceptibility of isolates to antibiotics such as Tetracycline,
Erythromycin, Ampicillin, Neomycin, Levofloxacin, Amoxicillin, Norflox,
Azithromycin, Amikacin, Cefixime, Doxycillin, Cephalosporin, Trimethoprim,
Streptomycin and Ciprofloxacin as well as for heavy metals such as Fe, Cu, Mn,
Ca, Al, Mg, Ni, Pb, K, Ba, Co, Mo, Li, Zn, S, Cr, Ag and Hg were tested. It is
cleared from this study that the methanolic extracts of Coriandrum sativum showed
better plasmid curing activity than the methanolic extracts of Cuminum cyminum
and Myristica fragrans Houtt. Coriandrum sativum showed least plasmid curing
activity against Pseudomonas spp. Curing percentage for Cuminum cyminum was
33.33-50%, for Coriandrum sativum was 10-75% while for Myristica fragrans
Houtt. was 20-72%. Methanolic extracts of orange, banana and pineapple peels
showed no curing activity against tested isolates. Rather these extract showed
overgrowth of bacteria and fungus other than tested isolates since it supplies
nutritional components to organisms. So active biomolecules from methanolic
extracts of seeds of Cuminum cyminum, Coriandrum sativum and Myristica
fragrans Houtt. may prove to be potential source for dissemination of multiple drug
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Int.J.Curr.Microbiol.App.Sci (2015) Special Issue-2: 302-316
ethidium bromide (Kumar et al., 2013).
Resistance to antibiotics by R plasmid are
due to conjugation(Kumar et al., 2013,
Shriram et al., 2010, Shriram et al.,
2013).Gram Negative microorganisms have
developed coping mechanisms in order to
survive in environments with high toxic
levels of metals. Therefore they can be used
as bio-indicators to detect heavy metal
pollution in the environment. The survival
of these organisms relies on the intrinsic
biochemical, structural, and physiological
properties and genetic adaptations. There are
generally four basic mechanisms used in
heavy metal resistance and include:
(i)exclusion of toxic heavy metal ions from
the cell by the alteration of membrane
transport systems involved in initial cellular
accumulation, (ii) extra- and intra-cellular
sequestration of metal binding components
similar to metallothioneins. (iii) cation/anion
efflux systems that are encoded by
resistance genes, and (iv) enzymatic
detoxification of heavy metals from toxic to
less toxic forms. Plasmid curing is a process
of completely removing plasmids from
bacteria by means of chemical agents or
now a day by means herbal extracts or
phytocompounds. Synthetic plasmid curing
agents like acridine orange, ethidium
bromide, sodium dodecyl sulfate are toxic
and mutagenic in nature. So, they are
unsuitable for therapeutic application. But
herbal extracts have more effective to use,
do not have toxicity and do not have
mutagenic effects; so they are safer to use as
a plasmid curing agents (Kumar et al.,
2013). Resistance genes play important role
in acquisition and dissemination by the
exchanges between plasmid(s) and the
bacterial chromosome and the integration of
resistance genes into specialized genetic
elements, called integrons (Carattoli, 2003).
Plasmids carry resistance genes which are
mobile. During plasmid borne antibiotic
resistance, the dosage and treatment is also
Introduction
The discovery of antibiotics in 1928 and
effective and efficient use of antibiotics to
control infectious agents begin in 1940
(Barbour et al., 2004). Due to overuse of
antibiotics to treat microbial infections,
microorganisms are becoming highly
resistant to available antibiotics. Bacteria
have evolved numerous defenses against the
antibiotics, susceptibility to antibiotics get
reduced now a days and drug resistant
pathogens are on the rise (Kumar et al.,
2013). Immense clinical problems arise in
immune compromised and cancer patients
due to MDR bacteria (Jasmine et al., 2011).
So Multiple Drug Resistance (MDR) is a
serious, global and future challenge to the
chemotherapy or drug discovery programs
(Shriram et al., 2010). Medical plants are
used in the preparation of herbal medicines
since ancient times, as they have antiviral,
antifungal and antibacterial activities
(Akrayi, 2011). With the development of
new technologies, study of plants, isolation
of active components, their role and
pharmacological effects become easier.
Medicinal plants contain wide variety of
active compounds such as terpenoids,
alkaloids, sulphur compounds, phenols,
esters, aldehydes, alcohols, glycosides,
thymol are responsible for antibacterial,
antifungal, anti-helminth, antioxidants, antiinflammatory activities (Bhattacharya et al.,
2013). Phytochemicals have a novel antibacterial action like inhibition of MDR
efflux pump, anti-antibiotic resistance
properties, R plasmid elimination. So,
elimination of R plasmid containing genes is
responsible for MDR is useful method
(Jasmine et al., 2011).Genes responsible for
resistance to antibiotics are present in
plasmid DNA. R plasmids contains variety
of genes encoding resistance to a broad
spectrum
antimicrobial
agents
like
antibiotics, heavy metals, disinfectant like
formaldehyde, mutagenic agents like
303
Int.J.Curr.Microbiol.App.Sci (2015) Special Issue-2: 302-316
important. Plasmids which have resistance
genes are in competition with other plasmids
that do not carry the resistance genes (Svara
et al., 2011).
analgesic activities. Nevertheless, no
evidence was found to support the
previously published reports of nutmeg s
hallucinogenic or other psychoactive
properties besides mild sedation. The major
compounds in the oil were sabinene
(21.38%), 4-terpineol (13.92%) and
myristicin (13.57%). On the other hand,
allylbenzene and propylbenzene derivatives
(myristicin, safrole, eugenol, and derivatives
thereof) were the predominant compounds
in nutmeg seeds (Parimala, 2013).
Cuminum cyminum belonging to Apiaceae
family is an annual plant which growing to a
height
of
15-30cm
according
to
environmental
conditions.
Cuminum
cyminum is stomachic, diuretic, carminative,
stimulant, astringent, emmenagogic and
antispasmodic. It is valuable in dyspepsia
diarrhea and hoarseness and many relieve
flatulence and colic. It is supported to
increase lactation and reduce nausea in
pregnancy. It has been shown to be effective
in treating Carpel tunnel syndrome as well
as indigestion and morning sickness.
Cuminum cyminum seeds have essential oils
containing a high proportion of myrtenal, pcymene,
pulgan,
cineol,
flanderin,
Benzaldehyde and small quantities of cTerpinene and 2 J-pinene (Abushama et al.,
2013)
Coriander (Coriandrum sativum L.)
an
annual of the Apiaceae family, is one of
valuable medicinal, seasoning and oliferous
plants. This species comes from the
Mediterranean region and it is grown all
over the world. The coriander fruit and the
essential oil isolated from it are used for
medicinal purposes (Duarte et al., 2012;
Mahendra et al., 2011; Sriti et al., 2011;
Chung et al., 2012). The coriander fruit
shows relaxant activity in the alimentary
tract; Coriander is used in the preparation of
many household medicines to cure bed cold,
seasonal fever, nausea, vomiting, stomach
disorders and also used as a drug for
indigestion, against worms, rheumatism and
pain in the joints. Many of healing
properties of coriander can be attributed to
its exceptional phytonutrients and hence, it
is often referred to as store house for
bioactive compounds. Coriander raw
material and oil are also used as an
aromatising agent in the pharmaceutical,
food, cosmetics and perfume industries.
Coriander seeds contain petroselinic acid,
linoleic acid, oleic acid and palmitic acid.
Major components of essential oil are
linalool, a-pinene, camphor and geraniol
(Bhuiyan, 2009).
Myristica fragrans Houtt. is an evergreen
tree, native of the E. Moluccas and
cultivated throughout Malaya. It is found
only as a specimen tree in Botanical
gardens. The seed of the plant is known as
nutmeg contains many volatile oils. These
oil constituents have a variety of individual
pharmacological effects, some of which
oppose others (Jellin et al., 2005). The fruit
contains ethereal oil-cells often with
phenolic and myristicin; the seed and the aril
are used for flavouring food. Nutmeg has
aromatic, stimulant, narcotic, carminative,
astringent, aphrodisiac, hypolipidaemic,
antithrombotic, anti-platelet aggregation,
antifungal,
antidysenteric,
and
antiinflammatory activities. The spice is used as
a remedy for stomach ache, rheumatism, and
vomiting during pregnancy Sonavanne et al.
(2002) explained that the n-hexane extract of
nutmeg seeds has anxiogenic, sedative and
Fruit peels of Orange, Pineapple and Banana
possess
antifungal
and
antibacterial
property. Therefore they are used in food
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Int.J.Curr.Microbiol.App.Sci (2015) Special Issue-2: 302-316
industry cosmetics and as biofertilizers.
Being a protective skin of fruit it has
property of tolerating harsh environment and
is also resistant to microbial population in
the environment.
Determination of antibiotic and metal
resistance
Isolates of Acinetobacer spp., Pseudomonas
spp., Proteus spp., E. coli and Klebsiella
pneumoniae were inoculated in sterile Luria
broth and kept it on rotary shaker for
overnight at 100rpm at 37°C. Dilutions of
antibiotics
such
as
Tetracycline,
Erythromycin,
Ampicillin,
Neomycin,
Levofloxacin,
Amoxicillin,
Norflox,
Azithromycin,
Rifampicin,
Oflaxacin,
Gentamicin, Cephalosporin, Trimethoprim,
Amikacin,
Streptomycin,
Doxycillin,
Cefixime and Ciprofloxacinand metals such
as Fe, Cu, Mn, Ca, Al, Mg, Ni, Pb, K, Ba,
Co, Mo, Li, Zn, S, Cr, Ag and Hg from 21024 µg/ml were prepared and each required
dilution were transferred on sterile Muller
Hinton Broth. After 24 h. Grid method was
performed for spot inoculations of
respective isolates on a respective dilution of
antibiotic and metal containing Muller
Hinton agar plates. The plates were
incubated at 37°C for 24 48h. The growth
of each isolate on respective dilutions of
antibiotic or metal containing Muller Hinton
agar
plates
was
monitored.
The
susceptibility of isolates to antibiotics such
as Tetracycline, Erythromycin, Ampicillin,
Neomycin, Levofloxacin, Amoxicillin,
Norflox and Azithromycin, Rifampicin,
Oflaxacin, Gentamicin, Cephalosporin,
Trimethoprim, Amikacin, Streptomycin,
Doxycillin, Cefixime and Ciprofloxacin as
well as to metals such as Fe, Cu, Mn, Ca,
Al, Mg, Ni, Pb, K, Ba, Co, Mo, Li, Zn, S,
Cr, Ag and Hg were tested.
Materials and Methods
Plant materials
Plant material (seeds) of Cuminum cyminum,
Corandrum sativum and Myristica fragrans
Houtt. were collected from an local shop as
well as fruit peels of orange, banana and
pineapple were collected from fruit juice
shop in Pune, Maharashtra
Extraction of plant materials
Seeds of Cuminum cyminum, Corandrum
sativum and Myristica fragrans Houtt. as
well as dried fruit peels of orange, banana
and pineapple were finely powdered with
auto-mix blender. 20g powder of seeds of
Cuminum cyminum, Corandrum sativum and
Myristica fragrans Houtt. as well as fruit
peel of orange, banana and pineapple were
extracted with 200ml methanol using
Soxhlet apparatus for a period of 5-6 h. The
extracts were concentrated using Vacuum
Rota Evaporator (SUPERFITTM) to obtain a
semisolid mass and 50°C oven dried to get a
dry form of extracts. Dried extracts were
stored in a crucible at room temperature.
Bacterial isolates
Previously cultured and identified isolates of
Acinetobacer spp., Pseudomonas spp.,
Proteus
spp.,
E.coli
and
Klebsiellapneumoniae were used. Plasmid
DNA was isolated by Kado and Liu (1983)
method from respective isolates. Plasmid
DNA bands were observed under UV
Transilluminator
by
agarose
gel
electrophoresis from all isolated strains.
Curing of plasmid mediated antibiotic
resistance
Methanolic extracts of seeds of Cuminum
cyminum, Corandrum sativum and Myristica
fragrans Houtt. were dissolved in 50ml
dimethyl sulfoxide (DMSO) and were used
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Int.J.Curr.Microbiol.App.Sci (2015) Special Issue-2: 302-316
further. Dilutions of all three methanolic
extracts from 2-1024µg/ml were prepared
separately and to each dilution 100µl
bacterial culture were added having O.D.
0.04-0.05 at 660nm. The tubes were
incubated at 37°C for 24 h. and after 24 h.
culture were plated on Muller Hinton agar
plates to obtain isolated colonies. Isolated
colonies were then replica plated to Muller
Hinton agar plates with antibiotics. The
colonies grew in Muller Hinton agar plates
but failed to grow in presence of antibiotics
were considered as cure derivatives. The
percentage curing efficiency was expressed
as number of colonies with cured phenotype
per 100 colonies tested (Shriram et al.,
2013).
Amoxicillin,
Levofloxacin
and
Azithromycin. The isolated strain of
Pseudomonas
spp.
showed
highest
resistance to almost all antibiotics except
Oflaxacin, Levofloxacin, Norflox and
Streptomycin. The isolated strain of
Klebsiella pneumonia and Proteus spp.
showed highest resistance to almost all
antibiotics except Oflaxacin, Gentamicin,
Ampicillin, Tetracyclin and Levofloxacin.
All isolated strains were resistant to all
metals such as Fe, Cu, Mn, Ca, Al, Mg, Ni,
Pb, K, Ba, Co, Mo, Li, Zn, S, Cr, Ag and
Hg. In our study, the methanolic extracts of
Coriandrum sativum showed better plasmid
curing activity than the methanolic extracts
of Cuminum cyminum and Myristica
fragrans Houtt. Coriandrum sativum
showed least plasmid curing activity against
Pseudomonas spp. Curing percentage for
Cuminum cyminum was 33.33-50%, for
Coriandrum sativum was 10-75% while for
Myristica fragrans Houtt. was 20 72%
(Table 2).
Results and Discussion
The extraction of all three plant materials
such as Cuminum cyminum, Coriandrum
sativum and Myristica fragrans Houtt. were
done successful by Soxhlet Extraction
method. Plasmid DNA was isolated by Kado
and Liu (1981) method from respective
isolates. Three plasmid DNA bands having
molecular weights 4000bp, 5000bp and
7000bp and one plasmid DNA band having
molecular weight 7000bp were observed
under UV transilluminator by agarose gel
electrophoresis
from
isolates
of
Acinetobacter spp., Pseudomonas spp.,
Klebsiella pneumoniae, E. coli and Proteus
spp. respectively. In our study the strain of
E.coli was isolated from sewage sample,
strain of Klebsiella pneumonia and Proteus
spp. were isolated from the hospital samples.
The isolated strain of Acinetobacer spp.
showed highest resistance to almost all
antibiotics except Rifampicin, Oflaxacin,
Ampicillin, Amoxicillin, Levofloxacin,
Norflox and Azithromycin. The isolated
strain of E. coli showed highest resistance to
almost all antibiotics except Rifampicin,
Oflaxacin,
Gentamicin,
Tetracyclin,
Using methanolic extracts of seeds of
Cuminum cyminum, Coriandrum sativum
and Myristica fragrans Houtt., all plasmid
DNA present in the Acinetobacter spp.,
E.coli and Proteus spp. were cured
completely. But, only one plasmid DNA
having molecular weight 7000 bp present in
Klebsiella pneumonia and Pseudomonas
spp. was cured. So, to get complete curing, it
is necessary to increase the concentration of
methanolic extracts of seeds of Cuminum
cyminum,
Coriandrum
sativum
and
Myristica fragrans Houtt. Methanolic
extracts of orange, banana and pineapple
peels showed no curing activity against
tested isolates. Rather these extract showed
overgrowth of bacteria and fungus other
than tested isolates since it supplies
nutritional components to organisms.
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Int.J.Curr.Microbiol.App.Sci (2015) Special Issue-2: 302-316
Table.1 Metal resistance of isolated strains
Name
of
Metal
Concentration of metals in µg per ml
2
4
8
16
32
64
128
256
512
1024
A B C D E A B C D E A B C D E A B C D E A B C D E A B C D E A B C D E A B C D E A B C D E A B C D E
Fe + + + + + +
Cu + + + + + +
Mn + + + + + +
Ca + + + + + +
Al + + + + + +
Mg + + + + + +
Ni + + + + + +
Pb + + + + + +
K
+ + + + + +
Ba + + + + + +
Co + + + + + +
Mo + + + + + +
Li + + + + + +
Zn + + + + + +
S
+ + + + + +
Cr + + + + + +
Ag + + + + + +
Hg + + + + + +
LegendsA: Acinetobacter spp.
C: E. coli
E: Proteus spp.
+: Antibiotic Resistance
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B: Pseudomonas spp.
D: Klebsiella pneumoniae
-: Antibiotic Sensitive
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-
Int.J.Curr.Microbiol.App.Sci (2015) Special Issue-2: 302-316
Table.2 Percent plasmid curing for all tested strains
Sr No.
Name of
Extracts
1.
Cuminum
cyminum
2.
3.
Coriandrum
sativum
Myristica
fragrans
Houtt.
Name of
Microorganisms
Number of
Colonies with
Cured
Phenotype
4
Acinetobacter
spp.
3
Pseudomonas
spp.
1
E.coli
2
Klebsiella
pneumonia
4
Proteus spp.
6
Acinetobacter
spp.
2
Pseudomonas
spp.
1
E.coli
1
Klebsiella
pneumonia
6
Proteus spp.
5
Acinetobacter
spp.
Pseudomonas
spp.
E.coli
Klebsiella
pneumonia
Proteus spp.
308
Total
Number of
Colonies
Percent
Plasmid
Curing
12
33.33%
7
42.85%
2
6
50%
33.33%
8
8
50%
75%
20
10%
2
2
50%
50%
12
9
50%
55.55%
8
11
72%
1
3
3
7
33.33%
42.85%
3
14
20%
Int.J.Curr.Microbiol.App.Sci (2015) Special Issue-2: 302-316
309
Int.J.Curr.Microbiol.App.Sci (2015) Special Issue-2: 302-316
Figure.1 Plasmid curing mediated antibiotic resistance reversal for Pseudomonas spp. by
methanolic extracts of seeds of Coriandrum sativum (a) Control plate, (b) Absence of colonies
on antibiotic Muller Hinton agar plates indictes putative cured derivatives
(a)
(b)
310
Int.J.Curr.Microbiol.App.Sci (2015) Special Issue-2: 302-316
Figure.2 Plasmid curing mediated antibiotic resistance reversal for Proteus spp. by methanolic
extracts of seeds of Myristica fragrancs Houtt.(a) Control plate, (b) Absence of colonies on
antibiotic Muller Hinton agar plates indictes putative cured derivatives
(a)
(b)
The extraction of Richardia brasiliensis was
done by Soxhlet Extraction method (Morias,
2013).
The
ethanolic
extract
of
Trachyspermum ammi was also prepared by
Soxhlet Extraction to separate the secondary
metabolites (Mobashshera, 2014) which is
similar to our study. The strain of Proteus
spp. was collected from Agharkar Research
Institute, Pune and Microbial Type Culture
Collection (MTCC), they have got only one
plasmid DNA band (Shriram, 2013) as well
as the strain of Proteus spp.(ATCC 25938)
is also contain only one plasmid DNA
(Bhattacharya, 2013) which are similar to
our study. The resistance of microorganisms occurs by several mechanisms
like efflux pump, production of enzymes
that inactivate the drug and change the target
of action of antibiotic. The resistance
mechanisms are transmitted through
exchange of genetic materials between
micro-organisms of the same species
(Oliveira, 2006). In the study carried out by
Ronald et al. (2002), high level of antibiotic
resistance was seen in all Gram negative
bacteria isolated from rivers of United
Status. The plasmid Profile of the selected
bacterial isolates revealed the presence of
plasmid/s in these isolates. In the study by
Ronald et al. (2002), over 40% of the
bacteria resistant to more than one antibiotic
had atleast one plasmid which is comparable
to our study. From Fever Hospital and Chest
Department, Zagczig University, Egypt, the
isolated strains of Klebsiella pneumoniae as
well as Pseudomonas aeruginosa showed
resistance to Amikacin, Trimethoprim and to
Gentamicin, Azithromycin respectively
(Azza et al., 2010) which are similar to our
study. In the study of plasmid profiling and
curing analysis, metal such as nickel shows
resistance against Pseudomonas aeruginosa
(Raja et al., 2009) which is similar to our
study. It is well known that there are no
currently acceptable concentrations of metal
ions which can be used to distinguish metal
resistant and metal sensitive bacteria (Malik
and Jaiswal, 2000). The curing efficiency of
methanolic
extract
of
Elettaria
cardamomumis in the range of 3-33%
(Akrayi, 2011) which is comparable with the
curing efficiency of Cuminum cyminum as
well as the curing efficiency of methanolic
extract of Piper longum fruitshows 64% of
plasmid curing efficiency (Kumar et al.,
2013) which is comparable with the curing
efficiency of Coriandrum sativum and
Myristica fragrans Houtt. It can be
311
Int.J.Curr.Microbiol.App.Sci (2015) Special Issue-2: 302-316
concluded from the present results that the
methanolic extracts of seeds of Cuminum
cyminum,
Coriandrum
sativum
and
Myristica fragrans Houtt. successfully
reversed the multiple drug resistance in
cured derivatives and making them sensitive
to antibiotics. This antibiotic resistance
reversal may be attributed to the curing of
R-plasmids harbored by these MDR
bacterial strains. These active biomolecules
from methanolic extracts of seeds of
Cuminum cyminum, Coriandrum sativum
and Myristica fragrans Houtt. proved to be
potential source for dissemination of
multiple drug resistant organisms.
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constituents from leaves and steams of
Korean Coriandrumsativum and their
immunotoxicity activity on the Aedes
aegypti
L.
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Acknowledgement
We are sincerely thankful to Dr. R. S.
Zunjarrao, the principle of Modern College
of Arts Science and Commerce Shivajinagar
Pune 5, for providing us with valuable
contribution
and
infrastructure
to
completeour project successfully. We are
also thankful to Dr. Mrs. Patwardhan, Head
of the Microbiology Department, H.V. Desai
College, Pune as well as Miss. Pratima
Shinde and Miss. Komal Chavan (M.Sc.
Part
II
students,
Department
of
Microbiology, Modern College of Arts,
Science and Commerce, Shivajinagar, Pune411005) for providing the isolated strains.
We would also like to thank the teaching
and non-teaching stafffor their cooperation
and for providing us the requirements
needed for our project.
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