Investigation of Biofilm Inhibition Activity and Antibacterial Activity of Psidium guajava Plant Extracts

17 Pages·2014·4.12 MB·English

Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 335-351
ISSN: 2319-7706 Volume 3 Number 9 (2014) pp. 335-351
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Original Research Article
Investigation of Biofilm Inhibition Activity and Antibacterial Activity of Psidium
guajava Plant Extracts against Streptococcus mutans Causing Dental Plaque
Ashok V.Gomashe, Anjali A. Sharma* and Ashwini Kasulkar
Department Of Microbiology, Shri Shivaji Science College Congress Nagar, Nagpur-12,
Maharashtra, India
*Corresponding author
A B S T R A C T
Dental caries is a localized, transmissible pathological infectious disease which
results in destruction of hard dental tissue. Dental plaque when allowed to
accumulate may lead to caries formation and discomfort due to the inflammation of
the gingival area. Both conditions are direct consequences of poor oral hygiene
measures of an individual. This begins with the formation of dental plaques which
is a structurally and functionally organized biofilm. Streptococcus mutans is a
Keywords pivotal bacterium in the formation of dental plaque and dental caries. Several
antibiotics are available to treat oral infections but these have several side effects.
Psidium An anti-plaque agent is an agent that causes an effect on plaque which results in a
guajava, reduction in caries and/or gingivitis. Thus there is an urgent call for alternative
Biofilm, prevention and treatment options that are safe equally effective and economical as
Streptococcus well. Hence the search for alternative products continues which diverts our interest
mutans, in natural phytochemicals isolated from plants used in traditional medicines and
Dental caries, considered as good alternatives to synthetic chemicals. Decoctions of various plant
Plaques, extracts have long been used in folklore practices to maintain the hygiene of the
Bacterial oral cavity since ancient times. The extracts of various parts of Psidium guajava
adhesion, plant were tested for their antibiofilm activity against the oral plaque forming
Exopolysacch bacteria Streptococcus mutans. It was found to significantly inhibit biofilm
aride formation. In the present study it was found that the extract from Psidium guajava
showed strong activity against Streptococcus mutans. The extract also prevents the
formation biofilm by the bacteria. The study suggested the possible benefits of this
herbal preparation which inhibit the biofilm formation by Streptococcus mutans an
oral pathogen. The three solvents (acetone, ethanol and methanol) based extracts of
Psidium guajava L. showed good activity but the acetone extract was highly active
against Streptococcus mutans. The acetone extract and ethanol extract (Psidium
guajava) is highly active against Streptococcus mutans.
Introduction
Over time the irrational use of the Increased prevalence of antibiotic resistance
antibacterial agents has produced strains of has led to the introduction of combination
multiple antimicrobial resistant bacteria. therapy which has increased treatment
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efficacy and contained drug resistance to India, nearly 60-70% of the child population
some extent, although combination therapy is affected by dental caries. (19) The biofilm
provided the answer to antibiotic resistance is an extracellular matrix that surrounds
for a while, there have been reports of microbial cells and is comprised of
emerging resistance to drugs in combination biological polymers such as
and multi-drug resistance in some bacteria . exopolysaccharide (EPS), protein, and
Oral biofilms are compositionally and DNA. The role of the biofilm is to attach to
structurally are complex bacterial abiotic surfaces, the epithelia of
communities. Dental caries is an infectious multicellular organisms, and interfaces such
microbial disease that results in localized as that between air and water. Surface
dissolution and destruction of the calcified adhesion of bacteria is an essential step and
tissues of the teeth [3]. Streptococcus mutans is required for the bacteria to arrange
are believed to be the principal etiological themselves favourably in their environment.
agent of human dental caries. It has
developed multiple mechanisms to colonize Fermentation of carbohydrate by acidogenic
the tooth surface and has become a oral bacteria is the key factor in
significant species in cariogenic development of dental caries. The acid
biofilm.Microbial biofilm cause a variety of released through microbial action leads to
microbial infections on human health. demineralization cavitation of tooth.
Dental plaque is a biofilm that grows in the Streptococcus spp. can colonize the tooth
oral cavity. It is the primary cause of dental surface and initiate the plaque formation by
caries and other oral infections and the less synthesizing extracellular polysaccharides
common peri-implantitis (similar to from sucrose.
periodontitis, but with dental implants),
however biofilms are present on healthy Biofilms enhance the virulence of the
teeth. Effective plaque removal procedures pathogen and have their potential role in
are expected to prevent the development of various infections. The biofilm forms those
these diseases. are more resistant to antimicrobial agents
and therefore more difficult to control,
Despite the ever increasing knowledge in remain largely unexplored. Their inherent
oral health care, the average adult in the resistances to antimicrobial agents are at the
country has between 10 and 17 decayed, root of many persistent and chronic bacterial
missing or filled permanent teeth. The infections. Biofilms have been reported to
majority of the population has at least minor be less susceptible to antimicrobial agents
gingivitis, an inflammation of the gums, and have reduced sensitivity to inhibitors.
with a much smaller percentage suffering The resistance shown by biofilm to various
from moderate to severe periodontitis. (16) antibiotics is a matter of concern. In recent
Dental plaque, which exists not only on the years, much of research has been focused in
tooth surface but also under the gums, is a identifying various alternative medicines to
diverse community of microorganisms in the treat infections caused by the drug resistant
form of a biofilm. The microorganisms bind bacteria. Various chemicals have been tested
tightly to one another and to the solid for their antibiofilm activities.
surface of tooth by means of an extracellular Unfortunately, those chemicals cannot be
matrix consisting of polymers of both host used as drug molecules to treat the diseases
and microbial origin. (12) It is more prevalent associated with the biofilm forming
in the Asian and the Latin countries. In bacteria.(6) The bacterial biofilms are
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creating problems as they attach to surfaces including gene expression, cell-cell
of some dental alloys, impression materials, signaling (ex. quorum sensing) (10) and
dental implants, restorative and cement antibiotic resistance. These specific cell-cell
materials. Biofilms are difficult or interactions have proven to be important in
impossible to destroy, particularly those dental plaque biofilm. (13) Biofilm
cells that form the deeper layers of a thick communities provide their members easy
biofilm. The insertion of orthodontic access to food and nutrients and allow the
appliances tends to create new surfaces cells inside, becoming more resistant to the
available for plaque formation and to body's natural antimicrobials as well as the
increase the level of microorganisms in the antibiotics which are administered. During
oral cavity. Gaps between synthetic and inhospitable conditions such as nutrient
natural elements are dangerous sites which starvation, microbes in biofilm communities
may be colonized by bacteria. (5) Orthodontic have the ability to enter into latent stages in
patients with fixed appliances frequently which organisms are viable but in non
have prevalence of Streptococcus mutans in culturable state.
plaque compared with untreated orthodontic
patients (6). Several agents are commercially available,
for example the antibiotics commonly used
These microorganisms have an important to treat oral infections i.e. Penicillins and
role in cariogenesis processes and Cephalosporins, Erthromycin, Tetracycline
consequently the study of the growth of this and derivatives, and Metronidazole have
particular biofilm is relevant in the control been documented [1]. These chemicals can
of plaque formation. The biofilm forming alter oral microbiota and have undesirable
bacteria are resistant to antimicrobial agents side effects such as vomiting, diarrhea, and
due to the lack of penetration of tooth staining [9]. Other antibacterial agents
antimicrobial agents. The EPS is a major used in the prevention and treatment of oral
feature of biofilms and is believed to play a diseases including Cetypridinium chloride,
major role in their resistance. (4) Biofilm Chlorhexidine, amine fluoride or products
formation is said to be under genetic control. containing such agents are reported to
HtrA gene has a significant role in surface exhibit toxicity, cause staining of teeth or in
protein expression and biofilm formation by the case if ethanol (commonly found in
Streptococcus mutans. The formation of mouth washes) have been linked to oral
dental plaque biofilms includes a series of cancer[5]. Given the incidence of oral
steps that begins with the initial colonization disease, increased resistance by bacteria, to
of the pellicle and ends with the complex antibiotics adverse effects of some
formation of a mature biofilm. antibacterial agents currently used in
dentistry and financial considerations in
They communicate via small diffusible developing countries, there is a need for
signaling molecules (e.g. competence- alternative prevention and treatment options
stimulating peptide, CSP; auto inducer); that are safe, effective and economical.
CSP induces both genetic competence and Hence the search for alternative products
acid tolerance in recipient sessile cells. The continues and natural phytochemicals
bacteria that become part of a biofilm isolated from plants used in traditional
engage in quorum sensing (14) medicine are considered as good alternatives
Communication can occur between cells in to synthetic chemicals [11]. It has been well
biofilm communities in a variety of ways, documented that medicinal plants confer
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antimicrobial activity towards oral bacteria. evaluate biofilm inhibition and antibacterial
Plant extracts or phytochemicals that inhibit activity of medicinal plant Psidium guajava.
the growth of oral pathogens, reduce the rate The plant material was authenticated from
of biofilms and dental plaque, influence the the Department of Botany of the college.
adhesion of bacteria to surfaces and reduce The study was carried out in the duration of
the symptoms of oral diseases can serve as August 2013 to February 2014 in The
alternatives in prevention and treatment of Department of Microbiology, Shivaji
dental caries. Science College Nagpur, Maharashtra,
India. In this study, there were following
Psidium guajava belongs to family steps which are as follows:
Myrtaceae with tough dark leaves that are
opposite, simple, elliptic to ovate and 5 15 Preparation of plant extracts
centimeters (2.0 5.9 in) long. The flowers
are white, with five petals and numerous All the plant materials (Leaves, flowers,
stamens. Medical research in laboratory barks and roots) were dried in open air
models, extracts from guava leaves or bark protecting that area from direct exposure to
are implicated in therapeutic mechanisms sunlight. The dried plant parts were crushed
against cancer, bacterial infections, in sterile pestle mortal and suspended in Hot
inflammation and pain. (5) It is a small tree, water, Acetone, Methanol, Ethyl acetate,
various parts of which exhibit antioxidant, Ethanol overnight in sterile conditions. The
hepatoprotective, antimicrobial and anti suspended extract was then filtered using
diabetic properties.[4] Present study is Whatman s filter paper no.1.
therefore focusing on identifying plants that The extracts were prepared as follows:
can be used as an herbal alternative to
chemical drugs to treat dental infections. We Preparations of Hot water extract (HWE)
are studying the efficacy of Psidium guajava
plant parts extracts against dental plaque Hot water extract was prepared by plain
forming Streptococcus mutans. decoction method. For this 30 gm of
powdered of each plant materials (leaves,
Materials and Methods
flowers, barks and roots) were taken into
beaker containing 300 ml of sterile distilled
Media used for antibacterial assay was
water. This was heated moderately in water
Muller Hinton Agar (HiMedia) and Media
bath till menstrum reduced to less than 1/4th
for biofilm formation and inhibition assay
of its original volume (approx. to 75ml). The
was Brain Heart Infusion medium which
water content from extract was evaporated
was purchased from HiMedia Laboratories,
off completely to achieve dried form of
Mumbai, India. Test organism used for the
extract and the resulting liquid was filtered
study was Streptococcus mutans MTCC 890
using Whatman s filter paper no.1.
procured from Institute of Microbial
Technology, Chandigarh, India. The activity
Preparation of Acetone / Methanolic/
of various parts (Leaves, Flowers, Bark, and
Ethyl acetate/ Ethanol Extract
Roots) of Psidium guajava plant was studied
in different solvents such as Hot water,
All the plant material was dried in open air
Acetone, Methanol, Ethyl acetate and
protecting that area from direct sunlight. The
Ethanol.
dried plant parts were crushed in sterile
pestle mortal as stated above. 100 mg of
The present research work was designed to
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each powdered plant material was extracted solution i.e., 0.85% NaCl 2-3 times to
with Acetone/ Methanolic/Ethyl acetate/ separate the cells adhered to glass surface.
ethanol by dipping in it for 24 hrs, resulting 1 % Crystal violet solution was added to
liquid was filtered using Whatman s filter observe for the adhered cells.
paper no. 1.
To study the biofilm inhibition activity
Antibacterial assay of plant extracts by
agar well diffusion method Brain Heart Infusion broth containing 1% D-
glucose was prepared and 30 µl of the broth
The extracts (Hot water, Acetone, Methanol, was transferred into the microtitre plates.
Ethyl acetate, Ethanol) were tested for The broth was then sterilized by autoclaving
antibacterial activity using agar diffusion on at121o C at 15 lbs pressure for 15 minutes.
Mueller Hinton Agar. The Media was The microtitre plate was then inoculated
sterilized by autoclaving at 121o C at 15 lbs with 3 µl of overnight grown culture of S.
pressure for 15 minutes. The molten agar mutans MTCC 890 strain. The extracts (Hot
was allowed to cool to 45 C and then 20ml water, Acetone, Methanol, Ethyl acetate,
of Mueller Hinton agar was poured Ethanol) containing leaves, flowers, bark,
aseptically into Petri plates. The agar was roots were added into each wells. The plate
allowed to set and harden. Agar test plates was then incubated at 37oC for 18 hrs. After
of each test organism were prepared. Using incubation the supernatant (non adherent
a sterile swabs lawn of the test organism was cells) was carefully decanted without
spread onto the Mueller Hinton agar plates. disturbing the adhering cells. The tube
The wells were punctured in the centre by containing biofilm were washed with saline
using a sterile cork borer. Then the wells (0.85% NaCl). Then 30 µl saline was added
were filled with various extract containing and mixed well to separate the cells adhered
leaves, flowers, bark, and roots. The plates to surface and to remove loosely attached
were incubated at 37oC for 24 hrs. After cells. 1 % Crystal violet solution was added
incubation the plates were observed for the to observe for the adhered cells. The optical
zone of inhibition. The zones were measured density was measured at 550 nm using a
using zone measuring scale. spectrophotometer.
Detection of biofilm formation Results and Discussion
Brain Heart Infusion broth containing 1% D-
Streptococcus mutans is the important
glucose was prepared and 3 ml of the broth
bacterium in the formation of dental plaque
was transferred into screw cap tube. The
and dental caries. Various extracts of a plant
broth was then sterilized by autoclaving at
Psidium guajava were tested for their
121o C at 15 lbs pressure for 15 minutes.
antibacterial activity against the oral plaque
The screw cap tubes were then inoculated
forming bacteria Streptococcus mutans.
with 30 µl of overnight grown culture of
They were found to significantly inhibit
Streptococcus mutans. The tubes were then
biofilm formation. In present study it was
tilted at an angle of 30o and incubated at
found that the extract from Psidium guajava
37oC for 18 hrs. After incubation the
showed strong activity against Streptococcus
supernatant was carefully decanted without
mutans. The extract also prevents the
disturbing the adhering cells. The tube
formation of biofilm by Streptococcus
containing biofilm were washed with saline
mutans. The study was carried out to
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Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 335-351
investigate the antibacterial activity and significant antibacterial activity and thus can
biofilm inhibition activity of Psidium be employed as an effective anti plaque
guajava plant parts extracts against agent and can be used in the prevention of
Streptococcus mutans. For this purpose, dental caries. The plant extract was
various extracts such as Hot water extracts, evaluated for antistreptococcal and
Acetone extracts, Methanol extracts, Ethyl antibiofilm activity against S. mutans.
acetate extracts, and Ethanol extracts of Psidium guajava was found to be active
parts of plant Psidium guajava were tested against Streptococcus mutans also has
against test organism Streptococcus mutans. potential antibiofilm activity. Currently,
Extracts of Psidium guajava showed the there is an increasing interest to investigate
potent antibacterial activity against S. the effect of natural compounds, especially
mutans studied by an agar diffusion assay. plants extracts, on the residence of the oral
The Table-1 showing the antibacterial cavity. It has been reported that Morus
activity of Hot water extract against S. alba,[9]Andrographis paniculata and
mutans. The zone of inhibition was found to Chinese black tea[6], cranberry [15] and
be more in leaves (20mm), in flowers Mikania sp. [16] exhibited potentially useful
(17mm), but less zone in bark (13mm) and antibacterial properties towards some oral
in roots (12mm) (Graph-1) (Figure 1-2) pathogens. The present study has shown that
The active plant extracts of the plant showed the extract of Psidium guajava exhibited
positive anti- adherence effect on the S. antibacterial activities. The selection of this
mutans biofilm formation on the glass plant was based on findings that their
surface in the presence of 1 % dextrose. extracts exhibited antibacterial activities
These active plant extract was found to against oral microbes grown under the
inhibit the biofilm formation on the glass planktonic state [13].The planktonic state
surface and showed decrease in the turbidity refers to the condition where the bacteria
when the OD was taken at 550 nm. (Table 6- were allowed to grow as suspension in the
10) (Graph 6-10) test tubes. P. guajava can significantly
disrupt the adhesion of the early plaque
Streptoccous mutans is the most important colonizers to the pellicle. This subsequently
oral bacteria which plays a major role in will interfere with the initial stage of biofilm
dental caries, bacteremia and consequently development. Similar observations have
bacterial endocarditis[14]. Application of also been reported by Percival et al [10]. who
antibiotics for prevention of dental caries is strongly acknowledged the importance of
not recommended, since there is risk of the salivary pellicle during the initial stages
development of MDR (multiple drug of biofilm formation. The property of the
resistance) strains. The use of plants and pellicle can be altered in the presence of
their extracts in the treatment of diseases certain plant extracts. The many positive
dates back to 460-370 BC when Hippocrates antimicrobial activities on oral bacteria
practiced the art of healing by use of plant exhibited by plant extracts provide great
based drugs. Different plants and their parts support in the promotion of such extracts as
(flowers, bud, leaves, stem, bark, fruits, pulp oral healthcare agents. Their use may help to
and roots) have been used for thousands of moderate the development of dental plaque
years[8] . S. mutans were found to form so that its texture is always thin and porous.
biofilm on the surface of screw cap tube in Besides, mouthwash sold in pharmacy stores
the presence of 1% dextrose. In this study or local supermarkets are either contains
plant extracts of Psidium guajava has many chemicals or alcohol-based which
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Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 335-351
may cause unwanted side effects to the but the acetone extract was highly active
consumers. against Streptococcus mutans [61]. The
results in the present study has showed that
Streptococcus mutans adheres by the acetone extract and ethanol extract
hydrophobic bond interaction to the enamel (Psidium guajava) is highly active against
surface. Therapeutic agents, which help to Streptococcus mutans. The similar
prevent hydrophobic bond formation, would antibacterial effect was described by Rathish
help to reduce the incidence of caries[7]. The Nair et al [12] where they reported that the
leaf extract of Psidium guajava showed a two solvents acetone and methanol based
broad spectrum antibacterial activity against extracts of Psidium guajava leaves showed
Gram-negative bacteria. The previous good activity but acetone extract was highly
findings that Gram-negative bacteria are active against S. mutans
hardly susceptible to the plant extracts in
doses less than 2 × 105 g/ml [61]. The Fathilah et. al. [2] has reported that the
variation of susceptibility of the tested aqueous extracts of P. guajava have caused
microorganisms could be attributed to their a reduction in the adhesion of the early
intrinsic properties that are related to the plaque settlers. The present study showed
permeability of their cell surface to the that Psidium guajava has excellent
extracts. The three solvents (acetone, antibacterial and biofilm inhibition activity
ethanol and methanol) based extracts of against Streptococcus mutans.
Psidium guajava L. showed good activity
Table-1: Antibacterial activity of Hot water extract against S. mutans:
Hot water extract Zone of inhibition in mm
Leaves 20
Flowers 17
Bark 13
Roots 12
Graph-1 Antibacterial activity of Hot water extract against S. mutans:
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Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 335-351

Figure-1 Figure-2
Antibacterial activity of Antibacterial activity
Hot water extract of of Hot water extract of
guava leaves against guava flowers against
Streptococcus mutans Streptococcus mutans
Table-2: Antibacterial activity of Acetone extract against S. mutans:
Acetone extract Zone of inhibition in mm
Leaves 28
Flowers 15
Bark 12
Roots R
Where, R= Resistant
Graph-2: Antibacterial activity of Acetone extract against S. mutans
30
m
m 25
n
n i 20
o
ti 15
bi
hi
n 10
of i 5
e
n
o 0
Z
leaves flower bark roots
Acetone extract

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Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 335-351

Figure-3 Antibacterial activity Figure-4 Antibacterial activity
of Acetone extract of guava of Acetone extract of guava
leaves against Streptococcus flowers against Streptococcus
mutans mutans
Table.3 Antibacterial activity of Methanol extract against S. mutans:
Methanol extract Zone of inhibition in mm
Leaves 15
Flowers 12
Bark 13
Roots 10
Graph.3 Antibacterial activity of Methanol extract against S .mutans:
16
m
14
m
n 12
n i 10
o
biti 8
hi 6
n
e of i 24
on 0
Z
leaves flowers bark roots
Methanol extract
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Figure-5 Antibacterial Figure-6 Antibacterial Figure-7 Antibacterial activity
activity of Methanol extract activity of Methanol extract of Methanol extract of guava
of guava leaves against of guava flowers against bark against Streptococcus
Streptococcus mutans Streptococcus mutans mutans
Table.4 Antibacterial activity of Ethyl acetate extract against S. mutans
Ethyl acetate extract Zone of inhibition in mm
Leaves 14
Flowers 18
Bark R
Roots 12
Graph.4 Antibacterial activity of Ethyl acetate extract against S. mutans
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