Abstract

1. Introduction
2. Staphylococcus aureus-a major problem in health control
3. Botanical/phyto chemicals:
i. Types of phytochemicals important in medicine
ii. Mechanism of Antimicrobial action of
Phytomedicines
iii. Effectiveness of plant herbal extracts
iv. Limitations of the use of herbal medicines
4. Antimicrobial drugs against S. aureus:
i. Antibiotics targeting S. aureus
ii. Mode of action of antibiotics
iii. Effectiveness of antibiotics
iv. Limitations of Antibiotics
5. Combination of plant extracts and antibiotics:
i. Need for creation of Synergy between plant extracts and
antibiotics
ii. Commonly used phytomedicines and antibiotics
combinations
6. Preparation of plant extract and antibiotic combination:
i. Preparation of Plant extract
ii. Checking the antimicrobial activity of plant
extracts
iii. Antibiotic sensitivity test of S. aureus strains
iv. Effect of combination of plant extracts and
antibiotics on S. aureus
v. Microscopic examination of S. aureus to
confirm the effect of synergism:
7. Conclusion
8. References.

ABSTRACT

Plant extracts have long been used for the treatment of urinary tract
infections, gastrointestinal infections, respiratory tract infections and skin
infections. They contain certain compounds like flavonoids, terpenoids,
alkaloids and phenols that exhibit antibacterial activities by targeting the

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plasma membrane of Staphylococcus aureus but they are less potent than
antibiotics. Staphylococcus aureus is pathogenic micro-organism for which
the synergistic effect of plant extracts and antibiotics are described.
Antibiotics like Oxacillin, Imipenem, Vancomycin, Norfloxacin, Tetracycline,
Sulphamethoxazole, Trimethoprim, Gentamicin, Carbenicillin and Linezolid
are the most commonly used drugs to control S. aureus but S. aureus
readily develop resistance against these antibiotics. In order to overcome
this problem, plant extracts also called botanical/phytomedicines are used
in combination with antibiotics. When plant extracts and antibiotics in
combination, ability of S. aureus to develop resistance against the
combination of plant extracts and antibiotics becomes difficult.

1. Introduction:
The increased resistance of S. aureus led to use the
antibiotics with other natural compounds that also show antimicrobial

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 activity. Staphylococcus aureus strains are the part of normal microbial
flora of the humans, animals and even birds. They are characterized as
opportunistic pathogens and cause serious illnesses as abscesses,
endocarditis, pneumonia and meningitis. The emergence of methicillin
resistant strains of Staphylococcus aureus is a major problem in the
development of antimicrobial drugs. MRSA have also developed resistance
against penicillins, aminoglycosides and macrolides. The development of
resistance is due to the presence of drug resistant genes on the
plasmids of S. aureus. Plant extracts consists of complex mixtures against
which the microbial development of resistance is rare. Moreover, they are
also non-toxic or have very little side effects. In sort, plant extracts
enhance the antimicrobial activity of antibiotics and are called as
modifiers of antimicrobial activity (Aqil et al., 2006).

2. Staphylococcus aureus-a major problem in health control:

Staphylococc
us aureus is an opportunistic pathogen and involved in several disease in
hospitalized patients or immunocompromised patients. This pathogen and
its strains are a serious problem in drug development and health control
because this pathogen develop resistance against antibiotics more readily
because of the presence of antibiotic resistant genes on the plasmids.
Methicillin resistant S. aureus strains are particularly important in health
control because they have developed resistance against Penicillins,
Vancomycin, Imipenem, Norfloxacin, Sulphamethoxazole, Trimethoprim,
Gentamicin, Carbapenems, and Linezolid. To overcome this resistance
problems, antibiotics are mixed with plant extracts that have increased
their antimicrobial activity. Hence, plant extracts are also called as
modifiers of antibiotic activity (Bosio et al., 2000).

3. Botanical/phyto chemicals:
Plants produce primary and secondary
metabolites during their normal metabolism. Primary metabolites include
fats and sugars that are present in almost all plants but secondary
metabolites are specific in their action (Stepanovic et al., 2003). These
secondary metabolites are phytochemicals that extracted from plants and
can be used for medicinal purposes as:
i. Types of phytochemicals important in medicine:
Phytochemicals are
actually secondary metabolites of plants. These secondary metabolites
are very important in medicine. They are generally divided into following
types as:
Flavonoids:

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 Flavonoids are pigment molecules that are responsible for the
color of flower petals. They also act as chemical messengers,
physiological regulators and cell cycle inhibitors. They are also involved in
the formation of nodules in leguminous plants.

Terpenoids:
Terpenoids are produced by a variety of plants especially
Conifers. Terpenoids contain phenolic compounds that are responsible for
their antimicrobial properties. Vitamin A is an example of terpene (Drago
et al., 2002).
Alkaloids:
Alkaloids are the chemical compounds produced by a variety of
plants. They are used as anesthetic and stimulants. They are used in
combination with quinolones against gram positive bacteria.
Phenols:
Certain spices naturally contain phenolic compounds like
carvacrol and thymol. Being organic in nature, these phenolic compounds
disrupt the plasma membrane of bacteria (Fernandes et al., 2003).
ii. Mechanism of Antimicrobial Action of Phytomedicines:
Phytomedicines
function by inhibiting the function of ATP synthase enzyme in the
plasma membrane of bacteria and thus affect the energy production. They
also affect the efflux pump of the plasma membrane. Some chemical
constituents within the plant extracts like thymol and carvacrol act as
membrane permeabilizers. In this way, they disrupt the membrane
permeability and enhance the uptake of antibiotics. There are several
mechanisms through which the plant extracts exert their antimicrobial
effect. They also enhance the activity of those antibiotics that act within
the cells like aminoglycosides because their hydrophobicity enhance the
activity of antibiotics (Banskota et al., 2001).
iii. Effectiveness of Plant Extracts:
Plant extracts are effective against
disease causing micro-organisms due to the following reasons as:
They have unique chemical structure.
It is very difficult for the microbes to develop resistance against
Phytochemicals.
They are especially important to treat those bacterial infections in
which bacteria develop resistance to most antibiotics.
They are relatively inexpensive.
It is not very difficult to synthesize the plant extracts.
They are not only direct antimicrobial agents, instead they are also
used as resistant modifying agents.
They also inhibit the action of Beta lactamases (Fernandes at al.,
2001).

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iv. Limitations of the Use of Herbal Medicines:
Although, they are very effective
in controlling the antibiotic resistant micro-organisms but still they have
certain limitations as:
They are less effective when used without antibiotics.
Methods to obtain plant extracts are not standardized.
Some chemical constituents present within the plant extracts are
harmful to human health and may cause allergic reactions.
They are susceptible to degradation.
Their in-vitro efficacy is greater than in-vivo efficacy.
There is a need to develop formulations of phytomedicines (Burdock,
1998).

4. Antimicrobial Drugs against S. aureus:

A wide range of antibiotics
can be used against S. aureus. But they gradually develop resistance
against these antibiotics due to the production of antibiotics resistant
enzymes especially penicillinases.
i. Antibiotics targeting S. aureus:
A variety of antibiotics can be used against S.
aureus as:

Antibiotics Mechanism

Oxacillin Inhibiting the cell wall synthesis

Vancomycin Inhibiting the cell wall Production

Norfloxacin Inhibiting the process of replication

Trimethoprim and Act as antimetabolite

Sulfamethoxazole

Methicillin Inhibiting the synthesis of cell wall

Tetracycline Inhibition of protein synthesis

Linezolid Inhibiting the protein synthesis

These antibiotics are very effective against different strains of S. aureus

(Fernandes et al., 1995).
ii. Mode of action of Antibiotics:
Penicillins like oxacillin and Methicillin function
by inhibiting the cell wall synthesis. They do this by binding to the
transpeptidase enzyme. Transpeptidase enzyme is involved in the

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formation of cross-links between linear peptidoglycan chains that form
major component of cell wall of bacteria (Wendakoon and Sakaguchi,
1995). Consequently, the cell wall is greatly weakened and cell undergoes
lysis due to osmotic pressure.

Vancomycin also inhibit the synthesis of cell wall but they do this at
earlier stage than penicillin. They prevents the phosphorylation of a
carrier protein called bactoprenol.

This carrier protein is involved in the transportation of N-acetyl

glucosamine and N-acetyl muramic acid to the outside of the cell.
Norfloxacin inhibits the process of replication by acting as inhibitor of
DNA gyrase and topoisomerase enzymes that unwind and relax the DNA
strands during the process of Replication (George and David, 2008).
Sulfamethoxazole and Trimethoprim work in synergism to inhibit the
synthesis of Folic acid. They act as inhibitors of Dihydropteroate
synthetase and dihydrofolate reductase. In this way, they inhibit the
synthesis of folic acid which acts as precursor for the synthesis of purines
and pyrimidines (Zuo et al., 2008). Their mechanism of action can be
represented by the following diagram as:

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 Tetracyclines and Linezolid act by inhibiting the synthesis of proteins.
Tetracyclines bind to 50S ribosomal subunit and prevents protein chain
elongation. Linezolid also binds to 50S ribosomal subunit but it inhibits the
formation of protein initiation complex (Hammer et al., 1999).
iii. Effectiveness of Antibiotics:
Antibiotics are very effective due to the following
reasons as:
Broad spectrum antibiotics are used to treat serious infections where
identification of pathogen is not possible.
Most antibiotics are target-specific and affect only the target
pathogenic cells.

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 They specifically target the Cell wall of S. aureus without affecting the
host cells because the host cells lack peptidoglycan (Garvey et al.,
2011).
iv. Limitations of Antibiotics:
The major limitations of antibiotics are as follows:
They also affect normal flora of the host.
They also produce side effects in humans.
Wide spread use of antibiotics results in the development of resistant
forms of pathogens.
They must be prescribed under severe cases.
S. aureus readily develop resistant against a wide range of antibiotics
because they produce a broad range of antibiotic resistant enzymes.
There are several factors that enforces the development of resistance
against antibiotics (Koo et al., 2002). Some of them are represented in
following diagram as:

So, in order to
overcome the
problems of

resistance, they are used in combination with other drugs.

5. Combination of Plant extracts and Antibiotics:

In order
to overcome the problems of microbial resistance against antibiotics, they
are mixed with other drugs such as phytomedicines.

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i. Need for creation of Synergy between plant extracts and
antibiotics:
S. aureus
is a part of normal microbiota of the body. It is an opportunistic pathogen
and cause severe infections in immunocompromised patients. S. aureus
first develop resistant against Methicillin. Emergence of Methicillin
resistant S. aureus presents a serious problem in health control. Gradually,
MRSA also develop resistant against lactams, aminoglycosides,
fluoroquinolones and macrolides because they carry multidrug resistant
genes on their plasmids and they also spread these genes to other
species during the process of conjugation. So, in order to overcome the
problem of resistance, antibiotics are used in combination with plant
extracts. Plant extracts contain chemical compounds that are of complex
chemical structure (Muroi et al., 2004). Due to the complexity of their
structure, development of bacterial resistance against them is very
difficult. When used alone, plant extracts are not very effective but when
they are used in combination with antibiotics, the microbes develop less
resistant against the combination. Plant extracts contain certain complex
chemical constituents against which the development of microbial
resistance is very slow whereas antibiotics affect their target cells. Recent
research has approved that synergy between plant extracts and
antibiotics is most effective against S. aureus as S. aureus develop less
resistant against combination of plant extracts and antibiotics (Nicolson
and Toole, 1999).
ii. Commonly used phytomedicines and antibiotics combination:
Some of the
combinations of plant extracts and antibiotics are effective against S.
aureus (Patterson, 2000) and can be represented in the following table as:

Antibiotics Plant extracts

Amoxacillin Lemon grass

Tetracycline Cardamom oil

Vancomycin Thymus vulgaricus extract

Gentamicin Esfand (Perganum harmala)

Chloramphenicol Spina christi (Egyptian tree)

Linezolid Oliveira

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 Methicillin Lemon grass

6. Preparation of Plant extract and antibiotic

Combination:
In
laboratory, plant extract and antibiotic combination can be prepared by
the following method as:
i. Preparation of Plant Extract:
First of all, take plant material and crush it to fine
powder. Then mix the plant extract with 70% methanol and after 48 hours,
filter the extract. Again, mix the filtrate with 70% methanol in order to re-
extract the plant material. Again, after 24 hours, filter the extract through
filter paper. The resulting filtrate will contain the plant extract (Santos et
al., 2002).
ii. Checking the antimicrobial activity of plant extracts:
The antimicrobial
activity of methanolic plant extract can be determined by using the Disc
diffusion assay. In this method, whatman filter paper is used. Whatman
filter paper is cut into small strips with the help of puncher. Strips are then
dipped in methanolic plant extract. Nutrient agar plates are prepared by
pouring the Nutrient agar into the petri dishes such that it occupies only
one third of their total volume. These nutrient agar plates are inoculated
with an overnight culture of S. aureus. After inoculation, strips which were
dipped in plant extracts, are placed on nutrient agar plates. Finally,
incubate the plates at 37 Celsius for 24-48 hours (Scheller et al.,
1999).The disc diffusion assay for S. aureus can be represented as:

iii. Antibiotic sensitivity of S. aureus strains:

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 Antibiotic sensitivity of S. aureus is
determined by using the disc diffusion method but in this case whatman
filter paper strips are dipped in different antibiotic solutions. S. aureus can
be collected from hospital acquired patients who have compromised
immune system. Suitable sites for sampling are skin, urine, eyes and even
burned tissues. Plates containing S. aureus culture and strips of different
antibiotics are incubated at 37 degree Celsius for 24-48 hours. After
incubation, hollow zones around the colonies are observed (Takaisi-Kikuni
and Schilcher, 1994). The antibiotics that are normally used against S.
aureus are as follows:
Amoxacillin
Tetracycline
Vancomycin
Norfloxacin
Sulfamethoxazole and Trimethoprim
Gentamicin
Chloramphenicol
Linezolid
Methicillin (Krol et al., 1993).
iv. Effect of combination of plant extracts and antibiotics on S.
aureus:
S. aureus is
now grown in the presence of both plant extracts and antibiotics. Now,
disc diffusion assay is performed by dipping the strips in the solution of
both plant extracts and antibiotics. Nutrient agar plates containing both S.
aureus and strips dipped in solution of both antibiotic and plant extracts
are incubated at 37 degree Celsius for 24-48 hours. After incubation, clear
zones around the colonies are noted in order to determine the most
effective combination of plant extracts and antibiotics (Kusumoto et al.,
2001).
v. Microscopic examination of S. aureus to confirm the effect of
synergism:
Tra
nsmission electron microscope can be used to visualize the effect of
antibiotic and plant combination on S. aureus (Taylor et al., 2002). In order
to visualize the cells in transmission electron microscope, cells are
specially prepared as:
Centrifugation of the sample containing S. aureus treated with
antibiotics and plant extracts.
The cells are then fixed with 1% osmium tetroxide.
Cells are then dehydrated with 75% ethanol.
The sample is embedded on carbon coated copper grids at 60 degree
Celsius.
Sample on copper grids is stained with uranyl acetate.

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 Finally, the sample is examined under transmission electron
microscope (Yang et al., 2005).
The effect of amoxicillin and lemon grass essential oil alone and in
combination on the growth of S. aureus can be represented under
transmission electron microscope as:

In the above figures, Symbols represent the following as:

A represents untreated MRSA
B represents MRSA after treatment with amoxicillin
C represents MRSA after treatment with lemon grass essential oil

A B

C D

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 D represents MRSA after treatment with both amoxicillin and lemon
grass essential oil (Sforcin et al., 2000).

7. Conclusion:
Antibiotics are no longer effective against S. aureus because
it has multidrug resistant genes on its plasmid. Due to the emergence of
resistant forms of S. aureus like Methicillin resistant S. aureus, it is very
difficult to control them by using the antibiotics alone. These antibiotic
resistant forms of pathogen are particularly important in hospital acquired
patients who have compromised immune system. In order to overcome
this problem, trend is shifting towards using the antibiotics in combination
with plant extracts. Plant extracts contain compounds of complex
chemical structure against which the development of microbial resistance
is slower. They are individually less effective against S. aureus but when
used in combination with antibiotics, they are comparatively more
effective than when used alone. The effect of combination on the growth
of S. aureus can also be visualized under transmission electron
microscope.

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