Introduction

In the past 60 years, antibiotics have been critical in the fight against
infectious disease caused by bacteria and other microbes1.
Today, there are about 4000 compounds with antibiotic properties.
Antibiotics are used to treat and prevent infections, and to promote growth
in animals. Antibiotics are derived from three sources: moulds or fungi;
bacteria; or synthetic or semi-synthetic compounds (2).
Antibiotics fight against bacteria by inhibiting certain vital
processes of bacterial cells or metabolism. Based on these processes, we can
divide antibiotics into five major classes:
1. Cell wall inhibitors, such as Penicillin and Vancomycin.
2. Inhibitors of nucleic acid synthesis, such as Fluoroquinolones, which
inhibits DNA synthesis, and Rifampin, which inhibits RNA synthesis.
3. Protein synthesis inhibitors, such as Aminoglycoside.
4. Anti-metabolites, such as the sulfa drugs.
5. Antibiotics that can damage the membrane of the cell, such as
PolymyxinB, Gramicidin and Daptomycin (2).

Antibiotic resistance
Antibiotics typically retard bacterial proliferation by entering in
microbes and interfering with the production of components needed to
form new bacterial cells. However, the use (and misuse) of antibiotics
encourages the evolution of bacteria toward resistance (3).
A number of factors contribute to antibiotic resistance including-
1) Misuse and overuse of antibiotics in humans, animals, and agriculture.
2) Patients’ demand for antibiotics when they don’t need them.
3) Failure to finish an antibiotic prescription (3).
Antibiotic resistance is responsible for great therapeutical and
economic burden in the treatment of infectious diseases and it may
threaten the success of antimicrobial chemotherapy. Single antibiotic
resistance itself is a great problem, however the appearance of multiple
antibiotic resistant (MDR) strains cause a more obstacles in treatment of
infections (4).
Resistant strains appeared soon after the introduction of an antibiotic
and initially showed up in hospitals, where most antibiotics were used,
causing clinical difficulties for nosocomial treatment. Antibiotic resistance
is unavoidable from an evolutionary perspective, since the antibiotic
pressure itself provides the potential for resistant bacteria to acquire
resistance. Discretionary uses of antibiotics lead to the spread of resistant
strains, which causes the most expressed problem at nosocomial
circumstances, mainly in immunocompromised patients (4).

Resistance Mechanisms
Broadly speaking, mechanism of antibiotic resistance can be divided
into genetic drug resistance, and phenotypic drug resistance.
Genetic resistance is due to chromosomal mutations or acquisition of
antibiotic resistance genes on plasmids of bacteria.
Phenotypic resistance is due to changes in the bacterial physiological state,
such as the stationary phase, antibiotic persisters, and the dormant state.

Genetic drug resistance

There are five major mechanisms of antibiotic drug resistance, which are
due to genetical mutations-
 Reduced permeability or uptake.
 Enhanced efflux.
 Enzymatic inactivation.
 Alteration or over-expression of the drug target.
 Loss of enzymes involved in drug activation.

Reduced Permeability or Uptake

The first mechanism is reduced permeability or uptake of the bacteria,
which Prevents entry of antibiotic in to the cell.
Gram-negative bacteria are relatively more resistant to antibiotics
because of the nature of their cell wall, which restricts absorption of many
molecules movements through openings called porins. Some bacterial
mutants modify the porin opening so that antibiotics are unable to enter
the periplasmic space.
eg. Neisseria gonorrhoea can acquire mutations that can cause resistance to
penicillin and tetracycline.
Enterobacter aerogenes, can acquire mutations that cause cephalosporin
resistance.

Increased Efflux Activity

Certain proteins in the plasma membranes of gram negative bacteria act as
pumps that expel antibiotics, preventing them from reaching an effective
concentration. This mechanism was originally observed with tetracycline
antibiotics, but it confers resistance to practically all major classes of
antibiotics. Bacteria normally have many such efflux pumps to eliminate
toxic substances.
eg. TetK serves as an example for an efflux-mediated Tetracycline
resistance. Under normal conditions, the efflux gene, TetK, is not
expressed, due to a suppressor bound to the promoter region. However, in
the presence of Tetracycline, it binds to the repressor, relieves the
suppression, and causes transcription and translation of the efflux pump,
thereby leading to Tetracycline resistance.

Enzymatic Inactivation
In many cases chemical alterations of antibiotics by some enzymes
inactivate their biological activity and lead to resistance, thus enzymatic
activity of such proteins encoded by genes destroys antibiotic if it gains
entry in to the cell.
eg. Beta-lactamases, which can cleave beta-lactam antibiotics and gives
resistance from it
Amino glycoside-inactivating enzymes, can add Acetyl, Adenyl, and
Phosphoryl groups to inactivate the antibiotic.

Alteration of Drug Target

Antibiotic resistance due to alterations in the target of the drugs in such a
way to counter their toxic effects is common in pathogens and
nonpathogens.
eg. Penicillin resistance, which is due to alterations in penicillin binding
proteins (PBP).
Mutations in DNA gyrase A and B subunits in quinolone resistance is
another example of an alteration of the drug target.

Loss of Enzymes in Drug Activation

In this case, the antibiotic itself acts as a prodrug, which has no direct
activity against the bacteria. Rather, it relies on the activation by a bacterial
enzyme, if such enzymes are altered or loosed, then drugs cannot activate
to kill that bacteria.
eg. Members of the nitrofuran family (nitrofurantion, nitrofurazone,
nitrofurazolidone, etc.), used in the treatment of urinary tract infections,
are reduced by cellular reductases encoded by nfsA and nfsB genes.
Mutations in these genes are associated with nitrofuran resistance (2, 5, 6).

Phenotypic drug resistance

Phenotypic drug resistance refers to the fact that when the bacteria
are not growing, they can become unsusceptible to antibiotics. Then, when
the bacteria are sub-cultured into a fresh media, and they begin to grow
again, they regain their antibiotic susceptibility. This complex mechanism
has been posing significant problems as in biofilm infections and
particularly for TB chemotherapy.
There are three major mechanisms of phenotypic drug resistance-
 Biofilm formation
 Salicylate induced drug resistance
 Bacterial persisters (2, 5, 6).

Biofilm formation
In contrast to the traditional notion that bacteria are free-living and
free-swimming organisms, many bacterial species in nature exist as
organized structures called biofilms, which consist of a self-produced
exopolysaccharide matrix in which bacterial cells (often cells of several
species or consortia) are embedded. They are highly organized; surface-
adherent structures and permit the transport of nutients and metabolic
waste in and out. Many clinical infections such as gingivitis, lung infections
(in cystic fibrosis patients), etc. are attributed to biofilms.
One of the characteristic properties of biofilms is their tolerance to
very high concentrations of antibiotic. The apparent antibiotic resistance of
biofilm associated cells are not due to mutations, since sensitivity
reappears when the biofilms are disrupted and the cells are returned to the
planktonic state. Moreover, it has been shown that biofilm-associated cells
are sensitive to several antibacterial agents (fluoroquinolones, metal
Oxyanions, etc.) (2, 5, 6).

Salicylate induced drug resistance

Another form of phenotypic drug resistance is mediated by salicylic
acid, which is the active component in aspirin. Different organisms have
been found to have Salicylate-mediated drug resistance. E. coli is the best
example, and additional ones include Klebsiella, Pseudomonas, and also M.
tuberculosis.
Let us examine the mechanism of Salicylate-induced antibiotic
resistance in E. coli. There is Multiple Antibiotics Resistance (MAR) operon
in the E.coli where the MarR is the repressor. Salicylate binds to this
repressor to release the suppression of the MAR operon. This operon
encodes a transcription factor, which in turn, activates the transcription of
the efflux pump as well as the membrane channel, which is required for the
functioning of the pump. Thus, the drug resistance mechanism is
conducted through increased efflux (2, 5, 6).

Bacterial persisters
The bacterial persisters are an important example of the phenotypic
resistance. Persistence was first discovered with penicillin in 1944. Joseph
Bigger demonstrated that penicillin can kill merely 99% of the bacteria. The
remaining 1% of the bacteria was persisters. When these persisters were
cultured to fresh media, they regained susceptibility to antibiotics (2, 5, 6).

Managing the Drug Resistance Problem

1. Limiting the Spread of Drug Resistant Bacteria

Several measures could be used to prevent the spread of drug resistant
bacteria. One strategy is to ensure that antibiotics are used only when
necessary. A second strategy is to ensure that they are used for the
appropriate amount of time; that is, that the treatment is not stopped
before it is completed. Patient compliance is a key problem in that respect.
A third strategy for limiting drug resistance is to use antibiotics
combinations. Unfortunately, while all these strategies seem sound in
theory, in reality, the problem persists (4).
2. Development of New Antibiotics
Another possibility is to develop new antibiotics. However, that is not an
easy task. The traditional approach of screening microbes for antibiotics is
not efficient. A second approach, which utilizes target-based screening,
became popular when genomics tools became available. However,
although the idea is appealing, in reality, it is extremely difficult (4).
3. Phage Therapy
Phage therapy can also be used to deal with antibiotics resistance. This
approach had already been used by the Russians during the Second World
War, and has been gaining popularity again in recent years. Phage can be
applied on the wounds of a patient to kill the bacteria, and has proven to be
quite effective. Of course, it cannot be used for internal infections, and the
bacteria might also develop phage resistance (4).

4. Mobilization of Host Defence Mechanisms

Yet another approach is to mobilise host defence mechanisms. This can be
achieved through the mobilisation of innate immunity such as defensins, or
through the development of vaccines, which make antibiotics less
necessary. The idea is to boast the immune response capability to control
the bacterial infection. Of course, that approach is not always successful (4).

5. The Use of Normal Bacterial Flora

Normal bacterial flora in host body can suppress some pathogens (4).

AN AYURVEDIC APPROACH TO MICROBIAL

INFECTIONS
Western allopathic medicine emphasizes the use of antibiotics and
other medicines and approaches to defend against “germs” or microbes
believed to be the primary cause of many health conditions and diseases.
Ayurveda recognizes the microbial approach to some degree, but
generally does not recognize microbes as the primary cause of disease.
According to the Ayurvedic approach, anyone who has developed an
imbalance in their bodily elements, or “doshas,” and has thereby weakened
their immune system, may be subject to a microbial infection which is
considered a symptom of that imbalance.
Ayurveda recognizes as useful anything that will save the patient in
an emergency, including antibiotics, but takes exception to the “magic
bullet” approach of preventing and treating microbial infections strictly
with antibiotics. Ayurveda recommends that balance be established in the
individual for the prevention and treatment of microbial infection (3).
Ayurveda is a traditional Indian medicinal system being practiced for
thousands of years. More than 1,200 species of plants, nearly 100 minerals
and over 100 animal products comprise the Ayurvedic Pharmacopoeia
Asava and Arishta are unique dosage form discovered by Ayurveda
having indefinite shelf life and it was said that the “older the better it is”.
Because this dosage form has an inherent attribute of continuous hydro-
alcoholic extraction and probably formation of natural analogues of the
chemical compounds present in the medicinal plants (7).

To combat bacterial resistance by Ayurvedic

medicines
Many attempts has been made to investigate the potential role of
Ayurvedic medicines and some active compounds for their efficacy to
combat the problems of drug resistance in bacteria by efflux inhibition,
inhibition of biofilm formations, interference in bacterial quorum sensing
etc. however this effect is much more pronounced when its crude extracts
or purified components are studied.
Most available data about the antimicrobial effect of a Ayurvedic
medicines are based on in vitro studies, but several herbs are used due to its
antimicrobial effects in the complementary- or ethnomedicine based on
empirical application in vivo.
The main disadvantage of the results of in vitro studies that it is
difficult to compare to each other because of the different test methods,
different methods of extraction, test assays and variation in chemical
phytoconstituents in plants due to different agro climatic conditions and
plant phenotype.
Useful antimicrobial compounds in ayurvedic medicines can be
divided into several categories, described below and summarized in
Table 2.

Table- 2) Major classes of antimicrobial compounds in Ayurvedic

medicines (11).
Class Example Mechanism
Simple phenols Catechol, Cinnamic enzyme inhibition by the
and phenolic acid, caffeic acid, oxidized compounds
acids. pyrogallol

Quinones Hypericin Bind to adhesins, complex

with cell wall, inactivate
enzymes
Tannins Ellagitannin Bind to proteins, Enzyme
inhibition, Complex with
cell wall, Membrane
disruption
Terpenoids and Camphor, Capsaicin Membrane disruption
Essential Oil
Alkaloids Berberine Intercalate into cell wall
and/or DNA
Lectins and Thionines, Fabatins Formation of ion channels in
Polypeptides the microbial membrane or
competitive inhibition of
adhesion of microbial
proteins to host
polysaccharide receptors.

Simple phenols and phenolic acids.

Some of the simplest bioactive photochemical consist of a single
substituted phenolic ring. Study on antimicrobial activity of a series of
dietary spices and herbal medicines in order to establish their antibacterial
effects against bacteria, of which infections are related to food poisoning.
Results shows that phenolic compounds significantly contributed to the
antibacterial activity of the studied herbs. Cinnamic acid, caffeic acid,
catechol and pyrogallol were shown to be toxic to microorganisms. The
mechanisms thought to be responsible for phenolic toxicity to
microorganisms include enzyme inhibition by the oxidized compounds,
possibly through reaction with sulfhydryl groups or through more
nonspecific interactions with the proteins.

Quinones.
Quinones are aromatic rings with two ketone substitutions. They are
ubiquitous in nature and are characteristically highly reactive. These
compounds, being colored, are responsible for the browning reaction in cut
or injured fruits and vegetables and are an intermediate in the melanin
synthesis pathway in human skin. Their presence in henna gives that
material its dyeing properties, often leading to inactivation of the protein
and loss of function. For that reason, the potential range of quinone’s
antimicrobial effects is great. Probable targets in the microbial cell are
surface-exposed adhesins, cell wall polypeptides, and membrane-bound
enzymes. 

Flavonoids
Flavonoids are hydroxylated phenolic substances. A great deal of
flavonoids are synthesised by plants to fight against bacterial infections.
Their activity is probably due to their ability to complex with extracellular
and soluble proteins and to complex with bacterial cell walls, as described
above for quinones. More lipophilic flavonoids may also disrupt microbial
membranes.
Catechins deserve special mention; catechins exert antibacterial
effects as well via DNA gyrase inhibition. Specific binding of selected
catechins were proved to bind to the N-terminal fragment of gyrase B.
Furthermore, catechins are able to restore the susceptibility of resistance
bacteria to different antibiotics e.g. tetracycline and beta-lactams, beta-
lactamase inhibitors.

Tannins
Tannins are polymeric water-soluble phenols. Tannins are known of
their free radical scavenging activity and have antibacterial effects.
Hydrolysable tannins have antibacterial potential against Helicobacter pylori
and it seems promising in the eradication of the bacterium without
affecting intestinal bacterial flora.

Terpenoids and Essential Oils

The fragrance of plants is carried in essential oil fraction. These oils are
secondary metabolites that are highly enriched in compounds based on an
isoprene structure. They are called terpenes, When the compounds contain
additional elements, usually oxygen, they are termed terpenoids.
Examples of common terpenoids are methanol and camphor
(monoterpenes) and farnesol and artemisin (sesquiterpenoids). Terpenenes
or terpenoids are active against bacteria, fungi, viruses, and protozoa. In
1977, it was reported that 60% of essential oil derivatives examined to date
were inhibitory to fungi while 30% inhibited bacteria.
The tea and petroleum extract of St John’s wort (Hypericum perforatum)
were proved to have antibacterial effect against methicillin resistant
Staphylococcus aureus strains, which are among the most common and most
problematic resistant strains to eradicate.

Alkaloids
Heterocyclic nitrogen compounds are called alkaloids. The first medically
useful example of an alkaloid was morphine, isolated in 1805 from the
Papaver somniferum; the name morphine comes from the Greek Morpheus,
god of dreams. Codeine and heroin are both derivatives of morphine.
Diterpenoid alkaloids, commonly isolated from the plants of the
Ranunculaceae family, are commonly found to have antimicrobial
properties. While alkaloids have been found to have microbicidal effects
(including against Giardia and Entamoeba species, the major antidiarrheal
effect is probably due to their effects on transit time in the small intestine.
Berberine is an important representative of the alkaloid group. It is
potentially effective against trypanosomes and plasmodia. The mechanism
of action of highly aromatic planar quaternary alkaloids such as berberine
is attributed to their ability to intercalate with DNA.

Lectins and Polypeptides

Peptides which are inhibitory to microorganisms were first reported in
1942. They are often positively charged and contain disulfide bonds. Their
mechanism of action may be the formation of ion channels in the microbial
membrane or competitive inhibition of adhesion of microbial proteins to
host polysaccharide receptors.
Among plant peptides, Thionines have toxic effects on different gram
positive and gram-negative pathogenic bacteria. Fabatins, which are
recently identified peptide molecules from fava beans appeared to inhibit
the growth of E. coli and P. aeruginosa.

Other Compounds
Many phytochemicals not mentioned above have been found to exert
antimicrobial properties. Oligomeric proanthocyanidines are mainly used
in vascular diseases and it is based on their ability to trap lipid peroxides
and free radicals and that they non-competitively inhibit xanthine oxidase,
which is a major generator of free radicals.
Most probably, hyperforin may be responsible for the beneficial
effects, which is one of the active agents responsible for the antidepressant
activity (4, 11).

Micro-organism’s used
Escherichia coli
Morphology
It is a Gram negative non-capsulated, short, bacilli 2-4 µ × 0.4-0.7 µ in
diameter and are motile. Spores are not formed.

Pathogenicity
Endotoxin produced by E. coli causes fever, leucopenia, etc. other
lesions produced are Gastroenteritis, Urinary tract infection, Pyogenic
infection, septicemia (10).

Pseudomonas aeroginosa
Morphology
It is slender, Gram negative, bacilli of 0.5×3.5 µ size, actively motile
flagellum. It is noncapsulated.

Pathogenicity
It is most troublesome agents causing nosocomial infections. It is
commonly encountered in secondary infection of burn, wounds, burns and
secondary infection and chronic ulcers of skin (9).

Klebsiella pneumoniae
Morphology
It is a Gram negative non-capsulated, short, bacilli 2-4 µ × 0.4-0.7 µ in
diameter and are motile. Spores are not formed.

Pathogenicity
It may causes pneumonia, urinary tract infection, and pneumonia (10).
Staphylococcus aureus
Morphology
It is ovoid or spherical, non-motile, rarely capsulated, non-sporing
Gram positive organism, arranged in clusters.

Pathogenicity
It causes majority of acute pyogenic lesions in man. Staphylococcal
diseases are mainly of 3 types-
A. Cutaneous lesions- Furuncles, boils, abscess, impetigo, carbuncles etc.
B. Deep infections- Staphylococcal septicemia, scalded skin syndrome,
toxic shock syndrome, pulmonary abscess, renal abscess etc.
C. Staphylococcal food poisoning (8).