Download Antimicrobial natural products

Science against microbial pathogens: communicating current research and technological advances
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A. Méndez-Vilas (Ed.)
Antimicrobial natural products
Kenneth G. Ngwoke1,4, Damian C. Odimegwu2,3 and Charles O. Esimone11
1
Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka Nigeria
Department of Molecular and Medical Virology, Ruhr University Bochum Germany
3
Faculty of Pharmaceutical Sciences, University of Nigeria Nsukka Nigeria
4
Queen’s University Belfast, United Kingdom
2
The efficiency of antibiotics in combating microbial infections was very promising shortly after their introduction. It was
even thought that the microbial war was as good as over as was declared by the Surgeon General of the United States.
However, resistance to these agents developed rapidly afterward and the problem of antibiotic resistance has remained a
menace threatening the benefits of antibacterial agents. As a result, a solution to the issue of antimicrobial resistance is a
matter of urgent importance. Natural products are viewed as a privileged group of structures which have evolved to
interact with a wide variety of protein targets for specific purposes. Also the same protein structure with little or no
variation serves different purposes in different organisms. As a result, it is anticipated that the search for antimicrobial
leads from natural sources will yield better results than from combinatorial chemistry and other synthetic procedures. This
explains why over 90% of antibiotics in clinical use are from natural origin. The same reasons however have been given to
explain the ease with which microorganisms adapt to and resist new antibacterial agents. Believing that pathogens will not
easily resist small synthetic compounds which are alien to nature, combinatorial chemistry and other synthetic methods
were deployed but the results has been dismal. Since compounds from bacteria and fungi are easily resisted and synthetic
chemistry are not yielding the desired result due to lack of ‘privileged structures’ in synthetic compounds, a compound
from natural origin which is not derived from bacteria or fungi but has the desired ‘privileged structure’ will prove to be
the ideal antimicrobial agent. Phytochemicals suit this description and have proven to be potent antimicrobial agents as
will be discussed later in this chapter.
Keywords: Antimicrobial agents; natural products; antibiotic resistance; synthetic chemistry; microoorganisms
Introduction
Extracts from herbs have been used by humans for a wide range of purposes including solving human health problems
[1]. For example, herbs have been used for their antimicrobial properties to treat infections and other diseases due to the
activities of the secondary metabolites contained within them [2-3]. Purified secondary metabolites such as Vinca
alkaloids are used for cancer chemotherapy [4]. Another secondary metabolite digitoxin, from the digitalis leaf, is used
for the treatment of heart failure [5]. Quinine, an alkaloid derived from the bark of the Cinchona tree, and its
derivatives such as chloroquine have been used for the treatment of malaria [6-7] as has artemisinin and its derivatives
from the bark of qinghaosu [8]. In fact, purified secondary metabolites have been the mainstay of malaria treatment [9].
In addition to their therapeutic uses, secondary metabolites possess various attributes which make them beneficial to
mankind. Benefits include food preservation which exploits the antioxidant properties [10] of secondary metabolites
such as phenolics [11] as well as their antimicrobial properties [12]. The presence of both antioxidant and antimicrobial
properties in a single molecule [13] makes them more effective and better suited as food preservatives. Secondary
metabolites from plants have also been used as flavours in food and as fragrances in cosmetics [14]. Technological
advances have influenced the use of herbal extracts for food preservation. For instance, the use of a technology that
combines essential oils with electrolyzed sodium chloride to effectively preserve fish has been reported [15]. Also
synergistic preservative effect resulting when plant principles were combined with metal chelators and organic acid has
been documented [16].
The applications and the potential uses of secondary metabolites are as numerous as there are secondary metabolites
in many species of plant on earth [17]. While some of these secondary metabolites may be by-products of plant
metabolism with unknown function to the plants, others are synthesized for specific purposes. Hydroxylated coumarins
have been reported to accumulate in carrots in response to fungal invasion [18]. Also the accumulation of
glucosinolates known for their antimicrobial properties [19] has been reported in Brassia rapa in response to fungal
infection [20]. Thus, antimicrobial compounds of natural product origin have developed out of these two cascades of
secondary metabolites expression in recorded plant species of importance.
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Development of antibiotics
There are two broad routes to drug discovery: the natural product and the synthetic route [21].
Natural route
The antibiotic discovery from natural source began with the discovery of penicillin from a mold by Alexander Fleming
in 1928 [21]. Natural drug discovery involves the exploration of natural sources such as soil, bacteria, mold and trees
for new chemical entities (NCE) which could be further developed and licensed for clinical use. Majority of the
antibiotics used worldwide are of natural origins [21-23]. In this method, natural products or extracts from the herbs,
bacteria and so forth are first tested for antibacterial activity, followed by purification and characterization of promising
candidates [24]. However, the ease with which pathogens acquire resistance to antibacterial compounds has resulted in
continuous search for a lasting solution.
Antimicrobial resistance
Epidemiology of resistance
The efficiency of antibiotics in combating microbial infections was very promising shortly after their introduction. It
was even thought that the microbial war was as good as over as declared by the Surgeon General of the United States
[25]. However, in his 1945 Nobel Prize speech, Fleming warned that the misuse of antibiotics would result in
resistance. He said ‘there may be danger, though, in under dosage. It is not difficult to make microbes resistant to
penicillin in the laboratory by exposing them to concentrations not sufficient to kill them and the same thing has
occasionally happened in the body’ [26].
A few decades after his speech, the issue of antimicrobial resistance became widespread and a major issue in
medicine. The first case of antimicrobial resistance was published in 1947. Barber reported the isolation of penicillin
resistant Staphylococcus pyogenes in 38 out of 100 patients with staphylococcal infections [27]. The many reports of
cases of resistance led to the search for new antibiotics. In 1957, the -lactam ring, 6-aminopenicillanic acid was purified
paving the way for semisynthetic penicillin. The synthesis of methicillin and ampicillin in 1960 and 1961 marked the
introduction of semisynthetic penicillin sub-group [28-29]. There were reports on some reduction in the incidence of
resistance as result of the introduction of the new antibiotics [29]. Methicillin and other semisynthetic penicillins
resistant to a strain of S. aureus were however isolated soon afterwards in 1961 [30]. By 1984, it was estimated that the
susceptibility of S.aureus to penicillin has reduced from 85% before 1946 to between 20% and 30% [31]. Furthermore,
a surveillance report from the Italian Epidemiology Observatory on the resistance of community acquired
pneumococcal infection to 21 antibiotics documented a total resistance of 14.3% to penicillins. The percentage was less
by 3% in children between 0-5 years compared to adults suggesting a link between usage and development of resistance
[32]. Within the same period in Taiwan (1998-2001), the resistance level was estimated to be about 76% in isolates
from invasive Pneumococcal infection patients [33]. Taiwan is a country where the control of antibiotic usage may be
less than it is in Italy. Antibiotic policy is more liberal in Asia (South east especially) than in Western Europe [34]. To
further examine the link between the level of resistance and usage, a survey carried out in Iceland found a
correlation between the consumption of antibiotics with epidemiology of resistance. The use of antibiotic
declined over a period of time with the percentage of resistant strains in a pool of isolates. On the other hand,
according to the report, while resistance to penicillin declined with decrease in penicillin usage, resistant to
macrolides increased due to increased usage [35]. As at 2003, it was reported that resistance to penicillin of
methicillin resistant Staphylococcus aureus (MRSA) from nasal swab of healthy subjects in Korea was 91%
[36] and 82.1% in Germany [37]. The high incidence in healthy subjects may again be connected with
excessive usage of antibiotics. A recent report from Canada estimated that the MRSA resistance to
fluoroquinolones was about 92% and 90% to clarythromycin [38]. In a separate publication, Jacobs and coworkers reported that the susceptibility of Streptocuccus pnemouniae to penicillins and clindamycin was less
than 49 % of all the isolates tested in the US in 2004 [39]. The proportion of MRSA in all isolates of S.
aureus in England and Wales increased steadily (Figure 1) between 1989 and 1997 [34].
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Figure 1. The proportion of MRSA in all isolates of S. aureus in England between 1989 and 1997 [34]
However a report from the Health Protection Agency (HPA) in the UK indicated that the continuous rise of MRSA
incidence peaked and plateaued at 42% between 2000 and 2002 after which a steady decline was experienced bringing
the incidence to about 38% in 2006 [40]. The decline may be due to greater awareness and stricter antibiotic policy in
force and stringent infection control programmes in the UK [41].
The trend of decline has not been found across the EU. While France experienced a steady decline within the review
period, Hungary saw a sharp increase from 19% in 2005 to 25% in 2006. There seemed to be a greater prevalence in
southern countries including the UK with incidence 25%. In contrast, countries in Central Europe recorded less than
22% prevalence. The northern countries still recorded below 4% at the time of the report [42]. Therefore the
epidemiology of antibiotic resistance varies from region to region and from country to country. While some countries
are recording a decline, others are experiencing an increase. However one thing was clear that whether there was
increase or decrease it has a direct linkage to the clinical use of antibiotics [43]. The exact influence of the veterinary
use of antibiotics on the prevalence of resistance in humans is still being debated [44-45].
Mechanism of bacterial resistance
Bacterial resistance to antibacterial agents can be intrinsic or acquired. Intrinsic resistance is due to natural
characteristics that are genetically encoded in the chromosomal DNA of bacteria which enables them to survive adverse
conditions [46-47]. Examples include the waxy cell wall of the Mycobacterium sp and the outer membrane permeability
barrier in Gram-negative bacteria that reduces access to especially hydrophilic antibiotics [48-51]. Such intrinsic
character is exhibited by all members of a strain and is vertically transferred from parent to offspring [52]. On the other
hand, acquired resistance results from mutation in the host DNA or through the acquisition of resistance encoded extrachromosomal materials in a horizontal transfer process. Plasmids and transposons are frequently involved in horizontal
transfer of resistance [53]. Either intrinsic or acquired, bacterial resistance to antibiotics is achieved by one or more of
the following mechanisms which include direct inactivation of the antibiotic molecule, modification of the antibiotic
target, changes in the permeability of the outer membrane to the drug, deploying of efflux pumps within the cells and
bypassing of drug targets [53-56].
Inactivation of antibiotic molecule
Inactivation of antibiotic molecule can occur through one of three known chemical processes namely hydrolysis, group
transfer and the redox process [57].
Hydrolysis
Some antibiotic molecules have hydrolysable bonds which when broken will lead to loss of activity. The amide bond in
penicillins and the cephalosporins is an example of such feature. These bonds are targeted by some amidases produced
mostly by Gram-negative bacteria such as Escherichia coli [58]. In -lactam antibiotics such as penicillins, studies of the
structure activity relationship have revealed that the integrity of the -lactam ring is necessary for bactericidal activity
against susceptible organisms [59]. Therefore the cleaving of the amide bond, as outlined in Figure 6, will render
the molecule inactive. Lactamase enzymes are divided into different groups primarily based on molecular
structure, functional characteristics and preferred substrates.
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Penicillin
R
C
S
HN
O
C
CH3
CH3
N
O
O
Amide Bond
OH
+
H2 O
-lactamase
R
C
S
HN
O
C
O
CH3
CH3
HN
O
OH
OH
6-aminopenicilloic acid
Figure 2. Equation showing the chemical process involved in the inactivation of -lactam antibiotics by -lactamase enzymes.
Molecular classification of -lactamase enzymes results in 4 groups of enzymes labelled A to D [60-61]. Group 1 has
cephalosporin as the preferred substrate, group 2 inactivates penicillins, cephalosporins and monobactams, group 3
inactivates most -lactam antibiotic including carbapenems while group 4 attacks only penicillins [54]. Although group 3
enzymes, the metallo--lactamases, are not effectively inhibited by most inhibitors, group 1 enzymes are not very
susceptible to clavulanic acid and group 2 enzymes are inhibited by active-site-directed inhibitors [61]. One of the two
main molecular strategies of the -lactamase enzyme is a nucleophilic attack on the active site by a serine based
nucleophile (groups 1, 2 and 4) which forms an intermediate that is then hydrolytically cleaved by a water molecule
(Fig. 2). The second is an attack on the ring resulting from active water molecule activated by the zinc centre of the
metallo-enzymes (group 3) [57]. There is also an extended spectrum--lactamases (ESBL) which can confer resistance to
all penicillins, third generation cephalosporins, carbapenems and aztreonams -lactamase enzymes can be coded on the
chromosomes and transferred vertically or horizontally through plasmids and transposons. The group 1 enzymes that
rapidly degrade cephalosporins are chromosomally encoded while the extended spectrum -lactamases can be plasmid
and transposon born [62]. The horizontal method of gene transfer has been viewed as being responsible for resistance
transfer from animal born pathogen to human‘s intestinal pathogens [63](Bates et al., 1993).
Other hydrolytic enzymes such as esterases and epoxidases have also been linked with resistance of bacteria to some
antibiotics. For instance macrolide esterases found in E.coli BM2195 has been linked with resistance to
erythromycin [64]. Epoxidases also hydrolyse the epoxide ring in epoxide antibiotics such fosfomycin [65].
Group Transfer
Resistance by group transfer is facilitated by a diverse group of enzymes that operate solely in the cytosol. These
enzymes induce resistance by chemically modifying the molecules through covalent processes requiring ATP, Acetyl
CoA, UDP-glucose as co-substrates [54]. This process leads to alteration in structure of the target site resulting in
impaired antibiotic-bacteria interaction. The enzymes achieve the chemical modification by O- and N-acylation [66-67]
O-phosporylation [68-69], O-glycosylation, O-ribosylation [70], O-nucleotidylation thiol transfer [71]. Antibiotics can
be inactivated by one or more of these chemical modifications. An example is chloramphenicol which is inactivated by
chloramphenicol acetyltransferases [72-73]. Some of these enzymes have been determined from Pseudomonas
aeuroginosa [74]. Virginiamycin a drug used as a growth promoter in animal husbandry is also susceptible to
inactivation by this group of enzymes which are plasmid encoded from S. aureaus a Gram positive coccus [75]. The
aminoglycosides, a class of antibiotics are susceptible to inactivation by phosphorylation at specific hydroxyl residues,
N-acylation or Nucleotidyltransferases [76].
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Redox Process
Higher mammals such as humans metabolise drugs and other xenobiotics via oxidation and reduction mechanisms,
resulting in detoxification and excretion of drugs [77-78]. Some bacteria also use the same mechanisms to some extent
to detoxify and resist antibiotic effects. TetX is an oxidative enzyme that degrades tetracycline through the redox
process. TetX gene is harboured in a transposon in Bacteriodes fragilis transposons. The enzyme catalyses the
hydroxylation of tetracycline, leading to changes at the magnesium ion binding site and subsequent loss of activity [7980]. Another enzyme, an NADPH -dependent reductase thought to be produced by Streptomyces virginiae catalyses the
inactivation of virginiamycin M1 by a reduction process [81].
Modification of antibiotic targets
Bacteria have developed resistance to antibiotics by simply altering the structure of the antibiotic targets which serves
as a pedestal for antimicrobials to elicit their inhibitory effect [82]. For a drug to elicit a desired effect, it must bind to a
target. For it to bind to the target, it must have an affinity for the target [83]. The level of affinity a drug has for a target
is dependent on the complimentarity of the drug and the target [84]. If there is any modification on the structural
mortise of the target molecule without a complimentary change on the antibiotic structure, the affinity will reduce or
disappear.
Modification of the target molecules may be achieved through mutation [82]. Mutation in DNA gyrase and
topoisomerase IV enzymes which are the targeted molecules of fluoroquinolones has been reported as being responsible
for resistance to this group of antibiotics. Reports suggested that mutations in gyrA and gyrB genes of DNA gyrase and
in the parC and parE genes of the topoisomerase enzyme confer resistance to susceptible bacteria against the quinolones
[85-88]. It has also been reported that the affinity of -lactam antibiotics to penicillin binding protein (PBP) reduces upon
mutation of the PBP genes. Resistance to macrolide-lincosamide-streptogramin like antibiotics due to target alteration
has been reported [89]. In this case, the target is modified by post-transcriptional methylation of the 23S ribosomal
RNA which is catalysed by erythromycin resistance methylase (erm) N-transferase enzyme encoded by erm gene.
These enzymes have been characterised from several organisms such as Gram-positive cocci and Bacillus spp [90-91].
Efflux pump mediated resistance
Some antibiotics that act in the cytosol need [92] to be transported across the cell membrane before they reach their
target [93]. They also have to achieve an effective concentration in the cytosol to exert inhibitory effects on
microorganisms. Antibiotics that work as protein synthesis inhibitors including macrolides and tetracyclines as well as
gyrase enzyme inhibitors such as fluoroquinolones will fall within this group.
Efflux pumps are membrane proteins that function by transporting antibiotic molecules from the cytoplasm of the
bacteria to the extracellular space. With this action, they effectively reduce the concentration of the antibiotic in the
cytoplasm to a sub-effective concentration [94]. This prevents accumulation and confers resistance to a wide variety of
antimicrobial. Some efflux pumps can be specific while others mainly confer multidrug resistance.
Multidrug resistance conferring efflux pumps can export a variety of related and non-related xenobiotics from
bacterial cells and several classes of multidrug efflux pumps have been identified [54]. These include the
drug/metabolite transporter (DMT) superfamily under which are subfamilies comprising the small multidrug resistance
(SMR) family; the multidrug and toxic compound extrusion (MATE) family and the ATP binding cassette (ABC)
family. The other families include major facilitator superfamily (MFS) and the resistance-nodulation-division (RND)
family [49, 54, 95-100]. In Gram-negative organisms, efflux pumps are mostly associated with the multidrug exporter
families. The RND family has been found to contribute mostly to clinical resistance especially in Gram negative
organisms. They combine with periplasmic membrane proteins, which are also called outer membrane factor that alters
permeability to antimicrobials to confer resistance [101, 102]. In Gram negative organisms these transporters mediate
the intrinsic resistances. For instance, Pseudomonas aeruginosa which is known to possess high intrinsic resistance to
many antibiotics is reported to have upward regulation of efflux transporters that is chromosomally encoded in the
presence of xenobiotics [49, 103]. Intrinsic multidrug resistance due to the multidrug efflux pump has also been
demonstrated in other Gram negative organisms e.g. E. coli [104]. This suggests that efflux transporters can play an
important role in multidrug intrinsic resistance in both Gram positive and Gram-negative organisms.
Inhibition of efflux pump transporters has been considered an avenue for combating antibiotic resistance. Some
inhibitors have been reported and optimization of activity has been an ongoing research activity [105-108].
Outer membrane impermeability
Gram negative bacterial cells are surrounded by outer membranes which are impervious to many drug compounds [98,
105-108]. Some hydrophilic antibacterial compounds access the cytoplasm via water filled channels created by porins
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which are membrane proteins that cross the cellular membrane and act as a pore through which molecules can diffuse
into the cytosol [109].
In the case of quinolones, in addition to diffusing through the porin pathway, they can also diffuse through the
bilayer by disorganization of the bilayer. The disorganization of the bilayer occurs as a result of the chelation of
divalent cations such as magnesium by the quinolones. Also, the aminoglycosides may cause perturbation of the bilayer
by the displacement of divalent cations enabling them to diffuse though the bilayer in addition to going through the
porin pores [110]. There is a correlation between the hydrophobicity of the antibiotic and resistance due to outer
membrane impermeability. This implies that hydrophilic molecules which are not small enough to diffuse through the
porin channels of Gram-negative microorganisms may not be able to pass through to the cytoplasm except if it has a
mechanism of disorganizing the lipid bilayer like the quinolones [111-112].
However, the resistance to antibiotic conferred on bacteria by outer membrane impermeability alone does not pose a
serious problem when it is not combined with other mechanisms of resistance. In combination with either efflux
transporters and or -lactamase enzymes, outer membrane impermeability enhances the resistance of bacteria to an
antibiotic [50, 102, 113-115]. The influx of antibiotics through the outer membrane varies depending on hydrophobicity
and size. The faster the influx, the quicker the accumulation of the drug in the periplasmic space so that the minimum
effective concentration (MEC) required for the activity of the antibiotic will be achieved. It has been mentioned
previously that the efflux pump in P. aeruginosa is up-regulated in the presence of antibiotics and coupled with its
unique outer membrane barrier it confers the intrinsic multidrug resistance character to the organism that is clinically
significant [51, 56, 103, 115].
Acquired resistance
Apart from the resistance an organism has due to its inherent characteristics such as the outer membrane barrier
discussed above, resistance to antibiotics could be acquired [116, 117]. In this case organisms which were susceptible to
an antibiotic become resistant leading to therapeutic failure in most cases. Acquired resistance could arise from
mutational adaptation or due to horizontal transfer facilitated by conjugational mobile genetic elements such as the
plasmids and the transposons [118].
Mutational Adaptation
Vertical transfer
Although an organism can undergo mutation spontaneously in the absence of an antibiotic (growth dependent mutation)
[119], adaptive mutation can be triggered by the continued presence of a non-conducive environment e.g. an antibiotic
used in a sub-lethal concentration. It has been shown that if an organism is exposed to a concentration below the
minimal inhibitory concentration of an antibiotic, the organism tends to mutate in order to adapt to the drug
environment.
Fleming warned that administration of inadequate dose antibiotics would teach the microorganisms to resist them
[26]. The result of the mutation is the production of another strain of the organism that is resistant to the activities of the
antimicrobial to which it was once susceptible. The survival of this resistant strain however depends on the continued
presence of the antibiotic especially if the therapeutic concentration that wipes out the susceptible strain is administered
[120]. If the environment is in appropriated conditions, the resistant strain can multiply and transfer the resistance
encoded genes to its new generations. This is known as vertical transfer resistance [119, 121].
Horizontal transfer
Horizontal transfer is another method through which bacteria can acquire resistance. In this mechanism, a resistance
gene is acquired from mobile genetic elements such as plasmids, transposons and integrons [122]. Phage viruses can
also serve as a conduit for resistance transfer when it invades susceptible bacteria [123-124]. Horizontal gene transfer
has been suggested as the route for the resistance transfer between different species of microorganism especially in
hospitals and between food animal and man [125].
Mobile genetic elements involved in horizontal transfer include plasmids, transposons and integrons. Plasmids are
extra-chromosomal DNA found in some bacteria with various types of genes that encode for varying functions which
are not normally found in bacterial chromosomes [126]. They contain genes that code for such functions as multidrug
resistance and enzymes that digest food and pollutant [127-128]. The loss or gain of a plasmid does not affect growth
and replication of bacteria. The R-plasmid (R= resistance) has been found in Gram negative bacteria. Plasmids are
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capable of independent replication [129] unlike the transposons. Transposons are DNA elements which in addition to
coding for resistance genes also codes for enzymes called transposase that catalyses transposition [128]. They are gene
sequences that can move from one location to another in a chromosome, as a result they are also called ‘jumping genes‘.
Transposons can be carried by plasmids or exist as part of bacterial nucleoids in which case they are called conjugative
transposons. Transposons cannot replicate independently [130-132]. Since they can transfer resistance genes from
plasmid to plasmid or from plasmid to chromosomes, they make it easier for susceptible organisms to acquire resistance
[133].
An integron is another genetic element involved resistance transfer. It has a provision or site in its structure for the
incorporation or integration of extra DNA which could be attached as gene cassette and also encodes for an enzyme that
facilitates the site-specific recombination event. Integrons were first described by Hall and Stokes [134]. The IntI sitespecific recombinase is called an integrase and has a promoter region for the expression of the integrated cassette [133136]. Multidrug resistance facilitated by integrons has been a cause of concern in clinical practice [137].
Any of the described mobile elements can be transmitted through one or more of the following mechanisms:
transformation, phage transduction and conjugation [135]. Resistance transfer by transformation involves the
acquisition of resistance genes from the growth medium. This gene may have been released by dead bacteria [138-139].
Phage transduction occurs when a bacteria DNA is packed into the phage genome on phage inversion. The bacteria
DNA could be transferred to another bacteria following inversion. Recombination could take place and if the segment
transferred is resistance encoded, resistance characteristic is thus transferred from one cell to another [142].
Conjugation is another mechanism by which bacteria transfer genetic materials. It is a sexual process which requires
a contact between two bacterial cells in which one designated F+ is the donor and the F- the receiver. The donor has the
fertility factor F [126]. The plasmid in Gram-negative bacteria carries the gene that encodes for sex pilus. During
conjugation, the donor extends its pilus, a hollow protein tube until it touches and fuses with the receipt cell [126].
The genetic material is transferred as a single stranded DNA when the bacterial cells are drawn together to create a
conjugational junction.
In Gram positive bacteria, conjugation does not involve sex pilus. It is believed to be initiated by special peptides
called pheromones [126]. In this case, it is the recipient that secrets the pheromones that stimulates the donor to secret a
cell surface component that enables mating and the transfer of plasmid. This has been demonstrated in Enterococcus
and Streptococcus species [143-144].
As the single stranded DNA enters the recipient, it is copied to form double stranded DNA. The classical
recombination mechanism that requires considerable homology between the recombining DNA molecules has been
blamed for the penicillin resistance in Streptococcus pneumonia and Neisseria gonorrhea that involves PBP gene
mosaic [129, 135]. Another recombination mechanism that involves transposable elements requires no homology
between the recombining DNA molecules. A third mechanism that involves the integrons and gene cassettes is a sitespecific recombination mechanism in which a recombinase enzyme facilitates the recombination of short homologues
[129, 135].
Semi-Synthetic route
The semi-synthetic route was ventured into due to antibiotic resistance to natural antibiotics and instability to acidic
medium [145]. Following the purification and identification of the pharmacophore of penicillin [28] and the
understanding of the mechanism of resistance by -lactamase enzymes [146-147], the pharmaceutical companies began
to modify the antibiotic molecules in such a way as to retain activity while resisting inactivation by microbial enzymes.
This method became possible when the structure of the beta-lactam ring which is the pharmacophore of penicillin was
determined. After it was discovered that the beta lactamase enzymes hydrolyse the -lactam bond to render the
pharmacophore inactive [147], attempts were made to introduce a moiety that would stabilize the pharmacophore
against the attack of the enzymes. Aminopenicillins and methicillin are also examples of derivatives produced by the
synthetic route [28]. The modification affected the properties of the compounds as it pertains to acid and alkaline
stability and stability to degradation by bacterial enzymes. Aminopenicillin is stable to stomach acid and can be
administered orally while methicillin is stable against the beta-lactamase enzyme. Thus methicillin (carboxypenicillin)
and ticarcillin were fashioned from penicillin for the treatment of infections due to resistant staphylococcus and
pseudomonas spp respectively [21].
The synthesis of other semi-synthetic antibiotics was also reported. Minocycline and doxycycline are both
tetracycline analogues [146]. Second and third generation cephalosporins were possible through semi-synthesis [149150]. Semi-synthesis was not, however, very effective in combating the dynamic resistance posed by the microbial
world. Cross resistance between different antibiotics of the same family was a major problem. It was thought that the
ease with which bacteria develop resistance to natural compounds could be due to co-evolution between the organism
and the antibacterial. The companies thought of introducing xenobiotics which are compounds that are not known to
nature [151].
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Synthetic route
The sulphonamides were the first set of antibiotics that were synthesized. Prontosil, a component of a dye which was
prepared for use in the textile industry was accidentally found to be active against microorganisms by a German chemist
[152]. The next product of pure synthesis did not arrive until 1962 when the quinolones were synthesized. It took about
40 years for another synthesized compound Linezolid (oxazolidinone) to be approved for clinical use [82]. The rapid
development and promises of the synthetic route was thought to pull the pharmaceutical industry away from the natural
drug discovery route.
Combinatorial chemistry has played a large role in the search for drug leads but it was not until 2005 when the first
product of combinatorial chemistry as mode of drug discovery was approved for clinical use. Sorafenib (Nexavar) was
approved by the FDA for the treatment of advanced renal cancer [153] and it received its marketing license in Europe in
2006 [154]. Otherwise combinatorial chemistry is regarded as a failure [24] when comparing the number of new drugs
from natural or synthetic route. It may underpin the reason why the interest in natural product lead discovery is being
renewed recently [145, 155-156].
Co-evolution which has been suggested as the reason why bacteria easily acquire resistance against antibacterial
molecules from natural origin is also the reason why it is more likely to get an antibacterial lead from nature than by
synthesis [157].
Natural products are viewed as a privileged group of structures which have evolved to interact with a wide variety of
protein targets for specific purposes [156]. Also the same protein structure with little or no variation serves different
purposes in different organisms [158]. Most of the proteins and structures are products of similar pathways in different
organisms. Taking labdane diterpenoids for example, many labdane diterpenoids have been isolated from different plant
families including the Zingiberaceae, Labiateae, Apocyanaceae and Euphorbiaceae [159]. Biological properties such as
antibacterial [160-161] have been attributed to the labdanes. It has been suggested that biological activity of diterpenes
may be derived from the fact that they share the same biosynthetic pathways as in other higher plants, bacteria and
humans [159]. Biosynthesis of diterpenes takes place through the Mevalonic acid (MVA) pathway in plants cell saps. A
MVA-independent pathway has been described in the plastids of plants [163-164]. In humans, MVA pathways have
important roles within cells regarding the synthesis of sterols and subsequently cholesterol which is an important
component of cell and body [165](Knight et al., 2009). The MVA pathway has been suggested as an important target
for cancer chemotherapy [166] and cytotoxic activities have been reported in diterpenes isolated from plants [167-168].
The isoprenoid pathways which could be MVA dependent or MVA-independent in plants and bacteria are also similar
at the early stages of synthesis [164] and many antibacterial diterpenes have been isolated [160-161, 169-170].
Therefore the similarity in these pathways shared in plants, humans and microorganisms may account for the
widespread biological activities inherent in diterpenoids. It has been suggested that the same protein binding target in
different organisms can perform different functions in different organisms and therefore natural products by the way of
molecular evolution has conserved the ability to preferentially bind to this targets [156].
Most of the antibiotics in clinical use though from natural origin are largely from bacterial and fungal cells which are
largely undifferentiated when compared to higher plants. Because co-evolution has been fingered as a reason for
resistance development and acquisition in bacteria, one could think that all antibiotics sourced from nature would easily
lose its potency against previously susceptible organisms. However, plant products have been used as antibiotics for
thousands of years and the same plants remedies passed on from generations to generations has remained effective
against traditional indications. It means that bacterial compounds have reduced ability to adapt to plant derived
antibacterial regimen. This is an advantage which regimens derived from higher plants have over the conventional
antibiotics derived from microorganisms. The plant derived regimen though they have the advantage of co-evolution,
conserved the ability to preferentially bind to specific targets as a result of their ‘privileged structures’ would have due
to divergence during the course of evolution acquired characteristics which has rendered them less susceptible to the
bacterial resistance mechanisms. As a result it is expected that plant derived antibacterial compounds will be able to
stem or at least slow down the continuous emergence of resistance to antibacterial agents.
Recent approaches in natural product research
It has been reported that out of 22 antibiotics approved by the FDA, more than half were derived from natural
antibiotics while all the remaining less one were derived from quinolones [171]. The failure of the synthetic routes to
deliver more leads and the recognition that natural products have drug-like properties [145, 172] has resulted in
renewed interest in the natural product discovery which has been known to have delivered most of the antibiotics in use
today.
Increasing efforts have been found in exploring different natural sources for new drug discovery. Some focus on
marine diatoms while others are in the search for antimicrobial peptides from insects [173-174] and amphibians [115,
175]. Antimicrobial peptides are collectively called defensins and can also be found in plants [176-177]. The traditional
search for antibiotic compounds from soil bacteria and fungi has also received renewed interest. In 2006,
plantensimycin was isolated from Streptomyces plantensis a soil organism from South African soil [178]. Also some
1018
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focus has been shifted to the marine actinomycetes as the possible source of NCE especially after the success of the
isolation of salinsporamide A, an inhibitor of proteasome from the Salinospra genus. New classes of terpenoids and
amino acid derived compounds have also been isolated [179-180].
Genomic technology and natural product discovery
Before the successful sequencing of the DNA genome of Haemophilus influenzae in 1995 [181-182], antimicrobial
sensitivity testing involved solely the testing of compounds against whole organisms. Presently, antimicrobial discovery
involves high throughput screening of low molecular weight molecules against a genomic target [183-184]. This means
that the target of an antimicrobial compound could be known before the screening. This process enables the screening
of a large number of molecules within a shorter time. However, the complex nature of natural extracts in the crude
preparation state may mean it will be difficult to design robust high throughput screening methods unless they are
purified as single compounds. Purification of single compounds from plant extracts is cumbersome but not impossible.
Evidence
Croton macrostachyus has been used for various medicinal purposes in traditional Africa such as the treatment of
malaria by the Zay people of Ethiopia [185] and the sap is used by the Kikuyus of central Kenya for wound healing and
ringworm treatment [186]. The latter use would have been possible due its possible antifungal and antibacterial
properties. However, Matu and Johannes [187] reported no antibacterial activity of the methanol, water and hexane
extracts of the specimen, but they reported a high level of anti-inflammatory activity on the root extract [187]. The
reason for the inactivity could be attributed to the extraction method and solvent.
Bridelia speciosa, Marattia fraxinea and Entada abysinnia are among the numerous plant extract effectively used for
traditional healing in Africa. B. speciosa is used in Cameroon for the treatment of malaria, urinary track and
gastrointestinal bacterial infection while M. fraxinea stem cutting is used in Cameroon for the treatment of infections
whilst the leaf stalk is also used for the treatment of infection in Tanzania [188]. E. abysinnia (abyssinica) is used for
the treatment of coughs, alleviation of arthritic pain and for the treatment of bronchitis in east Africa [189]. Kolavenol,
an anti-trypanosomal compound was isolated from the bark of E. abysinnia [190].
Antimicrobial activities observed in plant extracts are due to their secondary metabolites content. The knowledge of
this has led to the isolation of many secondary metabolites which has shown strong activities. A compound isolated
from a West African leguminous plant Helichrysum aureonitens has strong activity against gram-positive bacteria,
fungi and viruses including HSV-1 virus. The compound galangin is also active against cocsackie B virus.
Also from a West African legume, an isoflavone alpinumisoflavone, which prevents schistosomal infection
when applied topically was isolated and reported whereas phloretin isolated from some apples has strong
activity against a variety of microorganisms [13].
When an extract of Solanum nigrescens was compared with nystatin as a treatment for Candida albicans-induced
vaginitis, both given as intravaginal suppositories, in women with confirmed C. albicans vaginitis, the extract proved to
be as efficacious as nystatin. Also, cranberry juice has been shown to reduce the bacteria count in urine culture of
treated elderly women with urinary tract infection as compared to the untreated women. The efficacy and safety of
Virend® ( SP-303) which is a polyphenol isolated from a Euphorbiaceae shrub as a topical antiherpes agent has been
established in phase II studies [191-192].
Conclusion
There is therefore sufficient evidence to conclude that plants are a reservoir of antimicrobial substances which are not
only potent against target pathogens but also seems to stand a better chance to overcome the onslaught of microbial
resistance mechanisms.
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