Science against microbial pathogens: communicating current research and technological advances _______________________________________________________________________________ A. Mndez-Vilas (Ed.

Antimicrobial natural products

Kenneth G. Ngwoke1,4, Damian C. Odimegwu2,3 and Charles O. Esimone11
1 2

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 Queens University Belfast, United Kingdom 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 O O HN S CH3 CH3 O

Amide Bond
OH

+ -lactamase

H2 O

C O

HN

CH3 CH3 O

C O OH

HN

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 humans 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

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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.

References
[1] [2] ROJAS A, HERNANDEZ L, PEREDA-MIRANDA R, and MATA R. Screening for antimicrobial activity of crude drug extracts and pure natural products from Mexican medicinal plants. J. Ethnopharmacol. 1992; 35: 275-283 AKHONDZADEH S, NOROOZIAN M, MOHAMMADI M, OHADINIA S, JAMSHIDI AH and KHANI M. Melissa officinalis extract in the treatment of patients with mild to moderate Alzheimers disease: a double blind, randomised, placebo controlled trial. Journal of Neurology, Neurosurgery & Psychiatry. 2003; 74 (7): 863-866.

FORMATEX 2011

1019

Science against microbial pathogens: communicating current research and technological advances ______________________________________________________________________________ A. Mndez-Vilas (Ed.)

[3] [4] [5] [6] [7] [8] [9] [10]

[11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35]

RAJBHANDARI M, MENTEL R, JHA PK, CHAUDHARY RP, BHATTARAI S, GEWALI MB, KARMACHARYA N, HIPPER M and LINDEQUIST U. Antiviral activity of some plants used in Nepalese traditional medicine. Evidence-based Complementary and Alternative Medicine, 2009; 6 (4): 517-522. SAHENK Z, BRADY ST and MENDELL JR. Studies on the pathogenesis of vincristine-induced neuropathy. Muscle & nerve. 1987; 10 (1): 80-84. HOLLMAN A. Digoxin comes from Digitalis lanata. BMJ, 1996; 312 (7035): 912. BUCHER C, SPARR C, SCHEIZER WB and GILMOUR R. Fluorinated quinine alkaloids: synthesis, x-ray structure analysis and antimalarial parasite chemotherapy. Chemistry - A European Journal, 2009; 15 (31): 7637-7647. GREENWOOD D. The quinine connection. Journal of Antimicrobial Chemotherapy, 1992; 30 (4): 417-427. HAYNES RK, KRISHNA S. Artemisinins: activities and actions. Microbes and Infection. 2004; 6 (14): 1339-1346. WRIGHT C. Plant derived antimalarial agents: new leads and challenges. Phytochemistry Reviews. 2005; 4 (1): 55-61. VEERAPUR VP, PRABHAKAR KR, PARIHAR VK, KANDADI MR, RAMAKRISHANA S, MISHRA B, SATISH RAO BS, SRINIVASAV KK, PRIYADARSINI KI, UNNIKRISHNAN MK. Ficus racemosa Stem Bark 160 Extract: A Potent Antioxidant and a Probable Natural Radioprotector. Evidence-based Complementary and Alternative Medicine. 2009; 6 (3): 317-324. KOUAKOU-SIRANSY G, SAHPAZ S, IRI-NGUESSAN G, DATTE YJ, KABLAN, J, GRESSIER B and BAILLEUL F. Oxygen species scavenger activities and phenolic contents of four West African plants. Food Chemistry. 2010; 118 (2): 430435. DE MARTINO L, De Feo V and NAZZARO F: Chemical composition and in vitro antimicrobial and mutagenic activities of seven Lamiaceae essential oils. Molecules: 2009; 14: 4213-4230. COWAN M. Plant products as antimicrobial agents. Clinical Microbiology Review: 1999; 12 (4): 564-582. BOHLMANN J and KEELING CI. Terpenoid biomaterials. The Plant Journal. 2008; 54 (4): 656-669. MAHMOUD BSM, YAMAZAKI K, MIYASHITA K, SHIN I and SUZUKI T. A new technology for fish preservation by combined treatment with electrolyzed NaCl solutions and essential oil compounds. Food Chemistry. 2006; 99 (4): 656-662. ZHOU F, JI B, ZHANG H, JIANG H, YANG Z, LI J, LI J, REN Y and YAN W. Synergistic effect of thymol and carvacrol combined with chelators and organic acids against Salmonella typhimurium. Journal of Food Protection, 2007; 70: 17041709. GURIB-FAKIM A. Medicinal plants: Traditions of yesterday and drugs of tomorrow. Molecular aspects of medicine. 2006; 27 (1): 1-93. DARVILL AG and ALBERSHEIM P. Phytoalexins and their elicitors - a defense against microbial infection in plants. AnnuaL review of Plant Physiology and Plant Molecular Biolog. 1984; 35: 243-275. AL-GENDY AA, EL-GINDI OD, HAFEZ AS and ATEYA AM. Glucosinolates, volatile constituents and biological activities of Erysimum corinthium Boiss. (Brassicaceae). Food Chemistry. 2010; 118 (3): 519-524. ABDEL-FARID IB, JAHANGIR M, VAN DEN HONDEL CAMJJ, KIM HK, CHOI YH and VERPOORTE R. Fungal infection-induced metabolites in Brassica rapa. Plant Science. 2009; 176(5): 608-615. SINGH SB and BARRETT JF. Empirical antibacterial drug discovery-foundation in natural products. Biochemical Pharmacology. 2006; 71 (7): 1006-1015. CRAGG GM, NEWMAN DJ and SNADER KM. Natural products in drug discovery and development. Journal of natural products. 1997; 60 (1): 52-60. NEWMAN DJ, CRAGG GM and SNADER KM. Natural products as sources of new drugs over the period 1981-2002. Journal of Natural Products: 2003; 66 (7): 1022-1037. LI JWH and VEEDERAS JC. Drug Discovery and Natural Products: End of An Era or An Endless Frontier? Science. 2009; 325: 161-165. OVERBYE KM and BARRETT JF. Antibiotics: Where did we go wrong? Drug Discovery Today, 2005; 10 (1): 45-52. FLEMING A. 1945. Penicillin. Nobel Prize Lecture, http://nobelprize.org/nobel_prizes/medicine/laureates/1945/fleming-lecture.pdf (accessed20/01/010). BARBER M. Staphylococcal infection due to penicillin-resistant strain. British medical journal. 1947; 863-865. ROLINSON GN and GEDDES AM. The 50th anniversary of the discovery of 6-aminopenicillanic acid (6-APA). International Journal of Antimicrobial Agents: 2007; 29 (1): 3-8. WALDVOGEL FA. New resistance in Staphylococcus aureus. The New England Journal of Medicine: 1999; 340 (7): 556557. JEVONS MP. "Celbenin" - resistant Staphylococci. British medical journal. 1961; 1 (5219): 124-125. ATKINSON BA and LORIAN V. Antimicrobial agent susceptibility patterns of bacteria in hospitals from 1971 to 1982. Journal of Clinical Microbiology. 1984; 20 (4): 791-796. MARCHESE A, MANNELLI S, TONOLI E, GORLERO F, TONI M, SCHITO GC. Prevalence of antimicrobial resisitance in Streptococcus 97 pneumonia circulating in Italy: results of the Italian epidemiological observatory survey (1997-1999). Microbial Drug Resistance. 2001; 7 (3): 277-287. Siu LK, Chu ML, Ho M, Lee YS and Wang CC. Epidemiology of invasive pneumococcal infection in Taiwan: antibiotic resistance, serogroup distribution, and ribotypes analyses. Microb Drug Resist. 2002; 8: 201208. SMAC. 1998. The part to least resistance. UK standing medical advisory committe report, http://www.dh.gov.uk/en/Publicationsandstatistics/Publications/PublicationsPolicyAndGuidance/DH_4009357?ssSourceSiteI d=ab ARASON VA, SIGURDSSON JA, ERLENDSDOTTIR H, GUDMUNDSSON S. and KRISTINSSON KG. The Role of Antimicrobial use in the epidemiology of resistant pneumococci: a 10-year follow up. Microbial Drug Resistance. 2006; 12 (3): 169-176.

1020

FORMATEX 2011

Science against microbial pathogens: communicating current research and technological advances _______________________________________________________________________________ A. Mndez-Vilas (Ed.)

[36] [37] [38]

[39] [40] [41] [42] [43]

[44]

[45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63]

JEONG H, LEE J, CHOI B, SEO K, PARK S, KIM Y, BAEK K, LEE K and RHEE D. Molecular epidemiology of community-associated antimicrobial-resistant Staphylococcus aureus in Seoul, Korea (2003): Pervasiveness of multidrugresistant sccmec type ii methicillin-resistant s. aureus. Microbial Drug Resistance. 2007; 13 (3): 178-185. FLUEGGE K, ADAMS B, LUETKE VOLKSBECK U, SERR A, HENNEKE P and BERNER R. Low prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in a South Western region of Germany. European journal of pediatrics. 2006; 165 (10): 688-690. ZHANEL GG, DECORBY M, LAING N, WESHNOWESKI B, VASHISHT R, TAILOR F, NICHOL KA, WIERZBOWSKI A, BAUDRY PJ, KARLOWSKY JA, LAGACE-WIENS P, WALKTY A, MCCRACKEN M, MULVEY MR, JOHNSON J, THE CANADIAN ANTIMICROBIAL RESISTANCE ALLIANCE (CARA), and HOBAN DJ. Antimicrobial-Resistant Pathogens in Intensive Care Units in Canada: Results of the Canadian national intensive care unit (CAN-ICU) Study, 2005-2006. Antimicrobial Agents and Chemotherapy. 2008; 52 (4): 1430-1437. JACOBS MR, GOOD CE, BEALL B, BAJAKSOUZIAN S, WINDAU AR and WHITNEY CG. Changes in serotypes and antimicrobial susceptibility of invasive Streptococcus pneumoniae strains in cleveland: a Quarter Century of Experience. Journal of Clinical Microbiology. 2008; 46 (3): 982-990. LHPA. 2007. Antimicrobial resistance in England, Wales and Northern Ireland, 2006. LHPA report, http://www.hpa.org.uk/web/HPAwebFile/HPAweb_C/1216798080469 (accessed 10/02/010). PATEL D and MADAN I. Methicillin-resistant Staphylococcus aureus and Multidrug Resistant Tuberculosis: Part 1. Occupational Medicine. 2000; 50 (6): 392-394. EARSS. 2006. The European antimicrobial resistance surveillance system. EARSS Report, http://www.rivm.nl/earss/Images/EARSS%202006%20Def_tcm61-44176.pdf (accessed, 11/02/010. TACCONELLI E, DE ANGELIS G, CATALDO MA, MANTENGOLI E, SPANU T, PAN A, CORTI G, RADICE A, STOLZUOLI L, ANTINORI S, PARADISI F, CAROSI G, BERNABEI R, ANTONELLI M, FADDA G, ROSSOLINI GM and CAUDA R. Antibiotic Usage and Risk of Colonization and Infection with Antibiotic-Resistant Bacteria: a Hospital Population-Based Study. Antimicrobial Agents and Chemotherapy. 2009; 53 (10): 4264 - 4. ROLLO SN, NORBY B, BARTLETT PC, SCOTT HM, WILSON DL, FAIT VR, LINZ JE, BUNNER CE, KANEENE JB, HUBER JC Jr. Prevalence and patterns of antimicrobial resistance in Campylobacter spp isolated from pigs reared under antimicrobial-free and conventional production methods in eight states in the Midwestern United States. J Am Vet Med Assoc. 2010; 15: 236 (2): 201-10. PHILLIPS I. Withdrawal of growth-promotongantibiotics in Europe and its effects in relation to human health. International Journal of Antimicrobial Agents. 2007; 30: 101-107. LECLERCQ R and COURVALIN P. Intrinsic and unusual resistance to macrolide, lincosamide, and streptogramin antibiotics in bacteria. Antimicrobial Agents and Chemotherapy. 1991; 35 (7): 1273-1276. STRATEVA T and YORDANOV D. Pseudomonas aeruginosa - a phenomenon of bacterial resistance. Journal of medical microbiology. 2009; 58 (9): 1133-1148. WEISS J and BECKERDITE-QUAGLIATA SAEP. Resistance of gram-negative bacteria to purified bactericidal leukocyte proteins: relation to binding and bacterial lipopolysaccharide structure. The journal of clinical investigation. 1980; 65 (3): 619-628. POOLE K. Multidrug resistance in Gram-negative bacteria. Current Opinion in Microbiology. 2001; 4 (5): 500-508. NIKAIDO H. Outer membrane barrier as a mechanism of antimicrobial resistance. Antimicrobial Agents and Chemotherapy. 1989; 33 (11): 1831-1836. RUIZ N, KAHNE D and SILHAVY TJ. Advances in understanding bacterial outer-membrane biogenesis. Nat Rev Micro. 2006; 4 (1): 57-66. TENOVER FC. Mechanisms of antimicrobial resistance in bacteria. American Journal of Infection Control. 2006; 34 (5, Supplement 1): S3-S10. MCMANUS M. Mechanisms of bacterial resistance to antimicrobial agents. American Journal of Health-System Pharmacy. 1997; 54 (12): 1420-1433. DZIDIC S, SUSKOVIC J and KOS B. Antibiotic resistance mechanisms in bacteria: Biochemical and genetic aspects. Food Technology and Biotechnology. 2008; 46 (1): 11-21. 79. POOLE K. Mechanisms of bacterial biocide and antibiotic resistance. Journal of applied microbiology. 2002; 92 (s1): 55S64S. WANG Y, HA U, ZENG L and JIN S. Regulation of membrane permeability by a two-component regulatory system in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy. 2003; 47 (1): 95-101. WRIGHT GD. TetX Is a Flavin-dependent Monooxygenase Conferring Resistance to Tetracycline Antibiotics. Journal of Biological Chemistry. 2004; 279 (50): 52346-52352. KORSAK D, LIEBSCHER S and VOLLMER W. Susceptibility to antibiotics and {beta}-lactamase induction in murein hydrolase mutants of Escherichia coli. Antimicrobial Agents and Chemotherapy. 2005; 49 (4): 1404-1409. COENE B, SCHANCK A and DEREPPE J and VAN MEERSSCHE M. Substituent effects on reactivity and spectral parameters of cephalosporins. Journal of Medicinal Chemistry. 1984; 27: 694-700. MARCIANO DC, KARKOUTI OY and PALZKILL T. A fitness cost associated with the antibiotic resistance enzyme sme-1 -lactamase. Genetics. 2007; 176: 23812392. BUSH K, JACOBY G and MEDEIROS A. A functional classification scheme for beta-lactamases and its correlation with molecular structure. Antimicrobial Agents and Chemotherapy. 1995; 39 (6): 1211-1233. GARZA-RAMOS U and MARTNEZ-ROMERO EAJ. SHV-type extended-spectrum b-lactamase (ESBL) are encoded in related plasmids from enterobacteria clinical isolates from Mexico. Salud Pblica de Mxico. 2007; 49 (6). BATES SS, WORMS J and SMITH JC. Effects of ammonium and nitrate on growth and domoic acid production by Nitzschia pungens in batch culture. Can. J. Fish. Aquat. Sci. 1993; 50:1248-1254.

FORMATEX 2011

1021

Science against microbial pathogens: communicating current research and technological advances ______________________________________________________________________________ A. Mndez-Vilas (Ed.)

[64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] [89] [90] [91] [92]

BARTHELEMY P, AUTISSIER D, GERBAUDI G and COURVALIN P. Enzymic hydrolysis of erythromycin by a strain of Escherichia coli. The Journal of Antibiotics. 1984; 37 (12): 1692-1696. FILLGROVE KL, PAKHOMOVA S, NEWCOMER ME and ARMSTRONG RN. Mechanistic diversity of fosfomycin resistance in pathogenic microorganisms. Journal of the American Chemical Society. 2003; 125 (51): 15730-15731. VETTING MW, MAGNET S, NIEVES E and RODERICK SL and BLANCHARD JS. A bacterial acetyltransferase capableof regioselective n-acetylationof antibiotics and histones. Chemistry & Biology. 2004; 11: 565-573. ALLIGNET J and EL SOLH N. Diversity among the gram-positive acetyltransferases inactivating streptogramin A and structurally related compounds and characterization of a new staphylococcal determinant, vatB. Antimicrobial Agents and Chemotherapy. 1995; 39 (9): 2027-2036. YANG W, MOORE IF, KOTEVA KP, BAREICH DC, HUGHES DW and YAZAWA K, MIKAMI Y, MAEDA A, MORISAKI N and IWASAKI S. Phosphorylative inactivation of rifampicin by Nocardia otitidiscaviarum. Journal of Antimicrobial Chemotherapy. 1994; 33 (6): 1127-1135. 124. MATSUOKA M and SASAKI T. Inactivation of macrolides by producers and pathogens. Current Drug Targets - Infectious Disorders. 2004; 4 (3): 217-240. HUANG Y, LI H, HUTCHISON CE, LASKEY J, KIEBER JJ. Biochemical functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis. The Plant Journal. 2003; 33: 221-233. ETIENNE J, GERBAUD G, FLEURETTE J and COURVALIN P. Characterization of staphylococcal plasmids hybridizing with the fosfomycin resistance gene fosB. FEMS microbiology letters. 1991. 84 (1): 119-122. SCHWARZ S, KEHRENBERG C, DOUBLET B and CLOECKAERT A. Molecular basis of bacterial resistance to chloramphenicol and florfenicol. FEMS microbiology reviews. 2004; 28 (5): 519-542. DANG-VAN A, TIRABY G, ACAR JF, SHAW WV and BOUANCHAUD DH. Chloramphenicol resistance in Streptococcus pneumoniae: enzymatic acetylation and possible plasmid linkage. Antimicrobial Agents and Chemotherapy. 1978; 13 (4): 577-583. BEAMAN TW, SUGANTINO M and RODERICK SL. Structure of the hexapeptide xenobiotic acetyltransferase from Pseudomonas aeruginosa. Biochemistry. 1998; 37 (19): 6689-6696. ALLIGNET J, LONCLE V, SIMENEL C, DELEPIERRE M and EL SOLH N. Sequence of a staphylococcal gene, vat, encoding an acetyltransferase inactivating the A-type compounds of virginiamycin-like antibiotics. Gene. 1993; 130 (1): 9198. Boehr DD, DRAKER KA, Koteva K, Bains M, Hancock RE and Wright GD. Broad-spectrum peptide inhibitors of aminoglycoside antibiotic resistance enzymes. Chem. Biol. 2003; 10: 189196. TRICKER AR. Nicotine metabolism, human drug metabolism polymorphisms, and smoking behaviour. Toxicology. 2003; 183 (1-3): 151-173. KOLLOCK R, FRANK H, SEIDEL A, MEINL W and GLATT H. Oxidation of alcohols and reduction of aldehydes derived from methyl- and dimethylpyrenes by cDNA-expressed human alcohol dehydrogenases. Toxicology. 2008; 245 (1-2): 65-75. WANG H, LI W, LI J, RENDON-MITCHELL B, OCHANI M, ASHOK M, YANG L, YANG H, TRACEY KJ, WANG P and SAMA AE. The aqueous extract of a popular herbal nutrient supplement, angelica sinensis, protects mice against lethal endotoxemia and sepsis. Journal of Nutrition. 2006; 136 (2): 360-365. WHITTLE G, HUND BD, SHOEMAKER NB and SALYERS AA. Characterization of the 13-kilobase ermf region of the bacteroides conjugative transposon CTnDOT. Applied and Environmental Microbiology. 2001; 67 (8): 3488-3495. LEE C, MINAMI M, SAKUDA S, NIHIRA T and YAMADA Y. Stereospecific reduction of virginiamycin M1 as the virginiamycin resistance pathway in Streptomyces virginiae. Antimicrobial Agents and Chemotherapy. 1996; 40 (3): 595601. WALSH C. 2003. Antibiotics: actions, origin, resistance. ASM Press. HOLLENBERG MD. Receptor binding and agonist efficacy: new insights from mutants of the thrombin protease-activated receptor-1 (PAR-1). Molecular pharmacology. 2000; 58 (6): 1175-1177. SILVERMAN RB. 2004. The organic chemistry of drug design and drug action. 2nd edition edn. Elsevier Academic Press. TAVIO MDM, VILA J, RUIZ J, RUIZ J, MARTIN-SANCHEZ AM and DE ANTA, MTJ. Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli isolates. Journal of Antimicrobial Chemotherapy. 1999; 44 (6): 735-742. RAFII F, PARK M and NOVAK JS. Alterations in DNA Gyrase and Topoisomerase IV in Resistant mutants of Clostridium perfringens found after in vitro treatment with fluoroquinolones. Antimicrobial Agents and Chemotherapy. 2005; 49 (2): 488492. FRIEDMAN SM, LU T and DRLICA K. Mutation in the DNA gyrase a gene of escherichia coli that expands the quinolone resistance-determining region. Antimicrobial Agents and Chemotherapy. 2001; 45 (8): 2378-2380. MOON DC, SEOL SY, GURUNG M, JIN JS, CHOI CH, KIM J, LEE YC, CHO DT and LEE JC. Emergence of a new mutation and its accumulation in the topoisomerase IV gene confers high levels of resistance to fluoroquinolones in Escherichia coli isolates. International journal of antimicrobial agents. 2010; 35 (1): 76-79. ACKERMANN G, DEGNER A, COHEN SH, SILVA JJr and RODLOFF AC. Prevalence and association of macrolidelincosamide- streptogramin B 65 (MLSB) resistance with resistance to moxifloxacin in Clostridium difficile. Journal of Antimicrobial Chemotherapy. 2003; 51 (3): 599-603. ARTHUR M, BRISSON-NOEL A and COURVALIN P. Origin and evolution of genes specifying resistance to macrolide, lincosamide and streptogramin 67 antibiotics: data and hypotheses. Journal of Antimicrobial Chemotherapy. 1987; 20 (6): 783-802. WEISBLUM B. Erythromycin resistance by ribosome modification. Antimicrobial Agents and Chemotherapy. 1995; 39 (3): 577-585. Vila J and Pachn J. 2009. Antimicrobial Resistance and Therapeutic Alternatives Infectious Diseases and Pathogenesis, Acinetobacter Biology and Pathogenesis, pp. 1-22.

1022

FORMATEX 2011

Science against microbial pathogens: communicating current research and technological advances _______________________________________________________________________________ A. Mndez-Vilas (Ed.)

[93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] [104] [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] [117] [118] [119] [120] [121]

TENOVER FC. Mechanisms of antimicrobial resistance in bacteria. American Journal of Infection Control. 2006; 34 (5, Supplement 1): S3-S10. WEBBER MA and PIDDOCK LJV. The importance of efflux pumps in bacterial antibiotic resistance. Journal of Antimicrobial Chemotherapy. 2003; 51 (1): 9-11. 122 PAO SS, PAULSEN IT and SAIER MH Jr. Major facilitator superfamily. Microbiology and Molecular Biology Reviews. 1998; 62 (1): 1-34. VAN VEEN HW and KONINGS WN. The ABC family of multidrug transporters in microorganisms. Biochim Biophys Acta. 1998; 1365: 31-36. HIRAKATA Y, SRIKUMAR R, POOLE K, GOTOH N, SUEMATSU T, KOHNO S, KAMIHIRA S, HANCOCK REW and SPEERT DP. Multidrug efflux systems play an important role in the invasiveness of Pseudomonas aeruginosa. The Journal of Experimental Medicine. 2002; 196 (1): 109-118. DE KIEVIT TR, PARKINS MD, GILLIS RJ, SRIKUMAR R, CERI H, POOLE K, IGLEWSKI BH and STOREY DG. Multidrug efflux pumps: expression patterns and contribution to antibiotic resistance in Pseudomonas aeruginosa Biofilms. Antimicrobial Agents and Chemotherapy. 2001; 45 (6): 1761-1770. NARGOTRA A, SHARMA S, KOUL JL, SANGWAN PL, KHAN IA., KUMAR A, TANEJA SC and KOUL S. Quantitative structure activity relationship (QSAR) of piperine analogs for bacterial NorA efflux pump inhibitors. European journal of medicinal chemistry. 2009; 44 (10): 4128-4135. KOHLER T, EPP SF, CURTY LK and PECHERE J. Characterization of MexT, the regulator of the MexE-MexF-OprN multidrug efflux system of Pseudomonas aeruginosa. The Journal of Bacteriology. 1999; 181 (20): 6300-6305. SAIER MH Jr. A Functional-phylogenetic classification system for transmembrane solute transporters. Microbiology and Molecular Biology Reviews. 2000; 64 (2): 354-411. ZGURSKAYA HI and NIKAIDO H. Multidrug resistance mechanisms: drug efflux across two membranes. Molecular microbiology. 2000; 37 (2): 219-225. GOTOH N, TSUJIMOTO H, POOLE K, YAMAGISHI J and NISHINO T. The outer membrane protein OprM of Pseudomonas aeruginosa is encoded by oprK of the mexA-mexB-oprK multidrug resistance operon. Antimicrobial Agents and Chemotherapy. 1995; 39 (11): 2567-2569. VIVEIROS M, MARTINS A, PAIXO L, RODRIGUES L, MARTINS M, COUTO I, FHNRICH E, KERN WV and AMARAL L. Demonstration of intrinsic efflux activity of escherichia coli k-12 AG100 by an automated ethidium bromide method. International Journal of Antimicrobial Agents. 2008; 31 (5): 458-462. AMBRUS JI, KELSO MJ, BREMNER JB, BALL AR, CASADEI G and LEWIS K. Structureactivity relationships of 2aryl-1H-indole inhibitors of the NorA efflux pump in Staphylococcus aureus. Bioorganic & medicinal chemistry letters. 2008; 18 (15): 4294-4297. NARGOTRA A, SHARMA S, KOUL JL, SANGWAN PL, KHAN IA, KUMAR A, TANEJA SC and KOUL S. Quantitative structure activity relationship (QSAR) of piperine analogs for bacterial NorA efflux pump inhibitors. European journal of medicinal chemistry. 2009; 44 (10): 4128-4135. SANGWAN PL, KOUL JL, KOUL S, REDDY MV, THOTA N, KHAN IA, KUMAR A, KALIA NP and QAZI GN. Piperine analogs as potent Staphylococcus aureus NorA efflux pump inhibitors. Bioorganic & medicinal chemistry. 2008; 16 (22): 9847-9857. THOTA N, KOUL S, REDDY MV, SANGWAN PL, KHAN IA, KUMAR A, RAJA AF, ANDOTRA SS and QAZI GN. Citral derived amides as potent bacterial NorA efflux pump inhibitors. Bioorganic & medicinal chemistry. 2008; 16 (13): 6535-6543. BOLLA JM. Purification of Omp50, a Minor Porin of Campylobacter jejuni. Membrane Protein Protocols. 2003;131-138. BRYAN LE and BEDARD J. Impermeability to quinolones in gram-positive and gram-negative bacteria. European Journal of Clinical Microbiology & Infectious Diseases. 1991; 10 (4): 232-239. CHAPMAN JS and GEORGOPAPADAKOU NH. Routes of quinolone permeation in Escherichia coli. Antimicrobial Agents and Chemotherapy. 1998; 32 (4): 438-442. MARTIN NL and BEVERIDGE TJ. Gentamicin interaction with Pseudomonas aeruginosa cell envelope. Antimicrobial Agents and Chemotherapy. 1986; 29 (6): 1079-1087. WEISS J and BECKERDITE-QUAGLIATA SAEP. Resistance of gram-negative bacteria to purified bactericidal leukocyte proteins: relation to binding and bacterial lipopolysaccharide structure. The journal of clinical investigation. 1980; 65 (3): 619-628. SAIER MH Jr. A Functional-phylogenetic classification system for transmembrane solute transporters. Microbiology and Molecular Biology Reviews. 2000; 64 (2): 354-411. NIKAIDO H. Antibiotic resistance caused by Gram-negative multidrug efflux pumps. Clinical Infectious Diseases. 1998; 27: S32-S41. SUNDHEIM G and LANGSRUD S. Natural and acquired resistance of bacteria associated with food processing environments to disinfectant containing an extract from grapefruit seeds. International Biodeterioration & Biodegradation. 1995; 36 (3-4): 441-448. SOREK R, KUNIN V and HUGENHOLTZ P. CRISPR [mdash] a widespread system that provides acquired resistance against phages in bacteria and archaea. Nat Rev Micro. 2008; 6 (3): 181-186. ROBERTS MC. Acquired tetracycline and/or macrolidelincosamidesstreptogramin resistance in anaerobes. Anaerobe. 2003; 9 (2): 63-69. KRASOVEC R and JERMAN I. Bacterial multicellularity as a possible source of antibiotic resistance. Medical Hypotheses. 2003; 60 (4): 484-488. HAWKEY PM. The Origins and molecular basis of antibiotic resistance. British Medical Journal. 1998; 317: 657-660. MASCARETTI OA. 2003. Bacteria Versus Antibacterial Agents: An Integrated Approach. ASM press.

FORMATEX 2011

1023

Science against microbial pathogens: communicating current research and technological advances ______________________________________________________________________________ A. Mndez-Vilas (Ed.)

[122] SPIGAGLIA P, BARBANTI F and MASTRANTONIO P. Horizontal transfer of erythromycin resistance from Clostridium difficile to Butyrivibrio fibrisolvens. Antimicrobial Agents and Chemotherapy. 2005; 49 (12): 5142-5145. [123] MARRAFFINI LA and SONTHEIMER EJ. CRISPR Interference limits horizontal gene transfer in Staphylococci by targeting DNA. Science. 2008; 322 (5909): 1843-1845. [124] BRABBAN AD, HITE E and CALLAWAY TR. Evolution of foodborne pathogens via temperate bacteriophage-mediated gene transfer. Foodborne Pathogens and Disease. 2005; 2 (4): 287-303. [125] ZHANG Y and LEJEUNE JT. Transduction of blaCMY-2, tet(A), and tet(B) from Salmonella enterica subspecies enterica serovar Heidelberg to S. Typhimurium. Veterinary microbiology. 2008; 129 (3-4): 418-425. [126] FROST LS, LEPLAE R, SUMMERS AO and TOUSSAINT A. Mobile genetic elements: the agents of open source evolution. Nat Rev Micro. 2005; 3 (9): 722-732. [127] HARDY K. 1986. Bacterial Plasmids. Springer. [128] BRYAN LE. 1982. Bacterial resistance and susceptibility to chemotherapeutic agents. CUP Archive. [129] ALEKSHUN MN and LEVY SB. Molecular mechanisms of antibacterial multidrug resistance. Cell. 2007; 128 (6): 10371050. [130] KAISER GE. 2007. Kaiser's Microbiology. http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/plasmid.html (accessed 27/04/010). [133] SRUM H and L'ABE-LUND TM. Antibiotic resistance in food-related bacteriaa result of interfering with the global web of bacterial genetics. International Journal of Food Microbiology. 2002; 78 (1-2): 43-56. [134] SIMJEE S and GILL MJ. Gene transfer, gentamicin resistance and enterococci. Journal of Hospital Infection. 1997; 36 (4): 249-259. [135] OUELLETTE M and KNDIG C. Microbial multidrug resistance. International Journal of Antimicrobial Agents. 1997; 8 (3): 179-187. [136] HALL RM and STOKES HW. Integrons or super integrons? Microbiology. 2004; 150 (1): 3-4. [137] BENNETT PM. Integrons and gene cassettes: a genetic construction kit for bacteria. Journal of Antimicrobial Chemotherapy. 1999; 43 (1): 1-4. [138] DEPARDIEU F, PODGLAJEN I, LECLERCQ R, COLLATZ E and COURVALIN P. Modes and modulations of antibiotic resistance gene expression. Clinical microbiology reviews. 2007; 20 (1): 79-114. [139] HAVLICKOVA H, HRADECKA H, BERNARDYOVA I and RYCHLIK I. Distribution of integrons and SGI1 among antibiotic-resistant Salmonella enterica isolates of animal origin. Veterinary microbiology. 2009; 133 (1-2): 193-198. [140] JEON B, MURAOKA W, SAHIN O and ZHANG Q. Role of Cj1211 in natural transformation and transfer of antibiotic resistance determinants in Campylobacter jejuni. Antimicrobial Agents and Chemotherapy. 2008; 52 (8): 2699-2708. [141] CHEN I, CHRISTIE PJ and DUBNAU D. The ins and outs of DNA transfer in bacteria. Science. 2005; 310 (5753): 14561460. [142] SUMMERS AO. Genetic linkage and horizontal gene transfer, the roots of the antibiotic multi-resistance problem. Animal Biotechnology. 2006; 17(2): 125. [143] SINGLETON P. 2004. Bacteria in Biology,Biotechnology, and Medicine. John Wiley and sons. [144] CHANDLER JR, HIRT H and DUNNY GM. A paracrine peptide sex pheromone also acts as an autocrine signal to induce plasmid transfer and virulence factor expression in vivo. Proceedings of the National Academy of Sciences of the United States of America. 2005; 102 (43): 15617-15622. [145] CLARDY J, FISCHBACH MA and WALSH CT. New antibiotics from bacterial natural products. Nat Biotech. 2006; 24 (12): 1541-1550. [146] KIRBY WMM. Extraction of a highly potent penicillin inactivator from penicillin resistant staphylococci. Science. 1944; 99 (2579): 452-453. [147] MAJIDUDDIN FK, MATERON IC and PALZKILL TG. Molecular analysis of beta-lactamase structure and function. International Journal of Medical Microbiology. 2002; 292 (2): 127-137. [148] GILBERTSON-BEADLING, S., POWERS, E.A., STAMP-COLE, M., SCOTT, P.S., WALLACE, T.L., COPELAND, J., PETZOLD, G., MITCHELL, M., LEDBETTER, S., POORMAN, R., WILKS, J.W. and FISHER, C., 1995. The tetracycline analogs minocycline and doxycycline inhibit angiogenesis in vitro by a non-metalloproteinase-dependent mechanism. Cancer chemotherapy and pharmacology, 36(5), 418-424. [149] Phourcq F and Jarry C. Determination of third-generation cephalosporins by high-performance liquid chromatography in connection with pharmacokinetic studies. Journal of Chromatography A. 1998; 812 (1-2): 159-178. [150] BRYSKIER A, PROCYK T and LABRO MT. Cefodizime, a new 2-aminothiazolyl cephalosporin: physicochemical properties, toxicology and structure-activity relationships. Journal of Antimicrobial Chemotherapy. 1990; 26 (suppl_C): 1-8. [151] CASSELL GH and MEKALANOS J. Development of antimicrobial agents in the era of new and reemerging infectious diseases and increasing antibiotic resistance. JAMA: The Journal of the American Medical Association. 2001; 285 (5): 601605. [152] OTTEN H. Domagk and the development of the sulphonamides. Journal of Antimicrobial Chemotherapy. 1986; 17 (6): 689690. [153] PR newswire, 2005. http://www.thefreelibrary.com/FDA+Approves+Nexavar(R)+for+Treatment+of+Patients+with+Advanced...-a0139964558. [154] MEDICAL NEWS TODAY, 2007. Nexavar (Sorafenib) Launched in UK for hepatocellular carcinoma. Medical News Today, http://www.medicalnewstoday.com/articles/87657.php (accessed 20/1/010). [155] JI H, LI X and ZHANG H. Natural products and drug discovery. EMBO reports. 2009; 10 (3): 194-200. [156] KOEHN F and CARTER G. The evolving role of natural products in drug discovery. Nat Rev Drug Discov. 2005; 4(3): 206220. [157] STONE MJ and WILLIAMS DH. On the Evolution of Functional Secondary Metabolites (Natural- Products). Mol. Microbiol. 1992; 6: 29-34.

1024

FORMATEX 2011

Science against microbial pathogens: communicating current research and technological advances _______________________________________________________________________________ A. Mndez-Vilas (Ed.)

[158] [159] [160] [161] [162] [163] [164] [165] [166] [167] [168] [169] [170] [171] [172] [173] [174]

[175] [176] [177] [178] [179] [180] [181] [182] [183] [184] [185] [186] [187] [188] [189]

ANANTHARAMAN V, ARAVIND L and KOONIN EV. Emergence of diverse biochemical activities in evolutionarily conserved structural scaffolds of proteins. Current Opinion in Chemical Biology. 2003; 7 (1): 12-20. CHINOU I. Labdanes of natural origin-biological activities (1981-2004). Current medicinal chemistry. 2005; 12 (11): 1295-317. SHEN Y, LI R, XIAO W, XU G, LIN Z, ZHAO Q and SUN H. ent-Labdane Diterpenoids from Andrographis paniculata. Journal of natural products. 2006; 69 (3): 319-322. TATSIMO SJN, TANE P, MELISSA J, SONDENGAM BL, OKUNJI CO, SCHUSTER BM, IWU MM and KHAN IA. Antimicrobial principle from Aframomum longifolius. Planta Medica. 2006; 72 (2): 132-135. TANI H, KOSHINO H, SAKUNO E, CUTLER HG and NAKAJIMA H. Botcinins E and F and botcinolide from Botrytis cinerea and structural revision of botcinolides. Journal of natural products. 2006; 69 (4): 722-725. Rohmer M, Knani M, Simonin P et al. Isoprenoid biosynthesis in bacteriaa novel pathway for the early steps leading to isopentenyl diphosphate. Biochem J. 1993; 295: 517524. LIU D. 2008. Handbook of Listeria monocytogenes. CRC press. KNIGHT L, KURBACHER C, GLAYSHER S, FERNANDO A, REICHELT R, DEXEL S, REINHOLD U and CREE I. Activity of mevalonate pathway inhibitors against breast and ovarian cancers in the ATP-based tumour chemosensitivity assay. BMC Cancer. 2009; 9 (1): 38. SWANSON KM, HOHL RJ. Anticancer therapy: targeting the mevalonate pathway. Curr cancer drug targets: 2006; 6: 1537. JUNG WK, LIM JY, KWON NH, KIM JM, HONG SK, KOO HC, KIM SH and PARK YH. Vancomycin-resistant enterococci from animal sources in Korea. International journal of food microbiology. 2007; 113 (1): 102-107. PRABHAKAR REDDY P, RANGA RAO R, SHASHIDHAR J, SASTRY BS, MADHUSUDANA RAO J and SURESH BABU K. Phytochemical investigation of labdane diterpenes from the rhizomes of Hedychium spicatum and their cytotoxic activity. Bioorganic & medicinal chemistry letters. 2009; 19 (21): 6078-6081. STAVRI M, PATON A, SKELTON BW and GIBBONS S. Antibacterial diterpenes from Plectranthus ernstii. Journal of natural products. 2009; 72 (6): 1191-1194. CHAKRABORTY K, LIPTON AP, PAUL RAJ R and VIJAYAN KK. Antibacterial labdane diterpenoids of Ulva fasciata Delile from southwestern coast of the Indian Peninsula. Food Chemistry. 2010; 119 (4): 1399-1408. BUTLER MS and BUSS AD. Natural products the future scaffolds for novel antibiotics? Biochemical pharmacology. 2006; 71 (7): 919-929. SADOWSKI J and KUBINYI H. A scoring scheme for discriminating between drugs and nondrugs. Journal of medicinal chemistry. 1998; 41 (18): 3325-3329. TANG Y, SHI Y, ZHAO W, HAO G and LE G. Discovery of a novel antimicrobial peptide using membrane binding-based approach. Food Control. 2009; 20 (2): 149-156. FOGAA AC, LORENZINI DM, KAKU LM, ESTEVES E, BULET P and DAFFRE S. Cysteine-rich antimicrobial peptides of the cattle tick Boophilus 82 microplus: isolation, structural characterization and tissue expression profile. Developmental & Comparative Immunology. 2004; 28 (3): 191-200. CHE Q, ZHOU Y, YANG H, LI J, XU X and LAI R. A novel antimicrobial peptide from amphibian skin secretions of Odorrana grahami. Peptides. 2008; 29 (4): 529-535. MARCOS JF, MUOZ A, PREZ-PAY E, MISRA S and LPEZ-GARCA B. Identification and rational design of novel antimicrobial peptides for plant protection. Annual Review of Phytopathology. 2008; 46 (1): 273-301. THEVISSEN K, KRISTENSEN H, THOMMA BPHJ, CAMMUE BPA and FRANOIS IEJA. Therapeutic potential of antifungal plant and insect defensins. Drug Discovery Today. 2007; 12 (21-22): 966-971. WANG J, SOISSON SM, YOUNG K, SHOOP W, KODALI S, GALGOCI A, PAINTER R et al. Platensimycin is a selective FabF inhibitor with potent antibiotic properties. Nature. 2006; 441: 358-361. Jensen PR, Gontang E, Mafnas C et al. Culturable marine actinomycete diversity from tropical Pacific Ocean sediments. Environ Microbiol. 2005; 7:10391048. William Fenical and Paul R Jensen. Developing a new resource for drug discovery: marine actinomycete bacteria. Nature Chemical Biology. 2006; 2: 666 673. KARUDAPURAM S, ZHAO X and BARCAK GJ. DNA sequence and characterization of Haemophilus influenzaedprA+, a gene required for chromosomal but not plasmid DNA transformation. J Bacteriol. 1995; 177: 32353240. FLEISCHMANN R, ADAMS M, WHITE O, CLAYTON R, KIRKNESS E, KERLAVAGE A, BULT C, TOMB J, DOUGHERTY B, MERRICK J and AL E. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995; 269 (5223): 496-512. MILLS SD. When will the genomics investment pay off for antibacterial discovery? Biochemical pharmacology. 2006; 71 (7): 1096-1102. JAROCH S and WEINMANN H. Putting small molecules in the lead. Nat Chem Biol. 2005; 1 (4): 180-183. GIDAY M, TEKLEHAYMANOT T, ANIMUT A and MEKONNEN Y. Medicinal plants of the Shinasha, Agew-awi and Amhara peoples in northwest Ethiopia. Journal of ethnopharmacology. 2007; 110 (3): 516-525. NJOROGE GN and BUSSMANN RW. Ethnotherapeautic management of skin diseases among the Kikuyus of Central Kenya. Journal of ethnopharmacology. 2007; 111 (2): 303-307. MATU EN and VAN STADEN J. Antibacterial and anti-inflammatory activities of some plants used for medicinal purposes in Kenya. Journal of ethnopharmacology. 2003; 87 (1): 35-41. DE BOER HJ, KOOL A, BROBERG A, MZIRAY WR, HEDBERG I and LEVENFORS JJ. Anti-fungal and anti-bacterial activity of some herbal remedies from Tanzania. Journal of ethnopharmacology. 2005; 96 (3): 461-469. OLAJIDE OA and ALADA ARA. Studies on the anti-inflammatory properties of Entada abyssinica. Fitoterapia. 2001; 72 (5): 492-496.

FORMATEX 2011

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[190] FREIBURGHAUS F, STECK A, PFANDER H, BRUN R. Bioassay guided isolation of a diastereoisomer of kolavenol from Entada absyssinica active on Trypanosoma brucei rhodense. J. Ethnopharmacol. 1998; 61: 179-183. [191] OROZCO-TOPETE R, SIERRA-MADERO J, CANO-DOMINGUEZ C, KERSHENOVICH J, ORTIZ-PEDROZA G, VAZQUEZ-VALLS E, GARCIA-COSIO C, et al. Safety and efficacy of Virend for topical treatment of genital and anal herpes simplex lesions in patients with AIDS. Antiviral Res. 1997; 35: 91103. [192] GIRON LM, AGUILAR GA, CACERES A and ARROYO GL. Anticandidal activity of plants used for the treatment of vaginitis in Guatemala and clinical trial of a Solanum nigrescens preparation. J. Ethnopharmacol. 1988; 22: 307313.

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