1 ORUCHE, NELSON EBERECHUKWU PG/MSC/09/51294 EVALUATION OF ANTIBACTERIAL AND RESISTANCE MODIFYING POTENCY OF ETHANOLIC LEAF EXTRACTS OF FOUR MEDICINAL PLANTS Department of Microbiology Faculty of Biological Sciences Nwamarah Uche Digitally Signed by: Content manager s Name DN : CN = Weabmaster s name O= University of Nigeria, Nsukka OU = Innovation Centre
2 CHAPTER ONE INTRODUCTION AND LITERATURE REVIEW 1.0 INTRODUCTION The use of medicinal plants for treatment of various infectious and organic diseases in traditional communities has been an age-long global practice. The World Health Organization (WHO) estimated that 80% of the world population use herbal regimen for treatment and control of diseases (Hugo and Russel, 2003). The recognition of the therapeutic efficacies of herbs has therefore prompted many scientific investigations of these plants including the evaluation of their antibacterial potencies since the era of science and technology. Apart from the expensive costs of some antibiotics, many clinically important antibiotics have serious setbacks. A good number of standard antibiotics have been found to be neurotoxic, nephrotoxic and hypertensive, and few others cause severe damage to liver and bone marrow depression (Chong and Pagano, 1997).The primary benefit of using plant derived medicine is that they are relatively safer and cheaper than synthetic alternatives, offering profound therapeutic benefits and more affordable treatment (Aiyegoro and Okoh, 2009). In addition, herbal medicine is a complex mixture of different phytochemicals acting by different mechanisms, making it difficult for pathogens to develop resistance (Daferera et al., 2003) The high costs and toxic effects of many conventional antibiotics are not the only reasons why medicinal plants are being investigated in recent times as possible sources of alternative and supplements to the standard orthodox drugs. The problem of the ever increasing multidrug resistance (MDR), which has limited the use of cheap and old antibiotics, has necessitated a phenomenal increase in research for development of new
3 antimicrobial and resistance modifying agents of herbal origin (WHO,2002). It has been found that, in addition to the production of intrinsic antimicrobial compounds, some medicinal plants also produce multidrug resistance inhibitors which enhance the activities of antibiotics against antibiotic resistant bacterial pathogens (Stermitz et al., 2000; Adu et al., 2011; Ahmad and Aqil, 2007). Bacterial efflux pumps have been recognized to be responsible for considerable level of resistance to antibiotics in pathogenic bacteria (Mahamond et al., 2007; Stervri et al., 2006; Bambeke et al., 2010). These multi-drug resistance (MDR) pumps conferring resistance to a wide range of antibiotics have since been characterized in many bacterial pathogens especially in Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and more recently in mycobacteria (Sibanda and Okoh, 2007). The crude extract of many medicinal plants have been found to potentiate the in vitro activity of antibiotics by reducing the minimum inhibitory concentration (MIC) of an antibiotic to which resistance has occurred (Aiyegoro and Okoh, 2009). Many plant derived compounds have also been observed to augment the activities of antibiotics by inhibiting the multi-drug resistance (MDR) efflux systems in bacteria (Stervri et al., 2003). This could be of great benefit in combination therapy, perhaps facilitating the re-introduction of antibiotics that are no longer effective due to resistance. The screening of crude plant extract for synergistic interaction with antibiotics is also expected to provide ways for the isolation of possible multi- drug resistance inhibitors of plant origin. In this study, the antibacterial and resistance modifying potency of the ethanolic leaf extracts of the following four medicinal plants were investigated: Picralima nitida, Aspilia
4 africa, Chromolaena odorata and Hyptis suaveolens. Selection of these plants was based on the prior knowledge of their use in folkloric medicine in some parts of Eastern Nigeria for treatment and control of infection, sometimes in combination with conventional antimicrobial drugs. This research was therefore conducted with the following major aims and objectives: i. To evaluate the antibacterial activities of the ethanolic leaf extracts of the above mentioned medicinal plants ii. To investigate the ability of the plant extracts to modify the antibiotic resistance of drug resistant Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus to any of the following antibiotics: ciprofloxacin, norfloxacin, tetracycline, chloramphenicol and erythromycin. iii. To determine the phytochemical constituents of the crude extracts. 1.1 LITERATURE REVIEW 1.1.1 Botanic Description and Medicinal Properties of Picralima nitida Picralima nitida is a multi-stemmed savanna tree measuring up to 12 m or more in height, with an open canopy. The plant is geographically distributed in some African countries such as Kenya, Tanzania, Nigeria, and Ghana. It is commonly called Akuamma plant.
5 The plant is classified as follows: Kingdom : Plantae Family : Apocynaceae Subfamily : Rauvolfioideae Genus : Picralima Species : Picalima nitida
Fig 1: Picture of Picalima nitida 6
7 The stem, seed and roots of P. nitida have been reported by traditional medical practitioners to be effective in treatment of various infections and some conditions such as diabetes and hypertension (Nkere and Iroegbu, 2005). The leaves of the plant are also used for preparation of herbal mixture for treatment of various infections including malaria and typhoid fever in some parts of Eastern Nigeria. The antibacterial activities of the seed, stembark and root extracts of P. nitida has been investigated (Nkere and Iroegbu, 2005), but no scientific studies have been carried out on the leaf extract of the plant. 1.1.2 Botanic Description and Medicinal Properties of Aspilia africana Aspilia africana is a species of flowering plants commonly known as wild sunflower in English. It is widely distributed in Eastern Nigeria. The plant is classified as follows: Kingdom : Plantae Order : Asteralesae Family : Asteraceae Genus : Aspilia Species : Aspilia africana English name : Wild sunflower; haemorrhage plant Igbo name : Oramejina
Fig 2: Picture of Aspilia Africana 8
9 Historically, Aspilia africana has been used in some Igbo-speaking parts of Nigeria to prevent conception, suggesting potential contraceptive and anti-fertility properties (Kayodo et al., 2007). The leaf extracts of the plant is also used in traditional system of medicine in most South Eastern Nigeria to arrest bleeding in fresh wounds and to accelerate would healing process(oluyemi et al.,2004). Some herbalists reported using the leaf extract in combination with conventional antibiotics to treat septic wound that resisted common antibiotic treatment. 1.1.3 Botanic Description and Medicinal Properties of Chromolaena odorata Chromolaena odorata is a species of flowering plant in the sunflower family, Asteraceae. The plant is native to North America, from Florida and Texas, but has been introduced in Asia, West Africa, and Australia (Schmidt and Schilling, 2000). The plant is classified as follows: Kingdom : Plantae Order : Asterales Family : Asteraceae Genus : Chromolaena Species : Chromlaena odorata. Common names: Christmas bush; Siam weed, and Common Flower
Fig 3: Picture of Chromolaena odorata 10
11 The extracts of the leaf of Chromolaena odorata is used in some parts of Eastern Nigeria for treatment of wound and various skin infections. 1.1.4 Botanic Description and Medicinal Properties of Hyptis suaveolens Hyptis suaveolens is a pseudo cereal plant known as chan plant in English (Schmidt and Schilling, 2000). It measures up to 2 m high, having branches and long, white piliferous stems. Its flowers are purple or white and its leaves oval, wrinkled and pointed. The plant is richly distributed in many parts of Eastern Nigeria, where it is used for preparation of herbal mixtures for treatment of malaria, typhoid fever and diarrhea among other infections. The leaves are also used by rural dwellers as an insecticide to repel mosquitoes The plant is classified as follows. Kingdom : Plantae Order : Lamiales Family : Lamiaceae Genus : Hyptis Species : Hyptis suaveolens Common name: Chan plant
Fig 4: Picture of Hyptis suaveolens 12
13 1.2 The Mechanism of Antimicrobial Action of Plant Extracts Many plant extracts clearly demonstrate antibacterial activities due to various phytochemicals they contain. These phytochemicals are the secondary metabolites that offer the plants adaptive ability to adverse abiotic and biotic environmental conditions (Stefanovic and Comic, 2012). Although the mechanistic processes underlying the antimicrobial activities of these phytochemicals have not yet been fully understood, some researchers have succeeded in describing the mode of action of some of the common phytochemicals which are summarized in table 1a below. The terpeniod compound in the leaf extract of T. procumbens was found to exert antibacterial activities on both Gram positive and Gram negative by causing membrane disruption and inhibition of respiratory chain dehydrogenases of bacterial pathogens (Bama et al., 2012). Because the extracts of medicinal plants are complex mixtures of different phytochemicals acting by different mechanisms, microbial adaptability to the actions of these extracts becomes very difficult and so the chances of the pathogens developing resistance to the plant extract is very minimal.
Table 1a: Some major classes of antimicrobial compounds from medicinal plants and their mechanisms of action (Cowan, 1999) S/N Classes Examples Mechanism of action 1 Phenolics (Simple Phenols) Catechol Epicatechin Substrate deprivation and membrane disruption 2 Phenolics (Phenolic acids) Cinamic acid Membranes disruption 3 Phenolics (Quinones) Hypericin Bind to adhesins, complex with cell wall and inactivate enzymes. 4 Phenolics (Flavonoids) Chrysin Bind to adhesins and complex with cell wall. 5 Phenolics Abyssinome Inactivate enzymes, inhibit HIV reverse transcriptase 6 Phenolics (Tannins) Ellagitannins Binds to proteins and adhesins, enzyme inhibitor, substrate deprivation, complex with cell wall, membrane disruption and metal ion complexation. 7 Phenolics (Coumarins) Warfarin Interact with eukaryotic DNA (Antiviral activity) 8 Terpenoids, Essential oils Capsaicin Membrane disruption 9 Alkaloids Berberin and piperine Intercalate into cell wall and/or DNA. 10 Lectins and Polypeptide Mannose-specific agglutinin Block viral fusion or adsorption 14
15 1.3 Classes of Conventional Antibacterial Antibiotics and their Mechanism of Action An antibiotic was originally defined as a substance, produced by one microorganism, which inhibit the growth of other microorganisms (Hugo and Russell, 2003).The advent of synthetic methods, has however, led to modification of this definition, and an antibiotic now refers to a substance produced by microorganisms or to a similar substance (produced wholly or partly by chemical synthesis), which in low concentrations inhibits the growth of other microorganisms. The study of the mechanism of action of the orthodox antibiotics reveals the basis of their selective toxicity. The five broad targets sites of antibiotic action (cell wall, ribosome, chromosome, folate metabolism, and cell membrane) and the major classes of antibacterial antibiotics are briefly discussed in this section. A. Beta-lactam Antibiotics: These are the antibiotics that contain bata-lactam ring in their structure. Examples of β-lactam antibiotics include penicillin (eg: Penicillin G otherwise called benzyl penicillin, methicillin, oxacillin, ampicillin, amoxicillin, etc), cephalorporins and monobactams. Beta-lactam antibiotics block the final cross linking stage in the peptidoglycan biosynthesis which occurs in the cell wall. The liner, nascent glycan strands are cross linked via their peptide chains to the mature peptidoglycan in the cell wall by the activity of transpeptidase enzymes (penicillin binding proteins) in the reaction called transpeptidation. Beta-lactam antibiotics inhibit this transpeptidation by reacting with the transpeptidase enzymes through their β-lactam rings. Penicillins and cephalosporin are two major groups of β-lactam antibiotics that are commonly used in antibacterial chemotherapy. Many of the penicillin, such as pencillin G (benzylpenicillin), have a relatively narrow spectrum of activity, being most effective against Gram positive cocci, including Staphylococcus spp. Ampicillin, which is a chemically modified
16 form of penicillin G, has a broader spectrum of activity as it is active against many Gram negative organisms including E. coli, Shigella spp and Proteus spp. The broad spectrum activity of ampicillin is based on its ability to penetrate the outer membrane of the Gram negative bacteria to reach the site of action of transpeptidase enzymes unlike the penicillin G. Cephalosporins generally have broad spectrum of action, and many of them such as cefoxitin and cephalothin are resistant to many penicillase enzymes (β-lactamases), which degrade β- lactam rings of many penicillins. Cephalothin is often the antibiotic of choice for treating severe staphylococcal infections caused by β-lactamase producing Staphylococcus spp. B. Tetracyclines: These are group of antibiotics that are composed of four fused cyclic rings. They are obtained as by-products from the metabolism of various species of Streptomyces, although some semi-synthetic ones are now available. The following are common examples of tetracycline antibiotics: tetracycline, chlortetracycline, doxycycline, oxytetracycline, methacycline and minocycline. Tetracyclines interfere with protein synthesis by binding specifically to the 30s ribosomal subunit, apparently blocking the binding of aminoacyl-trna to the A site of the ribosome thereby preventing the addition of amino acids to the growing peptide chain and halting the protein synthesis. Tetracycline is actively transported into bacterial cells possibly in the form of magnesium complex. Mammalian cells do not take up the tetracycline, and it is this difference in uptake of tetracyclines that forms the basis for their selective toxicity. Tetracyclines are broad-spectrum antibiotics, having a wide range of activity against Gram positive and Gram negative bacteria. Although P. aeruginosa is less sensitive to
17 tetracyclines, it is generally susceptible to tetracyclines at concentrations obtainable in the bladder. Tetracyclines are bacteriostatic rather than bacteriocidal, and so should not be used in combination with β-lactams, which require cells to be growing and dividing to exert their lethal actions. Tetracyclines are no longer commonly used in the clinics as they were in the past due to the increase in bacterial resistance to tetracyclines. C. Aminoglycoside-aminocyclitol Antibiotics Aminoglycosides are antibiotics that contain amino sugars in their structure. Aminoglycoside antibiotics are produced by actinomycetes, for example streptomycin is produced by Streptomyces griseus. Common examples of aminoglycoside antibiotics include streptomycin, neomycin, kanamycin, genetamicin and tombramycin. Aminglycosides bind to the 30s ribosomal subunit of the 70s bacterial ribosome, blocking protein synthesis and decreasing the fidelity of translation of genetic codes. They disrupt the normal functioning of the ribosomes by interfering with formation of initiation complex, which is the first step in protein synthesis Aminoglycosides are used almost exclusively in the treatment of infections caused by Gram negative pathogens as their action against Gram positive bacteria is limited. The effectiveness of the aminoglycosides is enhanced by their active uptake by Gram negative bacteria. However, many of these antibiotics are toxic to mammalian cells. For example, streptomycin has serious effects on the eight cranial nerve, and so a prolonged use of the antibiotic causes deafness.
18 Despite its ototoxicity, streptomycin remains a front-line drug used against tuberculosis and in treatment of very serious bacterial infections such as endocarditis. Gentamicin, which is the most important aminoglycoside antibiotics, is active against many strains of Gram positive and Gram negative bacteria, including some strains of Pseudomonas aeruginosa. However, gentamicin is extremely toxic and should be used only in sever infections, particularly when the infecting bacteria are not sufficiently sensitive to other, less toxic antibiotics. D. Macrolides Macrolide antibiotics are group of antibiotics that are characterized by possessing molecular structure that contains large (12-16 membered) lactone rings linked through glycosidic bonds with amino sugars. Erythromycin, which is produced by Stretomyces erythreus, is the most important member of this group of antibiotics. Erythromycin selectively inhibits protein synthesis in a broad range of bacteria by binding to the 50s subunits of the ribosome thereby blocking translocation of the ribosome along the mrna. Erythromycin is most effective against Gram positive cocci such as Streptococcus pyogenes and staphylococci, and other bacteria such as Neisseria, H.influenza and Legionella pneumophila. The antibiotic is not active against Enterobacteriaceae, and Staphylococcus aureus is less sensitive to erythromycin than pneumococci or haemolytic streptococci. The new members of the macrolides are semisynthetic molecules that differ from the original compound by substitution pattern of the lactone ring system. Examples of the new macrolides antibiotics include erythromycin, clarithromycin, azithromycin, roxithromycin and dirithromycin.
19 E. Glycopeptide Antibiotics Two important glycopeptide antibiotics are vancomycin and teicoplanin. Vancomycin is an antibiotic isolated from Streptomyces orientalis. It is active against most Gram positive bacteria, including methicillin-resistant strains of Staphylococcus aureus and Staphylococcus epidermidis, Enterococcus faecalis, Clostridium difficile and Gram negative cocci. The Gram negative bacilli and mycobacteria are not susceptible to vancomycin. Vancomycin inhibits cell wall synthesis by binding directly to peptide portion of the peptidoglycan that is about to join to other peptide to form cross-linkage. Vancomycin binding prevents the formation of the cross-linkage needed for the cell wall to functionally protect the cell against osmotic shock. Teicoplanin is a naturally occurring complex of five closely related tetracyclic molecules. Its mode of action and spectrum of activity are essentially similar to vancomycin, although it is less active against some strains of coagulase negative staphylococci. F. Chloramphenicol Chloramphenicol is an antibiotic produced by Streptomyces venezuelae. Although chloramphenicol is bacteriostatic, it has a broad spectrum of activity against Gram positive and Gram negative organisms as well ass other bacteria such as mycoplasmas, rickettsia, and Chlamydia. It has the valuable property of penetrating into mammalian cells, and is therefore the drug of choice for treating infections caused by intracellular pathogens including Salmonella typhi. Chloramphenicol selectively inhibits protein synthesis in bacterial ribosome by binding to the 50s rrna. This prevents the binding of trna molecules to the aminoacyl and peptidyl biding sites of the ribosome.
20 Although chloramphemicol does not inhibit 80s ribosomes, the 70s ribosmes of mammalian mitochondria are sensitive to chloramphenicol, and therefore some inhibition occurs in rapidly growing mammalian cells with high mitochondrial activity. Chloramphenicol also causes dose-related aplastic anaemia, and this restricts the use of the antibiotic to cases where no effective alternative exists, eg. typhoid fever. G. Quinolone and Fluoroquinolone Antibiotics Quinolone consist of a group of antibiotics that act by blocking normal DNA replication. They selectively inhibit DNA gyrase (topoisomerase 11), which is not found in mammalian cells (Zakaria,2005). The gyrase is a highly versatile enzyme which is capable of catalyzing a variety of changes in DNA topology including negative supercoiling and removal of positive supercoiling, unknotting, and decatenation. Such activities ensure that the daughter chromosomes produced during replication can segregate in the cytoplasm prior to cell division. Quinolones bind to the A subunit of the gyrase at an exposed single stranded ends of the cut DNA chain. This makes the gyrase unable to reseal the DNA with the result that the chromosome of the bacterial cells becomes fragmented. Many quinolone antibacterial agents have been synthesized. Nalidixic acid is regarded as the progenitor of new quinolones. The commonly used newer quinolones belong to the group called fluoroquinolones, which are quinolones that are chemically modified by attaching fluorine compounds to the central ring system of the quinolone. Some common examples of fluoroquinolones include: ciprofloxacin, norfloxacin, lomefloxacin, sparfloxacins, etc. These newer members of quinolones are effective against a broad range of
Gram positive and Gram negative bacterial pathogens, including some pathogen like mycobacteria that are resistant to many antimicrobial agents. 21 H. Sulfonamides and Trimethoprin Sulfonamides and trimethoprin are structural analogues of the vitamin, paraaminobenzoic acid, and this makes them useful antibacterial agents. Para-aminobenzoic acid (PABA) is used for synthesis of folic acid in bacteria, which is an essential co-enzyme for the synthesis of purines and pyrimidine bases of the nucleic acids. Mammalian cells do not synthesize folic acid; they absorb it through an active transport system. Bacteria in contrast normally synthesize their required folic acid by making use of PABA, and are unable to take up folic acid from the surrounding through active transports. Sulfonamides, which are analogues of PABA, are effective competitor with natural substrate (PABA) for the enzymes involved in the synthesis of folic acid. The incorporation of the sulfonamides in place of PABA in the folic acid leads to formation of wrong coenzymes thereby arresting the formation of nucleic acid and causing bacteriostatic effect. 1.4 Mechanisms of Antibiotic Resistance in Bacterial Pathogens Antibiotic resistance is classified into two broad types namely, intrinsic (innate) and acquired resistance. A bacterial organism is said to possess an innate resistance to an antibiotic when inherent properties of the bacterial cell are responsible for preventing antibiotic action. For example, the Gram negative cell envelope is sufficiently impermeable, preventing certain antibiotics from reaching their intracellular targets.
22 Acquired resistance occurs when bacterial pathogens which were previously susceptible to certain antibiotic becomes resistant, usually, but not always, after exposure to the antibiotic concerned (Hugo and Russel, 2003). Intrinsic resistance is always chromosomally mediated, whereas acquired resistance may occur by mutations in the chromosome, or by acquisition of genes coding for resistance from an external source normally via a plasmid or transposon. Acquired resistance is a major threat in the spread of antibiotic resistance among bacterial pathogens. Resistance to antimicrobials is mediated by three main strategies namely: enzymatic inactivation of the drug, modification of target sites, and exclusion by efflux pumps (Sibanda and Okoh, 2007). While chemical modifications could be significant in antibiotic resistance, exclusion of unaltered antibiotics from within the bacterial cells, represents the primary strategy in denying the antibiotic access to its targets, and this is believed to enhance resistance in cases where modification is the main mechanism (Li et al., 1994; Poole, 2008; Stavri et al., 2007). (a) Alteration of Target Sites Chemical modifications in the antibiotic target site may result in reduced affinity of the antibiotic to its binding site (Lambert, 2005). For example, erythromycin and other macroclides bind to 50s bacterial ribosomal subunit and block protein synthesis. Some cases of resistance to macrolide develop due to mutation that modifies the target site of the antibiotic. In the case of erythromycin resistance, an enzyme, RNA methylase, adds a methyl group at a specific adenine group in the rrna of the 50s ribosomal subunit. The macrolide antibiotics are unable to bind to the methylated rrna.
23 The β-lactam antibiotics function by binding to the penicillin binding proteins (PBPs) and blocking the final stage of cell wall synthesis. In S. aureus and in Streptococcus pneumonia, resistance to β-lactams can be as a result of mutations that lead to production of PBP2a and PBP 2b respectively. The two enzymes have a reduced affinity for β-lactam antibiotics and yet they take over the function of the normal PBPs in the presence of inhibiting levels of β-lactams (Golami-Kotra et al., 2003; Golemi-Kotra et al., 2003). Quinolones bind to β-subunit of DNA gyrase, blocking the activity of this enzyme that is essential in DNA replication. Mutation in the gene coding for DNA gyrase results in production of a version of DNA gyrase that cannot bind quinolones. Acquisition of this mutant gene from the environment also makes the bacteria resistant to quinolones (b) Enzymatic Inactivation The production of hydrolytic enzymes and group transferases is a strategy employed by bacteria in attempt to render antibiotics ineffective. Genes that code for antibiotic degrading enzymes are often carried on plasmids and other mobile genetic elements (Wright, 2005). Beta-Lactams are inactivated by enzymes called β-lactamases which hydrolyze the cyclic amide bond of the four-membered β-lactam rings. Again, resistance to aminoglycosides in Gram negative bacteria is most often mediated by a variety of enzymes that modify the antibiotic molecules by acetylation, adenylation, or phosphorylation (Over et al., 2001). Many clinical bacterial isolates that are resistant to chloramphenicol have a plasmid that codes for chloramphenicol acetyltransferase, an enzyme that adds acetyl group to chloramphenicol. An acetylated chloramphenicol cannot bind to its target, the 50s ribosomal subunit of the bacterial ribosome, and subsequently it cannot stop protein synthesis.
24 (C) Efflux Pump Mechanism Efflux pumps are membrane proteins involved in the exclusion of toxic compounds (including virtually all classes of antibiotics) from within the cell into the external environment (Webber and Piddock, 2003). These proteins are found in both Gram negative bacteria and Gram positive bacteria as well as in eukaryotic cells. Efflux proteins act like bilge pumps by interacting with toxic compounds entering the cells and expelling them out into the external environment. Antibiotic efflux was first described in 1981, when it was discovered as a mechanisms for tetracycline resistance in enterobacteria (Zechini and Versace, 2009; Bambeke et al., 2010). It was later discovered in the course of time that efflux pump is the mechanism behind most of the multidrug resistance (MDR) phenomenon encountered in both Gram positive and Gram negative bacteria of clinical importance. In the prokaryotic organisms, there are five families of efflux transporter: MF (Major Facilitatator), MATE (Multidrug and toxic efflux), RND (Resistance nodulation-division), SMR (Small multidrug resistance) and ABC (ATP binding cassette). The efflux pumps have not evolved in response to the stresses of the antibiotic era. It has been estimated that 5-10 % of all bacterial genes are involved in transport and a large proportion of these genes encode efflux pump proteins either in the chromosomes or in the plasmid (Piddock and Webber, 2003). It is now recognized that the constitutive expression of the efflux pump proteins encoded by the house keeping genes in the bacterial genomes are largely responsible for the phenomenon of intrinsic antibiotic resistance (Lomovskaya and Bostian, 2006).
25 Although the efflux pump proteins are constitutively present in bacteria cells, the continued presence of their substrates induces over- expression of the genes encoding the proteins (Teran et al., 2003). This is a major risk associated with the exposure of pathogenic bacteria to sub-lethal doses of antibiotics that are efflux pump substrates, because this usually induces over-expressions of the efflux pumps. The broad substrate range of efflux systems is of great concern, because over- expression of a pump will result in cross-resistance to all other substrates of the pump including clinically relevant antibiotics. This is one of the ways through which efflux pump contribute to the phenomenon of acquired multidrug resistance in bacteria 1.5 Plants as Sources of Resistance Modifying Agents It has been proven that, in addition to the production of intrinsic antimicrobial compounds, plants also produce multidrug resistance (MDR) inhibitors which enhance the in vitro antimicrobial activities of antibiotics (Stermitz et al., 2003). Some isolated pure compounds of plant origin which have been reported to possess in vitro resistance modifying activities are provided alongside the antibiotics they potentiate in Table 1b. The discovery that some compounds of plant origin seem to inhibit antibiotic resistance process in bacteria has prompted serious research for such compounds from a variety of medicinal plants. Some of the compounds which have direct antimicrobial activity have also been shown to potentiate antibiotics when used at concentration lower than their minimal inhibitory concentration. If the isolation of resistance modifying compounds from plants is to be realistic, screening for such modulatory activities in crude plant extracts should be the first steps in identifying leads for isolation of such compounds. Some crude plant extracts have provided good indications as resistance modifiers in in vitro combination experiment with antibiotics
26 (Sibanda and Okoh, 2007). Aqueous extracts of tea (Camellia sinensis) have been shown to reverse methicillin resistance in methicillin resistant Staphylococcus aureus (Stapleton et al., 2004). The ethanolic leaf extracts of Chinese plants, Isatis tinetoria and Scutellaria baicalensis had synergistic activities with ciprofloxacin against antibiotic resistant S aureus (Yang et al., 2005).These are just a few examples among many plant extracts that have been found to potentiate the in vitro activity of antibiotics against antibiotic resistant bacteria Some synergies detected in the studies of antibiotics crude extract combination were not specific to any group of organisms or class of antibiotics (Nascimento et al., 2000 and Matias et al., 2012). This suggests that plant crude extract is a blend of compounds that enhance the activity of different antibiotics. Plants have been known to contain myriads of antimicrobial compounds such as polyphenols and flavonoid (Iwu et al., 1999). The antimicrobial and resistance modifying potentials of these naturally occurring flavonoids and ployphenolic compounds have been reported in several studies (Lamberts, 2005; Sato et al.,, 2004)
27 Table 1b: Resistance Modifying Agents of plant origin Compounds Plant source Antibiotic Potentiated Reference 1 Ferruginol Chamaecyparis lawsoniana 2 5-epipisiferol Chamaecyparis lawsoniana 3 2, 6-dimethyl-4- Jatropha phenyl-pyridine- elliptica 3, 5- dicarboxylic acid diethyl ester 4 Carnosic acid Rosmarinus carnosol officinalis 5 Ethyl gallate Caesalpinia spinosa 6 Epicatechin Camellia gallate sinensis 7 Epigallocatechin Camellia gallate sinensis 8 Methyl-1-α-14- Lycopus α-diacetoxy 7- europaeus α-hydroxy-8,15- Isopimara-dien- 18-o oxacillin, Smith et al. (2007) tetracycline, norfloxacin tetracycline Smith et al. (2007) ciprofloxacin, norfloxacin, Marquez et al.(2005) pefloxacin, acriflavine and ethidium bromide erythromycin Oluwatuyi et al. (2004) β-lactams Shibata et al. (2005) Norfloxacin Gibbons et al. (2004) Panipenem and β- Zhao et al. (2001) lactams tetracycline and Gibbons et al. (2003) erythromycin Source: Sibanda and Okoh (2007)
28 CHAPTER TWO MATERIALS AND METHODS 2.1 Collection and Identification of Plant Materials The plants used for this study (Picralima nitida, Chromolaena odorata, Hyptis suaveolens, and Aspili africana) were harvested from Ozubulu, in Anambra State within the period of May to June, 2011. The plants were identified by A.O. Ozioko, a former plant taxonomist at the herbarium section of the Department of Botany, University of Nigeria, and Nsukka. 2.2 Sample Preparations and Extraction The leaves of the test plants were plucked and rinsed with water to remove dirt. They were then air-dried under shade at room temperature for several days. The dried leaves were pulverized using a milling machine to obtain fine powder. The active ingredients were extracted by percolation method using ethanol. Briefly, 100 g of each leaf powder was added in 900 ml of 95% ethanol. The mixture was covered, shaken every 30 min for 6 h, and then allowed to stand for 48 h for extraction. The mixture was then separated by passing through Whatman s No 1 filter paper, after which the filtrate (containing the active ingredients) was evaporated to dryness under air pressure. The dried crude extracts were then put in sterile air-tight containers and stored in the refrigerator (4 o C) for subsequent use. 2.3 Collection and Identification of the Test Organisms The test bacterial organisms were the clinical and environmental isolates obtained from various laboratories in the Departments of Microbiology and Veterinary Pathology and Microbiology, University of Nigeria, Nsukka. The test bacterial organisms were at least three
29 isolates of each of the following bacterial pathogens: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus cereus and Salmonella spp. The identity of these isolates was confirmed by Gram staining and biochemical tests. 2.4 Determination of Antibacterial Activities of the Crude Plant Extracts One gram of each crude extract was dissolved in 5 ml of 20% dimethylsulfoxide (DMSO) to get a concentration of 200 mg/ml of the extract. This was serially diluted in two folds to obtain the following extract concentrations: 100, 50, 25, 12.5 and 6.25 mg/ml. The activities of the plant extracts on isolates were determined by using agar well diffusion techniques (Perez et al., 1990; Alade and Irobi, 1993; Nweze and Onyishi,2010). Eighteen hour old Mueller Hinton broth cultures of the test bacterial isolates were diluted to 0.5 McFarland turbidity standards using sterile normal saline. Using a sterile cotton-tipped swab, the standardized broth culture of the test isolate was evenly inoculated on dried surface of Mueller Hinton agar by streaking. The inoculated Mueller Hinton agar was then allowed to dry for about 5 min after which wells ware punched on the agar at equidistant positions using a sterile standard 6 mm cork borer. About 60 µl of different concentrations of the extract was separately introduced into the different wells that have been labeled, by using micropipette. About 60 µl of the 20% DMSO was introduced into the well bored in the centre of the agar medium as a control. This procedure was repeated in duplicate for all the test organisms and allowed to stay for 30 min on the bench after which they were incubated for 24h at 37 o C in an incubator. After the incubation, the inhibition zone diameters produced by the different concentrations of the crude extracts were measured in millimeter using transparent meter rule. Antimicrobial
activities were expressed as the mean inhibition zone diameters (mm) produced by the plant extracts in the duplicate experiment. 30 2.5 Determination of the Minimal Inhibitory Concentrations (MICs) of the Plant Extracts: The minimal inhibitory concentrations (MICs) of the extracts on the bacterial isolates were determined by macro broth dilution techniques following the recommendation of the Clinical and Laboratory Standard Institute (CLSI). The extract weighing 0.5 g was dissolved in 10 ml of 20% DMSO to get an extract concentration of 50 mg/ml. Then two-fold serial dilutions were made from this stock solution in tubes of 1 ml sterile Mueller Hinton broths to obtain various extract concentrations of 25 mg/ml, 12.5 mg/ml, 6.25 mg/ml, 3.125 mg/ml, 1.5625 mg/ml and 0.78125 mg/ml. An overnight nutrient broth culture of the test bacterial isolate was standardized to 0.5 McFarland turbidly standard (a suspension containing approximately 10 8 CFU/ml) by using sterile normal saline. Ten-fold dilutions of this suspension were additionally made in a sterile normal saline to obtain a final inoculum concentration of 10 6 CFU/ml. Then 1 ml of this adjusted inoculum was added to each tube of the Mueller Hinton broth containing different concentration of the crude extract. Each tube was mixed and incubated at 37 o C for 24 h in an ambient air incubator. This experiment was conducted in duplicate for all the test isolates. A tube of Mueller Hinton broth containing only the 1ml suspension of the test organism without extract and the tubes of Mueller Hinton broth containing different concentrations of the extract without the test organism were used as controls. The tubes were examined after 24h incubation. The MIC of the extract was taken as the lowest extract concentration that completely inhibited the growth of the test organisms in the tubes, as indicated by lack of visual turbidity.
31 2.6 Resistance Modulatory Assays Antibiotic resistant bacterial isolates were screened and selected for resistance modulatory assays using antibiotic discs prepared in the laboratory. The resistance modulatory assays were carried out by both disc diffusion test and macrobroth dilution method. 2.6.1 Preparation of Antibiotic Discs for Disc Diffusion Tests The following five antibiotics were used: ciprofloxacin, norfloxacin, chloramphenicol, tetracycline and erythromycin. The antibiotics were bought from a pharmaceutical shop in Nsukka, Enugu State. The antibiotic discs of each of these antibiotics were prepared in the laboratory in such a way that the disc potency (i.e. the antibiotic content of the disc) is equivalent to the standards specified by the Clinical and Laboratory Standard Institute (CLSI), formerly known as the National Committee for Clinical Laboratory Standards (NCCLS). A tablet of 500 mg of ciprofloxacin was pulverized using a sterile mortar and pestle to obtain fine powder. This was then dissolved in 100 ml of sterile distilled water to obtain a drug concentration of 5000 µg/ml. A 10-fold serial dilution was made to obtain a final drug concentration of 500 µg/ml. Using a 6 mm diameter perforator, 100 circular paper discs were cut out from Whatman s No 1 filter paper. These 100 paper discs were placed in Petri dish and oven sterilized at 60 o C for 2 h. One milliliter of the ciprofloxacin suspension (at a concentration of 500 µg/ml) was used to soak the 100 sterile paper discs so that each disc absorbs approximately 5 µg of the drug, which is equivalent to the potency specified by the CLSI. These antibiotic discs were then allowed to air-dry, after which they were stored briefly in the refrigerator for subsequent use. The same procedure was used to prepare other
32 antibiotic discs using appropriate solvents: norfloxacin (10 µg), chloramphenicol (30 µg), tetracycline (30 µg), and erythromycin (10 µg). 2.6.2 Screening for Antibiotic Resistance in bacterial Isolates Isolates of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were tested for resistance to the above antibiotic discs using Kirby-Baurer disc diffusion technique (Cheesbrough, 2000). The results of the reactions of the bacterial isolates to the antibiotics were interpreted as susceptible, intermediate or resistant, based on the criteria recommended by the Clinical and Laboratory Standard Institute (CLSI, 2011). At least three isolates each of the test organisms (S. aureus, E. coli and P. aeruginosa) that were resistant to more than two of the test antibiotic discs were selected as multidrug resistant isolates and stored for the resistance modulatory assays. 2.6.3 Determination of Minimum Inhibitory Concentration of the plant Extracts on the Resistant Bacteria The minimum inhibitory concentrations of the plant extracts on the resistant bacteria were determined by macrobroth dilution as previously described (section2.5). Then 1/4 th of the MIC was used as sub inhibitory concentration for the resistance modulation assay. 2.6.4 Evaluation of Resistance Modulatory Activity of the Plant Extracts by Antibiotic Disc Diffusion Test The resistance modifying potency of the plant extracts was determined by combining the sub inhibitory concentration of the extracts (1/4 th MIC) with the antibiotic discs against the resistant bacteria (Adu et al., 2011; Stavris et al., 2007). An appropriate dilution of
33 plant extracts in 10% DMSO is incorporated into a specific volume of molten Mueller Hinton agar at 50 o C to achieve the final extract concentration equivalent to 1/4 th MIC that has been predetermined for that particular resistant bacterial organism. The media was then poured into Petri plates and allowed to solidify. The plates were then inoculated with a standardized 18 h old broth culture of the test resistant bacterial isolate by using a sterile cotton tipped swab. The activities of the antibiotics on the test organism were then evaluated by Kirby-Baurer disc diffusion techniques using the same antibiotic discs prepared in the laboratory (section2.6.1). The plates without the plant extracts were also inoculated and tested as controls. The experiment was carried out in duplicates. The inhibition zone diameter of each antibiotic disc was measured after incubation at 37 o C for 24 h. The effects of the plant extract on the activity of the antibiotic discs against the resistant organisms was evaluated by comparing the size of inhibition zone diameters in plates containing plant extracts and in control plates without plant extract. 2.6.5 Evaluation of Resistance Modifying Activity of the Plant Extracts by Macrobroth Dilution Test The resistance modifying potency of the plant extracts was also evaluated using macrobroth dilution technique by determining the minimum inhibitory concentration (MIC) of the antibiotics on the resistant bacteria in the presence and in absence of sub inhibitory concentration (1/4 th MIC) of the plant extract (Mahamoud and Cheralier, 2007; Gibbons et al., 2007; Boamah et al., 2011). The MICs of the antibiotics were determined by macrobroth dilution method following the standard procedures of the Clinical and Laboratory Standards Institute (CLSI, 2006) and the methods of Andrews (2001). The antibiotic was reconstituted in appropriate
34 solvent and different ranges of two-fold serial dilutions were made from these stock solutions in tubes of 1 ml sterile Mueller Hinton broths. Depending on the value of the predetermined MIC of each crude extract on a particular antibiotic resistant organism, about 200-300 µl of a known concentration of the extract is added separately to 1ml tubes of Mueller Hinton broth containing the serially diluted antibiotic to obtain a final extract concentration equal to ¼th MIC of that extract. Then 1 ml of an overnight broth culture of the test resistant organism adjusted to 10 6 CFU/ml was added to each tube of serially diluted antibiotics in 1 ml of Mueller Hinton broth (the tubes which also contain ¼th MIC of the plant extract). Tubes of 1 ml Mueller Hinton broth containing the serially diluted antibiotics without the plant extract were also inoculated with the test resistant organism. The experiment was conducted in duplicate and incubated at 37 o C for 24 h. The following served as controls: (i) a tube of 1 ml Mueller Hinton broth containing the test organism without antibiotic and without plant extract. (ii) A tube of 1 ml Mueller Hinton broth containing only antibiotic and plant extract. At the end of the incubation, the tubes were examined for any difference in the MIC of the antibiotic containing sub-inhibitory concentration of crude extract and the MIC of the antibiotic containing no extract. 2.7 Phytochemical Screening of Plant Extracts The phytochemical screening of the extracts was carried out using the methods described by Trease and Evans (2004). 2.7.1 Test for Steroids and Terpenoids About 9 ml of 96% ethanol was added to 1.0 g of each of the samples and refluxed for 2 minutes and filtered. The filtrate was concentrated to 2.5ml on a water-bath and 5 ml of hot
35 water added. The mixture was allowed to stand for 1 h. Waxy matter observed was filtered off. The filtrate was extracted with 2.5 ml chloroform. The layers observed were separated using separating funnel. A volume of 1 ml of conc. H 2 SO 4 was carefully added to 0.5 ml chloroform extract and shaken to form lower layers. Reddish brown interface shows the presence of steroids. Another 0.5 ml of the chloroform extract was evaporated to dryness on a water bath at 40 o C. This was heated further with 3 ml conc. H 2 SO 4 for 10 minutes on a water bath at 40 o C. Grey colour indicated the presence of terpenoids. 2.7.2 Test for Phenols/ Tannins About 100 g of each of the sample was extracted in 10 ml of distilled water. The solution was heated in boiling water-bath for 3 minutes and filtered. Then 2 ml aliquots of the filtrate were placed in test tube and the following tests were performed: 1. Few drops of 10% neutral aqueous iron (iii) chloride were added to the aliquot of the diluted solution. Development of green to blue-black precipitates indicates the presence of tannins (Sofowa, 1982). 2. A volume of 1 ml of 10% lead acetate solution was added to the last portion of the initial aqueous extract, and homogenized. A coloured precipitate indicates the presence of phenols. 2.7.3 Test for Alkaloids About 20 ml of 5% sulphuric acid in 50% ethanol was added to about 2 g of each of the sample. This was heated on a boiling water bath for 10 minutes, cooled and filtered. The
36 filtrate was transferred into four test tubes, each containing 2 ml of the filtrate and used for the following tests. a. Few drops of Dragendoff s reagent (a solution of bismuth iodide in potassium iodide) were added to the last portion of the initial aqueous extract, and homogenized. A brick red precipitate indicates the presence of alkaloids. b. About two drops of Wagner s reagent (a solution of iodine in potassium iodide) were added to the second test tube and swirled for few seconds. A brownish-red precipitate indicated the presence of alkaloids. c. About two drops of Meyer s reagent (a solution of mercury iodide in potassium iodide) were added to the third test tube and homogenized for few seconds. A creamy (dirty white) precipitate indicated the presence of alkaloids. d. Two drops of picric acid (1%) solution was added to the fourth test tube containing 2 ml aliquot and homogenized for 30 seconds. A reddish precipitate indicated the presence of alkaloids. 2.7.4 Test for Saponins About 20 ml of distilled water was added to 0.25 g of each of the sample in a 100 cm 3 beaker and boiled gently on a hot water bath for 20 minutes. The mixture was filtered hot and allowed to cool. The filtrate was used for the following test. 1. Frothing test: A volume of 20 ml of distilled water was added to 5 ml of the filtrate in a test tube, and shaken vigorously. A stable froth (foam) upon standing for about 30 seconds indicates the presence of saponin. 2. Emulsion Test: Two drops of olive oil was added to 5 ml of the filtrate in the test tubes above. Formation of emulsion indicates the presence of saponin.
37 2.7.5 Test for Flavonoids About 10 ml of ethyl acetate was added to about 0.2 g of each of the sample and heated on a water bath at 40 o C for 3 minutes. The mixture was cooled, filtered and used for the following test: Ammonium test: About 4 ml of the filtrate was shaken with 1 ml of dilute ammonia solution. Layers were formed and allowed to separate. An intense yellow colour in the ammoniacal layers indicates the presence of flavonoids. To the yellow coloured solution, 3 drops of conc. Sulphuric acid was added. Disappearance of the yellow colour indicates the presence of flavonoids. 1% Ammonium chloride solution test: To 4 ml of the filtrate, 1 ml of 1 % ammonium chloride was added. A yellow colour indicates the presence of flavonoids. 2.7.6 Test for Oils About 500 mg of each of the samples was shaken in about 5 ml dilute sodium hydroxide (0.1 M) and filtered. Then 2 ml of dilute HCl was added to the filtrate. A white precipitate indicated the presence of volatile oil. 2.7.7 Test for Glycosides About 300 mg of each of the samples was dissolved in 10 ml of distilled water and the resulting solution was filtered. Five ml of equi-volume mixture of Fehling s solutions I and II was added to a 2 ml aliquot of the aqueous solution obtained above. The mixture was homogenized and heated in a water bath for not less than 5 minutes. Brick red precipitates indicate the presence of free reducing sugars.
38 2.8 Statistical Analysis All the data were subjected to one way analysis of variance (ANOVA) and the variant means were separated using Ducan multiple range test. Significance was accepted at P 0.05. The one way ANOVA test was used to determine if there was any statistically significant difference in the susceptibilities of the different test organism to each extract, and also to determine if there is any significant difference in the activities of the different plant extracts on each test organism.