Antibiotic To Develop A Combination Therapy Biology Essay


The first taxonomic description of Staphylococcus provided by Rosenbach in 1884 when he divided the genus into Staphylococcus aureus and Staphylococcus Albus, although Pasteur and Ogeston,1880, had observed spherical bacteria in abscess pus four year earlier (Gillespie and Hawkey, 2006). The staphylococci and a group of saprophytic tetrad-forming micrococci were placed into the genus Micrococcus and subsequently separated again. Later on, the systematic studies by using a range of factors like DNA-base technique (Silvestri and Hill 1965) and cell-well composition (Schleifer and Kandler 1972) have distinguished staphylococci from micrococci and other bacteria. This demonstrated that micrococci have G + C content of 63-73 mol%, compared with staphylococci which have a G + C content of 30-39 mol%, indicate that they are not significantly related (Ludwig et al. 1985; Stackebrandt et al. 1987).

2.1.2 Cell morphology and cultural characteristics

Staphylococcus aureus is uniformly a Gram positive in young (18 -24h) cultures and appear spherical with an average diameter of 0.5-1.5µm on light microscopy, and is frequently seen in grapelikeclusters. However, it is aerobic, facultatively anaerobic, nonmotile and non-sporulating organism. Also it grows well in a variety of commercial broth media, and is resistant to high concentrations of salt. Colonies on TSA are small, creamy and golden colored. Colonies on MSA are yellow and turns the media yellow.

2.1.3Cellular and secreted components:

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The cell membrane is typical lipid-protein bilayer composed mainly of phospholipids and proteins. The cytochromes and menaquinones bound to cell membranes are important of the electron transport system. Protein isolated from the membranes of S. aureus includes several penicillin-binding proteins (PBPs), which catalyse terminal reactions of peptidoglycan biosynthesis ( Hartman and Tomaze 1984). Iron-regulated cell-membrane protein are expressed under iron limitation (Domingue et al., 1989). Peptidoglycan and teichoic acid are the major components of the staphylococcal cell well, peptidoglycan makes up to 50-60% of the dry weight (Schleifer 1983). It is the main structural polymer in the wall and consists of a heteropolymer of glycan chain cross-linked by short peptides. Staphylococcal cell-wall teichoic acid is a water-soluble polymer that is covalently linked to peptidoglycan acid and amounts to about 30-50% dry weight ( Morath et al.,2002).

Adherence to soluble plasma component and/or extracellular matrix proteins is promoted by a range of S. aureus cell-wall-associated proteins (Patti et al., 1994). These adhesins may play an important role in the colonization of the host and during invasive disease. The best characterized of these are protein A SpA ( Forsgren et al.,1983; Hartleib et al., 2000), Two fibronectin-binding proteins FnBPA and FnBPB ( Greene et al.,1995), the fibrinogen-binding proteins clumping factors A & B (McDevitt et al., 1994) and a collagen-binding protein Cna (Patti et al .,1992). Also, S. aureus produce a group of functionally related pyrogenic toxins that cause fever and shock in its hosts. These toxins are classified as superantigens and include the staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-1 (TSST-1) which have a potent mitogenic activity for Tlymphocytes of several host species (Fleisher 1994). Moreover, S, aureus also produce four types of haemolysins known as α-, β-, γ-and δ-toxin which are considered as primary factors in the development of infection (Buerke et al., 2002). Several other extracellular components are secreted by S, aureus, including coagulase, staphylokinase, lipase, urease and hyaluronidase. However, Staphylococcus aureus produce an extracellular material called slime termed biofilm which is making them more resistant to the effect of antibiotics (Mack 1999).

2.1.4 Staphylococcus aureus genome:

S, aureus genome is composed of a single chromosome of around 2.8 Mb, which is predicated to encode approximately 2500 genes. The genome sequence of S, aureus strain was published in 2001 (Kuroda et al., 2001). It was observed that most of the antibiotic resistance genes were carried either by plasmids or by mobile genetic elements including a unique resistance island. Three classes of new pathogenicity island were identified: a TSST island family, an exotoxin island and an enterotoxin island.

2.1.5 Antimicrobial resistance of Staphylococcus aureus:

The emergence of resistance to penicillin, the emergence of and epidemic rise in methicillin resistance, the recognition of strains with intermediate resistance to vancomycin and the recent emergence of S.aureus that is fully resistant to vancomycin are the most significant events in the history of S, aureus antibiotic resistance.

Penicillin-resistant S, aureus strain emerged in the early 1940s, shortly after the introduction of penicillin into clinical practice. Resistant to methicillin and other β-lactamase-resistant penicillins was likewise observed soon after of methicillin was introduced into clinical use in Britain (Jevons 1961). At this time the methicillin-resistant strain isolated in Britain demonstrated heterogeneous resistance to methicillin were multiply antibiotic resistant and were isolated from hospitalized patients (Barber 1961). After the mid-1970s, large outbreaks of infections caused by MRSA were reported in many hospitals in Britain (Shanson et al., 1971; Cookson et al., 1988), Ireland (Cafferkey et al., 1985), the United States (Schaefler et al., 1981) and Australia (pavillard et al., 1982). Since then, many clones of MRSA associate with epidemic spread or sporadic infections have been described throughout the world.

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Approximately one-third of serious S, aureus infections in the UK are now caused by MRSA, although the figure varies considerably worldwide. Until recently, MRSA was mostly confined to the hospital setting and MRSA colonization of those discharged from the community rarely persisted long term except when associated with defects in the skin integrity or the presence of prosthetic material. However, community acquired MRSA associated with both colonization and infection is being increasingly recognized (Daum et al., 2002; Okuma et al., 2002). These strains are resistant to fewer non-β-lactam antibiotics than most of the previously defined MRSA strain.

The gene encoding methicillin resistance (mecA) is carried by the chromosome of MRSA and methicillin-resistant S. epidermidis (MRSE). mecA is part of a mobile genetic element termed staphylococcal cassette chromosome mec (SCCmec) (Katayama et al., 2000). Four type of SCCmec have been defined based on sequence analysis. Evaluation of 38 epidemic MRSA strains isolated in 20 countries showed that the majority possessed one of three typical SCCmec (types I-III) elements on the chromosome (Ito et al., 2001). However, SCCmec type IV was subsequently defined in community-acquired MRSA (Daum et al., 2002). Multiple MRSA clones carrying type IV SCCmec have been identified in community-acquired MRSA strains in the United States and Australia (Okuma et al., 2002).

The glycopeptide antibiotics vancomycin and teicoplanin prevent the transglycosylation and transpeptidation steps of cell-wall peptidoglycan synthesis by binding to the peptidyl-D-alanine termini of peptidoglycan precursors. Glycopeptides are very important in clinical practice to treat several MRSA infections. Since 1997, three categories of S. aureus resistances to vancomycin have been described: 1) S. aureus with intermediate-level resistance to vancomycin (VISA) which was first detected in Japan in 1996 (Hiramatsu et al. 1997b), 2) S. aureus with heteroresistance to vancomycin (hVISA) which was identified in Japan (Hiramatsu et al. 1997a) 3) and vancomycin-resistant S. aureus (VRSA) which was reported in Michigan in 2002, with second apparently unrelated case in Pennsylvania 2 month later (Centers for Disease Control and Prevention 2002a,b).

2.1.6 Clinical presentation and treatment:

Staphylococcus aureus is well known as a human opportunistic disease-causing organism responsible for a variety of diseases. Nosocomial infections caused by this pathogen are a major cause of morbidity and mortality. Some of the most common infections caused by S. aureus involve the skin and soft tissue, and they include cellulitis or furuncles, boils, impetigo, and postoperative wound infections. Some of the more serious infections produced by S. aureus are bacteremia, meningitis, cerebritis, acute endocarditis, pericarditis, myocarditis, osteomyelitis, pneumonia, abcesses, and scalded skin syndrome. However, food poisoning is another important syndrome associated with this bacterium. Also, toxic shock syndrome (TSS), has been attributed to infection or colonization with toxigenic S. aureus (Murray et al., 2003)

Methicillin-resistant Staphylococcus aureus (MRSA) emerged in the 1980s as one of the major clinical problems in hospitals (Oliveira et al., 2002). However, in the past decade, infections caused by MRSA have emerged in the community. Currently, CA-MRSA (Community-acquired MRSA) has emerged as an epidemic pathogen that is responsible for rapidly progressive, severe sepsis, fatal diseases including necrotizing pneumonia and necrotizing fasciitis (Boyle-Vavra, Daum, 2007). MRSA are almost resistant to all β-Iactams including penicillins, cephalosporins, monobactams and carbapenems, which are the antibiotics most commonly used to treat S. aureus infections. Recently, MRSA infections can only be cured with more toxic and more costly antibiotics like vancomycin, linezolid, and daptomycin, which are normally used as last-line agents. Since MRSA can spread easily among patients, hospitals over the world are confronted with the problem to control MRSA. In addition, it is very likely that even these drugs will soon encounter the resistance problems experienced by common antibiotics (Murray et al., 2005).

2.1.7 Drug target in staphylococcus aureus genome (adaB gene):

In the new pathway for the discovery or development of drugs to combat the increasing menace of drug-resistant microorganisms (Cohen, M.L., 2000), the crucial first step is to generate a set of target genes, from whole-genome data, that offer the potential for effective therapeutic intervention (Allsop, A.E.,1998; Rosamond, J. and Allsop, A., 2000). However, the ability to search for novel bacterial drug targets or validate theoretical targets has been revolutionized by genome sequence analysis and associated genetic techniques (Slonczewski and Foster, 2009). Moreover, the determination of complete genome sequence of staphylococcus aureus and the detection of bacterial genes that are essential for the survival of the pathogen and non-homologous to human genes represent a promising means of identifying novel drug targets (K.R. Sakharkar et al. 2008). As known, bacterial cells are not defenseless against genetic damage. However, DNA repair mechanisms appear to be present in all living organisms as a defense against environmental damage (Mims et al., 2004). Therefore, DNA repair gene (adaB) of S. aureus that encodes protein which is involved in the synthesis of methylated DNA-protein cysteine methyltransferase is selected as a target protein in the present study. The enzyme is produced by this gene play an important role to protect the genome of S. aureus cell against both spontaneously occurring and induced mutational damage. Whether damage is introduced by mutagens or by inaccurate DNA synthesis, microbial survival depends on the ability to repair DNA (Nester et al., 2004). One of DNA repair processes is direct repair which is either reverses or simply removes the damage. This may be regarded as 'first line' defense. Excision repair ,the 2nd process, where damage in a DNA strand is recognized by an enzymatic 'housekeeping' process and excised, followed by repair polymerization to fill the gap using the intact complementary DNA strand as a template. This is also a primary form of defense since the goal is to correct damage before it encounters and potentially interferes with the moving DNA replication fork. Other process is 'Second line' repair which operates when DNA damage has reached a point where it is more difficult to correct. When normal DNA replication processes are blocked, permissive systems may allow the interfering damage to be inaccurately corrected, allowing errors to occur but improving the probability of cell survival. In other instances, where damage has passed the replication fork, post-replication or recombinational repair processes may 'cut and paste' to construct error-free DNA from multiple copies of the sequence found in parental and daughter strands (Mims et al., 2004).


2.2 Escherichia coli

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Escherichia coli, which belong to the family Enterobacteriaceae, is Gram-negative, facultative anaerobic, oxidase negative, and non-sporulating (Edwards and Ewing, 1972). These bacteria are capable of fermenting a wide variety of carbohydrates with production of both acid and gas (Baylis et al., 2006). However, rapid fermentation of lactose is a characteristic feature of E.coli. Cells are typically rod-shaped and are about 2 μm long and 0.5 μm in diameter, with a cell volume of 0.6 - 0.7 μm3 (Kubitschek, 1990). E. coli is commonly found in the lower intestine of human and other warm-blooded animals, and can live on a wide variety of substrates. They can also occur in water, food and soil. E. coli is also an important human pathogen. It is the bacterial species most frequently isolated from pathological materials (Kayser, 2005).

In acute urinary tract infections, E. coli is the causative organism in 70-80% of cases and in chronic. However, E. coli causes about 15% of all cases of nosocomial sepsis. The most important other E.coli infections are including wound infections, infections of the gallbladder and bile ducts, appendicitis, cholecystitis, peritonitis, meningitis in premature infants, neonates, and very elderly patients. They also have the ability to form K antigen, capsular polysaccharides that contribute to biofilm formation. Biofilm-producing E. coli are recalcitrant to immune factors and antibiotic therapy and are often responsible for chronic urinary tract infections (Ehrlich et al., 2005). Nevertheless, some particular strain, such as serotype O157:H7, can cause serious food poisoning in humans, and are occasionally responsible for product recalls (Vogt RL, Dippold L, 2005).

E. coli that cause intestinal infections are now classified in five pathovars with differing pathogenicity include Enteropathogenic E. coli (EPEC), Enterotoxic E. coli (ETEC), Enteroinvasive E. coli (EIEC), Enterohemorrhagic E. coli (EHEC) and Enteroaggregative E. coli (EAggEC). EPEC and EAggEC frequently cause diarrhea in infants. ETEC produce enterotoxins that cause a choleralike clinical picture. EIEC cause a dysenterylike infection of the large intestine. EHEC produce verocytotoxins and cause a hemorrhagic colitis as well as the rare hemolytic-uremic syndrome (Kayser, 2005; Baylis et al., 2006). Moreover, antimicrobial resistance is common among ETEC and EPEC strains isolated from outbreaks in the United States (Donnenberg, 1995; Dalton et al., 1999), and since the early 1990s, 0157 and other strains have demonstrated slowly increasing levels of resistance to certain antibiotics include streptomycin, sulfonamides, and tetracycline (Cheryl et al.,2003).

2.3 Klebsiella pneumoniae

Klebsiella pneumoniae is nonmotile, straight bacilli, 0.3-1 µm x 0.6-6 µm, lactose fermenting, facultative anaerobic, gram-negative bacterium with a prominent polysaccharide capsule, found in the normal flora of the skin, mouth, and intestines (Ryan and Ray, 2004, William et al., 2001). This capsule encases the entire cell surface, accounts for the large appearance of the organism on gram stain, and provides resistance against many host defense mechanisms. Its DNA mol. % G + C is 53-58 (Anthony, 2006)

K. pneumoniae is the member which responsible for most human infections among other members of the Klebsiella genus of Enterobacteriaceae. It is opportunistic pathogen found in mammalian mucosal surfaces and also in the environment (Anthony Hart, 2006; Mims et al., 2006). The principal pathogenic reservoirs of infection are the patients' gastrointestinal tract and the hospital personnel's hands. However, K. pneumoniae can spread rapidly, often leading to nosocomial outbreaks (Obiamiwe and Berkowitz, 2009). Common sites include surgical wound sites, urinary tract, lower respiratory tract, and biliary tract. The spectrum of clinical syndromes includes urinary tract infection (UTI), bacteremia, community-acquired pneumonia, meningitis, upper respiratory tract infection, diarrhea, osteomyelitis, wound infection, and cholecystitis (William et al., 2001; Obiamiwe and Berkowitz, 2009).

However, K. pneumonia has a high mortality rate of approximately 50% even with antimicrobial therapy. The mortality rate approaches 100% for persons with alcoholism and bacteremia. Nevertheless, extensive use of broad-spectrum antibiotics in hospitalized patients has led to both increased carriage of klebsiellae and the development of multidrug-resistant strains that produce extended-spectrum beta-lactamase (Anthony, 2006; Obiamiwe and Berkowitz, 2009).

2.4 Natural product (Calligonum comosum):

Both the idea and the practice of using medicinal plants as remedies for human diseases are as old as the human civilizations. In ancient Chinese medicine, Cures for infection using plants likely contained "natural antibiotics" properties began to be described over 2,500 years ago (Lindblad, 2008 ). Many other ancient cultures, including the ancient Greeks, ancient Egyptians and medieval Arabs already used plants and molds to treat infections (Forrest, 1982; Wainwright 1989; Slonczewski and Foster, 2009). For example, ancient Egyptian medicine of 1000 B.C. is known to have used C. comosum and other herbs for medicine (Jaber S, 2008). Recently, the World Health Organization (WHO) estimated that about 80 per cent of the world's population are known to use the traditional medicines derived from medicinal plant. Moreover, in some developed countries, the use of herbal medicines is steadily growing with approximately 40% of population reporting use of herb to treat medicinal illnesses within the past years (Bent and Ko 2004; Dubey et al., 2004; Kamboj, 2000). Some plants are known as medicinal because they contain active substances that cause certain reactions, from relenting to the cure of diseases, on the human organism (Silva Junior et al. 1994).

. Calligonum comosum (Arta), belongs to family Polygonaceae, is a desert woody shrub, approximately reaching 1-3 m in height, much branched from the base (Jaber S, 2008). Its general distribution and growing goes from the North African deserts to the desert sands of the Arabia and as far east as Pakistan (Lipscombe Vincent, 1984). The young branches are dark green, articulate and very quickly lose their small linear leaves (chaudhary, 1999). This shrub grows in arid, sandy ecosystems where the annual rainfall does not exceed 100 mm. Bedouins dry the young shoots to prepare a nourishing meal and use its branches which make excellent firewood (Lipscombe Vincent, 1984).

It has been used by healers in the treatment of stomach ailments. The stems and leaves of C. comosum are chewed for curing toothache (Ghazanfar SA, 1994, Liu et al., 2001). The young shoots and leaves are picked in spring, prepared as a powder and used externally as an ointment. However, it is used for gastric problems and is frequently used to treat scabies in dromedaries. In Saudi Arabia, C, comosum is used for tanning (Jaber S, 2008). Also, its root decocting is used for gum sores (Zoget and Al-Alsheikh, 1999). Munton (1988) pointed to the high levels of crude protein, potassium and calcium in its vegetative parts. Its flower can be eaten as it is high in sugar and nitrogenous components. Jaber S (2008) reported that Calligonum comosum extract has an antimicrobial activity against several types of bacteria and worms. EI-Hawary and Kholief (1990) found that the extract of C, comosum produced a hypoglycemic effect. Moreover, the extract of C. comosum aerial parts possesses anti-inflammatory properties and has shown significant anti-ulcer and cytoprotective effects against gastric ulcers (Liu et al., 2001).


2.5 Antimicrobial agents:

Antimicrobial drugs can be subclassified as antibacterial, antifungal, and antiviral agents. These agents include natural compounds, called antibiotics, as well as synthetic compounds produced in laboratories. Antibiotics were drugs that had actions against bacteria. The term antibiotic ,from the Greek, which means "against life" was introduced as a descriptive name of the phenomenon exhibited by these drugs, and has traditionally referred to natural metabolic products of microorganisms like bacteria and fungi that kill or inhibit the growth of other microorganisms (Calderon and Sabundayo, 2007; Mims et al., 2006). The earliest use of antibiotics was probably in the treatment of skin infections with moldy bean curd by many ancient cultures including, the ancient Chinese, the ancient Egyptians, ancient Greeks and medieval Arabs (Forrest, 1982; Wainwright 1989; Lindblad, 2008; Slonczewski and Foster, 2009). The development of modern antibiotics can be traced to the work of Louis Pasteur, one of the first pioneers in this field who observed that the in vitro growth of one microbe was inhibited when another microbe was added to the culture (Brenner and Stevens, 2006). Several decades later, the modern antibiotic revolution began with the discovery of penicillin in 1928 by Alexander Fleming when he observed that the growth of his staphylococcal cultures was inhibited by a Penicillium contaminant (Slonczewski and Foster, 2009). His observations eventually led to the isolation and use of penicillin for treating bacterial infections. The discovery of penicillin stimulated the discovery and development of a large number of other antibiotics, the use of which has revolutionized the treatment of infectious diseases.

Antimicrobial drugs are usually classified based on their site and mechanism of action and are subclassified on the basis of their chemical structure. There are five main target sites for antibacterial action includes cell wall synthesis inhibitors (e.g. β-lactams and the glycopeptides), protein synthesis inhibitors (e.g. aminoglycosides, tetracyclines and chloramphenicol), metabolic and nucleic acid inhibitors (e.g. quinolones, rifampicins and sulfonamides), and cell membrane inhibitors like polymyxins (Calderon and Sabundayo, 2007; Mims et al., 2006; Slonczewski and Foster, 2009). Moreover, the antimicrobial activity of a drug can be characterized in terms of its bactericidal or bacteriostatic effect, its spectrum of activity against important groups of pathogens, and its concentration- and time-dependent effects on sensitive organisms. Antibiotics which target the bacterial cell wall, or cell membrane, or interfere with essential bacterial enzymes are usually bactericidal in nature. Those which target protein synthesis, such as the aminoglycosides, tetracyclines usually are bacteriostatic (Finberg et al., 2004). The spectrum of antimicrobial activity of a drug is the primary determinant of its clinical use. Antimicrobial agents that are active against a single species or a limited group of pathogens (e.g., gram-positive bacteria) are called narrow-spectrum drugs, whereas agents that are active against a wide range of pathogens are called broad-spectrum drugs (Brenner and Stevens, 2006).

The emergence of antimicrobial resistance is a matter of degree, and is an evolutionary process that is based on selection for pathogens that have enhanced ability to survive doses of antibiotics that would have previously been lethal (Mims et al., 2006; Cowen, 2008). Some species are innately resistant to some families of antibiotics either because they lack a susceptible target or because they are impermeable to the antibacterial agent. The Gram-negative rods with their outer membrane layer exterior to the cell wall peptidoglycan are less permeable to large molecules than Gram-positive cells. However, within species that are innately susceptible, there are also strains that develop or acquire resistance ((Mims et al., 2006).

2.6 Methods:

2.6.1 Polymerase chain reaction (PCR)

PCR Polymerase Chain Reaction (PCR) is a technique developed by Kary Mullis in the 1983 (Mullis, 1990) to amplify a specific DNA sequence across several orders of magnitude, generating millions of copies of a particular DNA sequence in vitro within a few hours (Saiki et al., 1985, 1988; Mullis and Faloona, 1987) is one of the most important techniques in molecular biology today. This technique provides rapid, simple, and sensitive detection of bacterial genes (James R. Johnson, 2000).

The principle of PCR is based on the repetitive cycling of three simple steps which are depend on heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. The first step in each PCR cycle is denaturation, which increases the temperature within the sample vial to about 94 °C, causing the double-stranded dDNA within the sample to separate into two pieces. The second step, annealing, is completed when the temperature reduces to approx. 55 °C, which allows the two specific oligonucleotide primers bind to the DNA template complementarily. The final step is the synthesis portion of the reaction, wherein the temperature is raised to about 74 °C, the optimum temperature for the catalytic functioning of Taq DNA polymerase. In this step, target DNA is extended, replicating to form additional copies of the target DNA. After extension, two single template DNA strands and two synthesized complementary DNA strands combine together forming two new double strand DNA copies and the reaction will repeat above steps. Within 30 cycles, over a million copies of the original target DNA can be reproduced.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus, which inhabits hot springs where temperatures exceed 90â-¦C (Saiki et al., 1988). Taq polymerase remains active despite repeated heating during many cycles of amplification. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotide, by using single-stranded DNA as a template and DNA oligonucleotides, which are required for initiation of DNA synthesis, and the most important part, primers containing sequences complementary to the target region along with a DNA polymerase are key components to enable selective and repeated amplification. However, the temperatures used and the length of time which are applied in each cycle depend on a variety of parameters.

The discrimination of pathogenic from non-pathogenic strains and detection of infectious agents by virtue of specific genes is one of the basis for PCR diagnostic applications in microbiology (Newton and Graham, 1997) as in the heterogeneous nature of the methicillin resistance, testing of the presence of mecA gene by PCR remains the most sensitive method for identification of methicillin-resistant S. aureus isolates (Tomasz et al., 1989). . Because of the exquisite sensitivity it offers, PCR has rapidly become a standard method in diagnostic microbiology. More recently, reagent kits and various instrument platforms have added speed, flexibility, and simplicity (Tang et al., 1997; Fredricks and Relman, 1999; Tang and Persing, 1999). Moreover, PCR is used whenever the exact sequence of DNA building blocks needs to be determined: e.g. in other genome sequencing projects, in gene research, in the investigation of genomic changes, in the search for targets, etc. However, PCR also plays an important role in the area of drug research which depends on potential target genes.

In addition to the above applications, PCR also can be used as confirmatory tool as with the Sa442 DNA fragment, which originally described by (Martineau et al., 1998) is a popular DNA target for identification of Staphylococcus aureus by PCR (Grisold et al., 2002; Tan et al., 2001).

2.6.2 Reverse transcription-polymerase chain reaction (RT-PCR)

Reverse transcription (RT)-PCR is a laboratory technique commonly used in molecular biology to amplify RNA targets. Because DNA polymerase requires a double-stranded DNA template, RNA must be transcribed into complementary (c) DNA prior to PCR by the enzyme reverse transcriptase (RT). The cDNA then serves as the template for the first PCR temperature cycle. The combined use of RT and PCR to amplify RNA targets was first described in 1987. This is the most useful and sensitive technique for mRNA detection and quantitation that is currently available (Wang and Brown, 1999). RT-PCR is mostly used to detect viruses and the viability of microbial cells through examination of microbial mRNA (Larrik, 1992). It is also an important technique in the diagnosis of infectious and genetic diseases and is the key procedure used to detect and quantify RNA as a measure of gene expression. Also, RT-PCR can be used to characterize patterns of mRNA expression, todiscriminate between closely related mRNAs and to analyses RNA structure.

Two reverse transcriptase enzymes commonly used are Moloney murine leukemia virus (M-MuLV) reverse transcriptase and avian myeloblastosis virus (AMV) reverse transcriptase. Both enzymes have the same fundamental activities but differ in some characteristics, including temperature and pH optima. In addition to M-MuLV and AMV, other variants of this enzyme are available for use in the molecular diagnostic laboratory. These enzymes are available in preoptimized RT-PCR kits.

In vitro reverse transcription is primer directed. A single primer is used to generate cDNA and can be one of the primers used in the subsequent PCR reaction (sequence-specific) or a random oligonucleotide. Specificity is not required of reverse transcription. Random oligonucleotides are convenient in that one RT kit or reaction can be used for all RNA targets. Traditionally, the RT step has been performed in a separate tube containing only components necessary for reverse transcription. After RT, an aliquot is removed, added to a PCR reaction tube, and subjected to amplification. Drawbacks of the separate tube method include inconvenience and cross-contamination risk. More recently, single-tube RT-PCR assays, either two-enzyme or single-enzyme, have been described.

2.6.3 Gel Performance:

Gel electrophoresis is a technique used for the separation of DNA, RNA or protein, in electrophoresis, an electric field is generated to separate charged molecules that are suspended in a gel. Negatively charged molecules move toward the anode, on one side of the gel, and positively charged molecules move toward the cathode on the other side.

The gel is immersed within an electrophoresis buffer (running buffers) that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value. The most frequently used electrophoresis buffers for agarose gel electrophoresis are Tris-acetate with EDTA (TAE) and Tris-borate with EDTA (TBE) (Voytas, 2000). The pH of both buffers is greater than 7.0, meaning that the phosphate backbone of DNA has a net negative charge and migrates toward the anode during electrophoresis. TAE has less buffering capacity but greater ability to resolve high-molecular-weight DNA fragments. It is often used when DNA is to be isolated from the gel or for resolution of larger DNA fragments (>12 kb). The interaction of TBE with agarose results in a smaller apparent pore size producing better resolution of small DNA molecules (<1 kb) and reducing the tendency of DNA bands to broaden due to dispersion and diffusion.

Before adding DNA samples to a gel, loading buffer is added in order to increase the density of the sample so it sinks to the bottom of the well and to add color to the sample. In turn, this color serves as a marker to simplify the loading process and to allow the progress electrophoresis to be monitored, based on the movement of dye(s) through the gel (Sambrook et al., 2005). After electrical separation, the DNA can be subjected to a fluorescent dye that binds the gel into a series of bands that can show which kinds DNA molecules are present. Then, the bands observed can be compared to known molecular weight size markers in order to determine their size.

2.6.4 DNA Sequencing

The term of DNA sequencing is refers to sequencing methods for determining the order and sequential arrangement of the nucleotides in a molecule of DNA. DNA sequencing techniques are key tools in many fields, and are promoting new discoveries that are revolutionizing the conceptual foundations of many fields (Francna et al., 2002). However, fundamental to the development of PCR-based gene detection assays is the knowledge of the nucleotide sequence of the gene of interest. Currently, DNA sequencing can be done routinely by automated DNA sequencing systems. In fact, DNA is sequenced for several reasons such as determine the sequence of the protein encoded by the DNA, the location of sites at which restriction enzymes can cut the DNA, the location of DNA sequence elements that regulate the production of mRNA, alterations in the DNA by mutation and to detect of the genetic mechanisms of drug resistance in microorganisms (Shamputa et al., 2004). The appropriate DNA sequences usually can be located by searching the GenBank databases ( with keywords that include the gene name and species (James, 2000).

2.6.5 Gram staining

Gram staining, first step in the identification of a bacterial organism, is by far the most widely used procedure of differentiating the species of bacteria into two large groups: Gram-positive and Gram-negative. The variation in the staining outcome of these two groups reflects a fundamental difference in the physical and chemical properties of their cell walls (Bergey et al., 1994; Nester et al., 2004). This method is named after the 19th century, the Danish scientist Hans Christian Gram, who developed the technique in 1882 and published it in 1884 to discriminate between two types of bacteria with similar clinical symptoms (Beveridge TJ, 2001).

In gram staining the bacterial cells are first stained with a purple dye called crystal violet. Then the preparation is treated with alcohol or acetone. This washes the stain out of Gram-negative cells. Then to view them requires the use of counter stain of a different color (e.g., the pink of safranin). Bacteria that are not decolorized by alcohol wash are Gram-positive.

2.6.6 Biochemical test:

Growth characteristics on culture media can narrow down the number of possible identities of an organism, but biochemical tests are generally necessary for a more conclusive identification (Nester et al., 2004). There are two important tests for S.aureus Catalase and Coagulase.

Catalase test is one of the main tests used by microbiologists to differentiate between bacterial species in the lab. For example, this test is used to differentiate streptococci (catalase-negative) from staphylococci which is catalase-positive (Rollins DM, 2000). The presence of catalase enzyme in the test isolate is detected using hydrogen peroxide. If the bacteria possess catalase (i.e. are catalase positive), when a small amount of bacterial isolate is added to hydrogen peroxide, bubbles of oxygen are observed (Struthers and Westran, 2003).

Coagulase is an enzyme that is able to clot plasma in a fashion similar to the thrombin-catalayzed conversion of fibrinogen to fibrin. The test is important to differentiating S. aureus from the coagulase-negative staphylococci such as Staphylococcus epidermidis, which are common skin commensals (Struthers and Westran, 2003).

2.6.7Antimicrobial Susceptibility Testing: Kirby-Bauer disc diffusion method:

Named for its inventors, the Kirby-Bauer disc diffusion susceptibility test is one of the standardized methodologies for determining the susceptibility or resistance of a given organism to various antimicrobial compounds. A stander concentration of a bacterial strain is first uniformly spread on the surface of an agar plate. Then discs impregnated with different specified concentrations of selected antimicrobial drugs, are placed on the surface of the medium. As the bacteria multiply during overnight incubation, the antimicrobial drug diffuses into the agar. A clear zone of inhibition around an antimicrobial disc reflects, in part, the degree of susceptibility of the organism to the drug. The zone size is influenced by characteristics of the drug including its molecular weight, stability and concentration in the disc. Special charts have been prepared correlating the size of the zone of inhibition to susceptibility of an organism to the drug. Based on the size of the zone, organisms can be described as susceptible, intermediate or resistant to the drug (Struthers and Westran, 2003; Nester et al., 2004). The Kirby-Bauer disk diffusion method is rigorously standardized as recommended by the Clinical and Laboratory Standards Institute (CLSI) and the standardized media used for this test is Mueller-Hinton agar (MHA) (Wikler, M. A., et al. 2006; Slonczewski and Foster, 2009). In addition, it is also a preferred method to be used for synergy test (Joyce Elaine Cristina Betoni et al., 2006). Minimum Inhibitory Concentration (MIC):

Quantitative tests determine the lowest concentration of a specific drug that prevents the growth of an organism in vitro. This concentration is called the minimum inhibitory concentration (MIC). The MIC is fundamental test in determining the ability of a bacterial strain to grow in broth cultures containing different concentrations of the antimicrobial (Turnidge et al., 2003). Serial dilutions generating decreasing concentrations of the drug are first prepared in tubes containing a suitable growth medium. Then, a known concentration of the organism is added to each tube. The tube are incubated for at least 16 hours and then examined for visible growth or turbidity. The lowest concentration of the drug that prevents growth of the microorganism is the MIS (Struthers and Westran, 2003; Nester et al., 2004). However, the MIC does not tell whether a drug is bacteriostatic or bactericidal (Slonczewski and Foster, 2009). In fact, MIC is an important method in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents (Andrews, 2001). Bactericidal Concentration (MBC):

The minimum bactericidal concentration (MBC) is the lowest concentration of a specific antimicrobial drug that kills 99.9% of a given strain of bacteria. The MBC is determined by assaying for live organisms in those tubes from the MIC test that showed no growth. A small sample from each of those tubes is transferred to fresh, antibiotic-free medium. If growth occurs, then living organism remained in the original tube. Conversely, if no growth occurs, then no living organisms remained, indicate that the antimicrobial was bactericidal at that concentration (Nester et al., 2004). The bacteria that survive in the tubes that are more than two dilutions above the MIC are considered tolerant to the antimicrobial. Antimicrobials are usually regarded as bactericidal if the MBC is no more than four times the MIC (French GL, 2006). However, determining both MIC and MBC gives precise information regarding organism's susceptibility (Struthers and Westran, 2003).

2.6.8 Synergy test:

When antimicrobial drugs are given in combination, they can exhibit antagonistic, additive, synergistic, or indifferent effects against a particular microbe. Combinations of antimicrobial agents are considered to be synergistic if the effect of the combination is greater than the effect of either agent alone or greater than the sum of the effects of the individual agents. Antagonism results if the combination provides an effect worse than the effect of either agent alone or worse than the sum of the effects of the individual agents. Also, they are considered to be additive if the combined effect is equal to the sum of the independent effects; and to be indifferent if the combined effect is similar to the greatest effect produced by either drug alone (Brenner and Stevens, 2006). There are many analytical methods established for examining the effect of drug combinations, and all are dependent upon the definition of additivity. Nowadays, in the infectious diseases research literature, there are two laboratory methods commonly used to determine synergism. The first utilizes fractional inhibitory concentrations (FICs) and FIC indices which are determined by either broth or agar checkerboard techniques. For an antibiotic combination to be synergistic by this method, there must be an at least fourfold reduction in the MIC of each antibiotic when the two agents are combined compared with the MIC of each antibiotic tested by itself. The other method uses time-kill curves to compare differences in colony counts of an organism over a predetermined time interval (Cappelletty and Rybak, 1995).However, Kirby-Bauer disc diffusion method is also a preferred method can be used for synergy test (Joyce Elaine Cristina Betoni et al., 2006). Research on synergism between extracts and antimicrobial drug is very limited and few studies have been reported (Nascimento et al. 2000, Aburjai et al. 2001, Aqil et al. 2005).

2.6.9 Gas chromatography-mass spectrometry (GC-MS):

Gas chromatography-mass spectrometry (GC-MS) is a method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. Medicinal properties of herbs used in traditional systems of medicine are attributed to the presence of various types of biologically active molecules. Any variation, either qualitative or quantitative, in the chemical profile of the herb can lead to the total loss of medicinal properties. Therefore, it is essential, to identify and determine the quantity of each of the active principles by applying a suitable method, which allows on-line detection of molecules present in the herbal extract. Nowadays, with the advent of modern hyphenated techniques, it is possible to obtain comprehensive chemical profiles of herbal medicine preparations or extracts. GC-MS and LC-MS are now being used quite extensively for direct on-line analysis of components present in the herbal preparations and for ensuring the quality of the herb.

With MS as the preferred detection method, and single- and triplequadrupole, ion trap and time-of-flight (TOF) mass spectrometers as the instruments most frequently used, both LC-MS and GC-MS are the most popular hyphenated techniques in use today (Wilson et al., 2003). GC-MS, which is a hyphenated technique developed from the coupling of GC and MS, was the first of its kind to become useful for research and development purposes. Mass spectra obtained by this hyphenated technique offer more structural information based on the interpretation of fragmentations. The fragment ions with different relative abundances can be compared with library spectra. Compounds that are adequately volatile, small, and stable in high temperature in GC conditions can be easily analyzed by GC-MS. Sometimes, polar compounds, especially those with a number of hydroxyl groups, need to be derivatized for GC-MS analysis. In GC-MS, a sample is injected into the injection port of GC device, vaporized, separated in the GC column, analyzed by MS detector, and recorded. The time elapsed between injection and elution is called''retention time'' (tR). The GC separates the components of a mixture in time and the MS detector provides information that aids in the structural identification of each component (Sarker and Nahar, 2006).

. GC-MS has been demonstrated to be a valuable analytical tool for the analysis of mainly nonpolar components and volatile natural products. These techniques have been used in the traditional Chinese medicine (Schaneberg et al., 2003). However, Chen et al. (1987) described a method using direct vaporization GC-MS to determine approx 130 volatile constituents in several Chinese medicinal herbs. Also, Quinolizidine alkaloids, the main class of alkaloids found in the family Leguminosae, have been analyzed by GC-MS recently (Kite et al., 2003). As saponins are highly polar compounds and difficult to volatilize, the application of GC-MS is mainly restricted to the analysis of aglycones, known as sapogenins or saponins (Sarker and Nahar, 2006). By GC-MS, the components, predominantly monoterpenes, of the volatile oil oleoresin have been analyzed recently (Delazar et al., 2004). GC-MS also can be used to identify fatty acids in a mixture, without direct comparison with standards (Su et al., 2002). Moreover, GC-MS has been used for the dereplication of extracts of sweet-tasting plant species (Chung et al., 1997). Kite et al. (2003) described the application of GC-MS in the chemotaxonomic studies based on quinolizidine alkaloid profile in legumes.

2.6.10Cytotoxicity (MTT) assay

Treating cells with a cytotoxic compound can result in a variety of cell fates. The cells may undergo necrosis, in which they lose membrane integrity and die rapidly as a result of cell lysis. The cells can stop actively growing and dividing (a decrease in cell viability), or the cells can activate a genetic program of controlled cell death. Cytotoxicity (MTT) assay is performed following the method described in 1987 by Carmichael et al. (Carmichael et al., 1987), and percentage of cell viability is determined by spectrophotometric determination of accumulated formazan derivative in treated cells at 560 nm in comparison with the untreated ones. However, cytotoxicity is tested against L5178Y mouse lymphoma, H4IIE rat hepatoma or C6 rat glioma cell lines using the MTT assay (Ebada et al., 2008).

The MTT assay has been used for numerous medical, microbiological, and toxicological tests. The main advantage of this functional colorimetric assay is its simplicity and independence of the radiolabelling (Stec et al., 2007). It has been reported on the suitability of the MTT assay for the cytotoxicological evaluation of several natural products on various cell line cultures (Lewis et al., 1999; Robb et al., 1990). However, the origin of natural product source has opened way for potentially greater advances in the field for identifying suitable targets for multidrug resistant pathogens like MRSA (Stec et al., 2007). As in the majority of the MTT tests, the human cell lines are used to determine the cytotoxicity of natural products. The principle of this assay is as follows: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, is a yellow-colored compound that is converted by mitochondrial reductases into a blue formazan derivative(Ebada et al., 2008).