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Mycobacteria is a species of the genus Mycobacterium, the single genus family within the Mycobacteriaceae group. Mycobacteriaceae belong within the order of Actinomycetales. They are defined as being Gram positive, nonmotile, aerobic bacteria which are acid alcohol fast and catalase positive, lacking endospores and capsules. Actinomycetales represent a wide variety of diverse nicroorganisms and so all species within the mycobacteria family are characterised on the basis of their phenotypic characteristics. The morphology of the bacteria are observed as being straight or curved rods which are between 0.2 - 0.6µm wide and 1.0-10 µm long. Mycobacteria also contain a unique cellular wall which is waxy, hydrophobic and rich in mycolic acids, allowing them to be more easily distinguishable (Rastogi et al, 2001).
Mycobacteria currently contain 60 recognised or proposed species within the genus, which can be categorized into two groups on the basis of their growth rate, slow growing and fast growing species. Like the majority of other genres of bacteria, Mycobacteria contain both pathogenic and saprophytic strains. The slow growing species of the bacteria require a time period of greater than seven days to produce visible colonies on a solid agar medium and are often the pathogenic form of the species to humans and animals. The alternative fast growing species of the bacteria can produce visible colonies in a time period of less than seven days and are usually the nonpathogenic strain of the family (Shinnick and Good, 1994).
Mycobacteria have the ability to colonize and occupy hosts and host cells without displaying any adverse signs or effects. Asymptomatic infections of the pathogenic mycobacteria species Mycobacteria Tuberculosis and Mycobacteria Leprae are present among billions of people worldwide. Infections resulting from Mycobacteria are often extremely difficult to treat due to the composition and complexity of the bacterial cell wall. This cell wall, which displays characteristics of being both gram positive and gram negative, is a distinguishable and unique feature of the bacteria. It is of significant value for cell survival on exposure of the bacteria to harsh environments and unfavorable conditions. Mycobacteria are naturally resistant to numerous antibiotics, including penicillin, which act by disrupting cell wall biosynthesis. Antibiotics to which the bacteria are susceptible include clarithromycin and rifamycin, however resistant strains to these have become prominent.
The virulent strains of Mycobacteria possess the ability to disrupt the phagosomal membranes of macrophages and also the capability to inactivate mitochondrial membranes of phagocytes. These characteristics are a survival technique of the bacteria, allowing the bacteria to multiply and reproduce within phagocytes therefore causing infection.
The complexity of the composition of the mycobacterial cell envelope adds to the uniqueness and differentiation of the Mycobacterium species from the majority of other prokaryotes. The composition and properties of the components in the mycobacterial cell envelope provides the bacteria with an efficient permeability barrier. This characteristic allows Mycobacteria to control and mediate the substances which enter the cell from the surrounding environment and also enables them to be resistant to a wide variety of drugs (Crick et al 2010). Knowledge of the biosynthesis, composition and arrangement of the Mycobacterial envelope is required in order to understand the biological activities of each component and to determine how these activites and roles contribute to the pathology of Mycobacterial diseases. The cell envelope is composed of a plasma membrane, a capsule and a cell wall. The significant biomolecules which form each of these structures is known, however it is the distribution of the smaller and minor components of the bacteria which is poorly understood. The plasma membrane resembles that of a typical bacterial membrane. It is a vital component of the bacteria however it is believed it has a limited role within the pathological processes. The polysaccharide-rich capsule is also an important component in the structure of Mycobacteria, although the chemical composition has only recently been recognized. It is believed to be composed of polysaccharides and proteins with traces of lipids also being present. The arrangement of these elements forming the structure is imperfectly understood. The cell wall resembles a gram positive wall but also contains gram negative characteristics. The wall, unusually, includes a layer of lipids or mycolate esters arranged in such an order to from a permeability barrier to polar molecules (Daffé and Draper, 1998).
Structural analysis studies have been implemented in order to gain knowledge and understand the components of the mycobacterial cell wall. This information and increased knowledge provides the basis for biosynthesis and functional studies in the development of more effective and advanced drugs and the possibility of vaccines. Mycobacteria, including the pathogenic strains which cause Tuberculosis and Leprosy, produce low permeability cell walls which enable them to be resistant to therapeutic agents. The highly complex and organized structure of the cell wall, which is unique to Mycobacteria, therefore provides the most appropriate target for antimycobacterial agents. Knowledge of enzymes involved in the synthesis of cell wall components and pathways utilized by the components could result in potential targets for the generation of new drugs (Chatterjee, 1997). Mycobacteria cell walls contain vast amounts of C60-C90 fatty acids, mycolic acids, which are covalently linked to an arabinogalactan molecule. The structures of arabinogalactan have been examined along with extractable cell wall lipids including trehalose-based lipooligosaccharides, phenolic glycolipids, and glycopeptidolipids. The hydrocarbon chains of these lipids combine and assemble together to produce an extremely thick asymmetric layer. Analysis of this structure indicates that the fluidity is restricted in the innermost section of the bilayer, but gradually increases toward the outer surface of the cell. Fluidity and permeability of the bilayer may be influenced as a result of differences associated with mycolic acid structure. These variations may also be responsible for the different levels of sensitivity of numerous mycobacterial species to lipophilic inhibitors. Hydrophilic nutrients and inhibitors however, can travel through the cell wall with the use of porin channels (Brennan and Nikaido, 1995).
Gram positive bacteria contain a permeable cell wall which permits the penetration of antimicrobials. Resistance to antimicrobials as a result of restricted penetration can occur when resistant strains of bacteria develop or produce thickened cell walls. The high lipid content of Mycobacterial cell walls is believed to present an extremely effective permeability barrier to the penetration of antimicrobial agents. Transport pathways through this membrane remain unknown for a large variety of drugs. Hydrophobic antibiotics including Rifampicin and Fluoroquinolones have the ability to traverse the cell wall by diffusion and cross through the mycolic acid and glycolipid composed hydrophilic bilayer. Hydrophilic nutrients and antibiotics such as beta lactam antibiotics and chloramphenicol however, are unable to diffuse across the bilayer. These small and hydrophilic components are thought to have adapted to utilize porin channels and pathways which are present in numerous species of Mycobacteria. The presence of porins within the lipid bilayer indicates that the cell wall of Mycobacteria contains an outer membrane which is analogous to that of gram negative bacteria. Mycobacterial porins however, are much less abundant in comparison to those present in the gram negative outer membrane. As a result, the rate of uptake of small hydrophilic nutrients and antibiotics is significantly lower. Structural analysis has identified that some molecules such as erythromycin, kanamycin and vancomycin are too large to utilize porin pathways and so permeability remains an issue. Mutations in the porin pathways resulting in reduced porin efficiency and lower levels of porin expression are thought to be likely mechanisms for pathogenic Mycobacteria to acquire resistance to antibiotics or compounds to which they are susceptible (Danilchanka et al, 2008) (Lambert, 2002).
Viruses are powerful molecules which are useful in order to investigate and manipulate the hosts that they infect. The presence and recognition of the vast variety of bacteriophages within the biosphere has led to renewed interest in identifying their morphological and genetic diversity. Mycobacteriophages are viruses which infect mycobacterial host molecules. Due to the increasing importance of mycobacterial infections to human health, mycobacteriophages are appealing models for discovery, genomic characterization and manipulation. Mycobacterial genetic advancements have been due to the isolation and characterization of mycobacteriophages. Initially interest in mycobacteriophages began in the 1940s when phages infecting Mycobacterium smegmatis and Mycobacterium tuberculosis were isolated. Nowadays new phages are being discovered and isolated from environmental sources using Mycobacterium smaegmatis as a host. There are now more than seventy complete genomic sequences available from which the information will aid with two main objectives. These include providing an array of genetic tools for furthering understanding of mycobacterial hosts and providing insights into viral diversity of the virus. Mycobacteriophages which have been characterized are evident as being double stranded DNA tailed phages belonging to the Caudovirales. The phage can be further classified based on their tails into the families myoviridae containing contractile tails, siphoviridae containing long, flexible non contractile tails and less frequently the podoviridae containing short stubby tails. The majority of these mycobacteriophages have isometric heads and contain genome lengths ranging from 42 to 150 kbp. The first sequenced mycobacteriophage and the most useful to date are namely the L1, L5 and TM4 phage. The TM4 phage has been used in order to construct novel shuttle phasmids which permit the introduction of foreign genes into Mycobacteria. The phasmids have proven invaluable in the processes of transduction, transposon delivery and diagnostic introduction of reporter genes in the manipulation of mycobacteriophage genomes. They have also facilitated the characterization of high efficiency transformation mutants in Mycobacterium smegmatis and the use of antibiotic selectable markers through temperate phage L1 shuttle phasmids. With the completion and availability of fifty mycobacteriophage genome sequences, the knowledge of mycobacteriophage types and mycobacteriophage genes is far from complete. It is becoming increasingly probable that newly discovered mycobacteriophage will share similarities with the currently characterized phage while phage containing novel genes will also be discovered. Knowledge of mycobacteriophage genomics currently available will require expansion in order to enhance understanding of the associated diversity and evolution. The unknown functions of greater than 1000 new sequence phamilies indicates that insights into currently available mycobacteriophage genomics is restricted. Functional genomic approaches are now being adapted with the potential of providing information regarding the roles of these mycobacteriophage. (Hatfull, 2010) (Hatfull et al, 2008)
Mycobacterium Tuberculosis and Mycobacterium Smegmatis
Similar to the majority of bacterium, the Mycobacterium genus include both saprophytic and pathogenic species. Mycobacterial pathogenic species include Mycobacterium leprae, the causative agent of leprosy and Mycobacterium tuberculosis, the causative agent of tuberculosis. Both of these infectious strains are a major contributor to the mortality rates in developing and developed countries. Tuberculosis is responsible for the death of one person every twenty seconds, the equivalent to five thousand people per day. It is therefore becoming increasingly important to identify and study the virulence determinants of these bacteria species. These attempts however are hampered and restricted due to the slow growth rate of both species, as Mycobacterium leprae does not grow in vitro and Mycobacteria tuberculosis with a generation time of twenty four hours, requires weeks to yield visible colonies on a Petri dish. As a result, fast growing saprophytic Mycobacteria are used as a model for studies of mycobacterial biology in cases where results can be utilized for pathogenic strains. Mycobacterium smegmatis, a non pathogenic species of the mycobacterium family is commonly used as a comparative model for Mycobacterium tuberculosis due to the short generation time. Similarities which are identified as being conserved in both species could be studied in Mycobacterium smegmatis, enabling insights to be gained into varying aspects of physiological adaptation within a shorter time frame than is achievable in Mycobacterium tuberculosis. The sequencing and assembly of Mycobacterium smegmatis has allowed for preliminary comparisons to be made between the species. From the virulence genes currently identified from Mycobacterium tuberculosis, twelve of the nineteen share closely related homologues with those of Mycobacterium smegmatis. Initially identified as being specific to Mycobacterium tuberculosis, the presence of these genes has led to the conclusion that they are involved in the role of virulence. If these genes are characterized as being common mycobacterial housekeeping genes, then Mycobacterium smegmatis can be utilized advantageously in order to determine the functions of the genes. Two component genetic systems which play a regulatory role in Mycobacterium tuberculosis have also been studied. Six of eleven of the two component systems and five of seven orphan response regulatory kinases have been identified as sharing homologues in Mycobacterium smegmatis. Genes which encode sigma factors have been researched within this study. These factors provide the bacterial transcription machinery with the necessary specificity, of which nine of the thirteen Mycobacterium tuberculosis sigma factor genes have had homologues detected in the unfinished smegmatis genome. For these reasons, Mycobacterium smegmatis has been adapted as an appropriate model for the study of pathogenic properties of Mycobacteria (Reyrat and Kahn, 2001) (Tyagi and Sharma, 2002).
Antibiotic resistance among bacteria is often plasmid mediated or as a result of resistance genes encoded by transposable elements. The pathogenic forms of Mycobacteria, including Mycobacterium tuberculosis and Mycobacterium leprae, are resistant to most common antibiotics. The intrinsic resistance to many antibiotics results in limitations in the chemotherapeutic options available for the treatment of the infectious diseases. Resistance of the bacteria has become attributed to the cell surface structure, due to the permeability characteristics of the multi layer cell envelope to the penetration of antibiotics (Nguyen and Thompson, 2006). The envelope of the bacteria is believed to be a dynamic structure as it undergoes alterations depending on whether the bacteria is growing or persisting within the host. Permeability through the cell wall is restricted to lipophilic antibiotic molecules such as Rifampicin. Therefore, in order to treat mycobacterial infections, a combination of antinbiotics is required including, Rifampicin, Isoniazid, Pyrazinamide, and Ethambutol. Overuse of antibiotics can result in the production of resistant strains of bacteria and the increase of antibiotic resistance. There are numerous mechanisms which are involved with these processes which are continuously being studied. Systems that function together with the cell permeability barrier provide intrinsic resistance through neutralizing the toxicity of antibiotics in the cytoplasm. This is achieved with the aid of antibiotic responsive regulatory proteins and corresponding resistance genes. Antibiotic modifying activities including aminoglycoside acetyltransferases and phosphotransferases which have been identified in both fast and slow growing mycobacteria are also thought to contribute to antibiotic resistance. Drug resistant mycobacterium tuberculosis was initially discovered in 2006 and is now detected worldwide. One in four patients suffering from tuberculosis infection have a form of the disease which is unresponsive to standard drug products. Natural compounds are therefore becoming a prominent phenomenon in the treatment Mycobacterium tuberculosis (Martin et al, 1990)(McCarthy and O'Mahony, 2010).
Herbal Compound Antimicrobial Effects
The use of antibiotics in the treatment and control of infectious diseases led to a decrease in mortality rates within the last decade. However the explosive use of antibiotics resulted in an increase in antibiotic resistance and bacteria developing resistant strains which are becoming increasingly difficult to treat. Alternative treatments are now being utilized in an effort to control this problem (Alanis, 2005). While the majority of drug products currently available are synthetically engineered, natural compounds are becoming a prominent choice in order to fight infection. Plant extracts have been proven to display inhibition which is comparable to that of commonly used products such as Streptomycin. Mainstream medicine has adapted the use of drugs derived from botanical sources as approximately half of the pharmaceutical agents currently prescribed contain at least one ingredient which has been derived from a plant source (McCarthy and O'Mahony, 2010).
Common mullein weed, also known as Verbascum thapsus, is a member of the Scrophulariaceae. This family aquired its name as a result of a member of the family known as Scrophularia, which was used historically to treat scrofula, a tuberculosis infection of the lymph nodes in the neck. The name can be traced back five hundred years and can be derived from Latin to mean 'breeding sow'. The derivative Mullein can also be translated from Latin. The term 'malanders' was the old term for leprosy, while the term 'malandre' applied to lung diseases for which the plant was used as a remedy, thus acquiring its name. The establishment of the link of mullein weed to leprosy and tuberculosis caused by Mycobacteria resulted in the plant historically being used as a remedy for the infectious diseases. Mullein was cultivated on a large scale in Ireland before the introduction of amtimycobacterial drugs, and the plant is now being re examined for its use in treatment. Extracts of Mullein leaf have proven to be effective against both gram positive and negative bacteria while also possessing antitumour, antiviral, antifungal and antibacterial properties. Verbascum species contain a diverse range of compounds, with similar compounds containing antimycobacterial properties. Mullein has been identified as possessing large amounts of lipophilic molecules and so provides great potential to search for anti tuberculosis agents (McCarthy and O'Mahony, 2010).
Mentha pulegium, also known as pennyroyal, is a plant commonly found across Europe which belongs to the family Lamiaceae. This plant has primarily been used as an insect repellant and so it is no surprise that the name was derived from the latin term 'pulex irritans' meaning flea. The flowering segments of the Mentha pulegium Labiatae plant have a history of traditional medical use for the treatment of many infectious diseases as a result of its antiseptic properties. The antiseptic properties of this plant have recently been examined by determining antimicrobial activity against numerous microorganisms. Using a disc fusion method and a microbroth dilution assay, significant antimicrobial activity has been reported against Gram positive bacteria, therefore suggesting its use in the treatment of infections by these bacteria. Mentha extracts have also been identified as possessing antibacterial and antifungal properties. It is believed that the provision of this extract can reduce the number of lung tumors and possibly aid in the treatment of mycobacterium tuberculosis infections although further research will be required to evaluate the full potential for therapeutic applications (Mahboubi and Haghi ,2008).
Glycyrrhiza glabra is a plant which is commonly known as Liquorice. It is a member of the family Fabacaea and the genus Glycyrrhiza which contains over twenty species that are native to southern Europe. Liquorice is the root of the Glycyrrhiza glabra plant, which is used to extract the sweet flavour commonly used in food products. The name Glycyrrhiza is derived from the ancient greek term 'glykos' meaning sweet and the term 'rhiza' meaning root. The plant has a history of medical use, commonly being used as a remedy for coughs, chest and lung infections and most importantly consumption also known as Tuberculosis. Recent studies have begun to examine the antimicrobial potential of Glycyrrhiza glabra roots. Antimicrobial activity using these roots has been reported against both Gram positive and Gram negative bacteria. Following from these studies, the effect of Glycyrrhiza glabra against Mycobacteria was investigated. Antimycobacterial activity has been detected at a concentration of 500mg/mL. The compound glabridin has been identified using phytochemical analysis as being active against strains of Mycobacterium tuberculosis, therefore suggesting the potential of Glycyrrhiza glabra or indeed liquorice as an antitubercular agent (Gupta et al, 2008).