Microbial Production of Non-Beta Lactam Antibiotics - Assignment Example

Microbial Production of Non-Beta Lactam Antibiotics Actinomycin Actinomycetes are phylogenetically related from the evidence of 16S ribosomal cataloguing and DNA:rDNA pairing studies and they are gram-positive bacteria, with high guanine (g) plus cytosine (c) ratio in their DNA above 55% by mole (Goodfellow and Williams, 1983). “Actinomycetes” came from the Greek “atkis” (a ray) and “mykes” (fungus), and that has features of both Bacteria and Fungi (Das et al., 2008). They are soil organisms which have same characteristic as those for bacteria and fungi and yet possess significant distinctive features to delimit them into a distinct category in the strictly taxonomic sense. Actinomycetes are bound with bacteria in the same class of Schizomycetes but confined to the order Actinomycetales (Kumar et al., 2005). The actinomycetes are a group of bacteria which possess many important as well as interesting features. They have substantial value as producers of antibiotics and of other therapeutically useful compounds. They exhibit various ranges of life cycles which are unique among the prokaryotes and appear to play a major role in the cycling of organic matter in the soil ecosystem (Veigaet al., 1983). Thus it holds a prominent position due to their diversity and proven ability to produce new compounds, because discovery of novel antibiotic and non-antibiotic lead molecules through microbial secondary metabolite screening is becoming increasingly important. They are unicellular likewise bacteria, but they also produce mycelium which is non septate (coenocytic) and more slender, like true bacteria they do not have distinct cell wall and their cell wall is without chitin and cellulose (commonly present in the cell wall of fungi). Unlike slimy distinct colonies of true bacteria which grow quickly on culture media actinomycetescolonies show slow gowth, show powdery consistency and firmly stick to agar surface. They produce hyphae and conidia or sporangia like fungi. Some Actinomycetes whose hyphae undergo segmentation resembles bacteria, both by morphologically and physiologically. Actinomycetes are numerous and widely distributed in soil, compost etc and are next tobacteria in abundance. Plate count estimates gives value ranging from 10^4 to 10^8 per gram of soil. They are sensitive to acidity or low pH (optimum pH range is within 6.5 to 8.0) and waterlogged soil conditions. As we go deep into the soil, the population of actinomycetesincreases. They are aerobic (requires oxygen), heterotrophic (cannot make its own food) and mesophilic (25-30ºC) organisms and some species are commonly present in compost and manures are thermophilic growing at 55-65ºC temperature (eg. Thermoactinomycetes,Streptomyces). The common genera of actinomycetes in the order of abundance in soils are streptomyces (nearly 70%), Micromonosporaand Nocardiaalthough Actinomycetes,Actinoplanes, Streptosporangiumand Micromonosporaare also generally encountered. Based on the study conducted by Lam (2006), indigenous marine actinomycetes exist in the oceans and these are widely distributed in different marine ecosystems. There is tremendous novelty and diversity among the marine actinomycetes present in marine ecosystem. Progress is been made to isolate novel actinomycetes from samples that are collected from different marine environments and habitats. Different types of newly discovered secondary metabolites are being produced by these marine actinomycetes produces. Many of these metabolites have the potential to be developed as therapeutic agents as they possess biological activities. Marine actinomycetes are fruitful but underexploited source for the discovery of novel secondary metabolites. Devi et al. (2008) conducted a study where 73actinomycetes strains were isolated from five areas of Bay of Bengal. Fourteen of these showed antagonistic activity against seventeen human pathogens of gram positive and gram negative bacteria and two fungal species. Six of them showed broad spectrum activity. Six broad spectrum actinomycetes were found according to morphological and biochemical characteristics that belonged to the genera Streptomyces, Saccharopolyspora.,Micromonospora, Nocardia, Actinopolysporaand Actinomadura. Also by synergistic activity and heat killing activity, these six actinomycetes showed clear zone of inhibition in all selected human pathogens. The antibacterial antagonistic activities produced by these strains clearly indicated that marine environment harbours many broad spectrum antibiotic producing actinomycetes. According to Gopalakrishnan et al. (2010), actinomycetes are potential sources for the production of antibiotics. Six eight actinomycete strains were isolated from marine sediment samples of Indian EEZ and were screened for the anti-vibrio activity. The pathogenic vibriosselected for study were Vibrio harveyi, V. cholerae, V. proteolyticus, V.nereis, V. parahaemolyticus, V. alginolyticus, V. splendidus, V. Mediterranean and V. fluvialis. Kirby-baur disc method was employed for testing bioactivity. Out of the 68 strains isolated, 29 strains showed anti-vibrio activity. Most of the strains (25 strains) showed activity against human pathogens such as V. choleraeand V. parahaemolyticus. Among all these strains S26 had showed maximum antagonistic activity (70%) against these vibrios. So the broth culture of this strain was taken and subjected to solvent extraction by Ethyl Acetate, Hexane and 1-butanol. extracts were then tested for their activity against the vibriosand was found that 1-butanol Extract of strain S26 inhibits all the vibriosused for testing, hexane extract of S26 strain showed inhibition towards V. alginolyticus, whereas Ethyl acetate extract of S26 strain showed inhibition against V. cholera, V. harveyi and V. parahaemolyticus. Adel et al. (2005) carried out a study to inspect the factors affecting the antifungal production of four actinomycetes species i.e. Streptomyces lydicus, S. antimycoticus,S.erumpens and S.ederenis. Glycerol was used which is the best nitrogen source for antifungal production by S.erumpens and S.lydicus, ammonium sulphate for S.ederensis, while soyabean form S.antimycoticus. The optimum temperature for antifungal production by for S.antimycoticusand S.erumpuswas 28ºC while S.lydicusand S.ederensiswas 24 ºC. The optimum pH for antifungal production by the four selected species was 7.0. It was then observed that S. lydicus, S. erumpensand S. antimycoticuseach produced four active components, while S.ederensisproduced only three active components. After the chemical analysis of the culture filtrates, the species revealed the presence of 13 chemical compounds in the culture filtrates of S. lydicus, 12 chemical compounds in the culture filterate of each of S. erumpensand S. ederensis, 11 chemical compounds in the culture filterate of S. antimycoticus. It was then concluded from the experiment that actinomycetes play an important role in antagonizing both human and plant pathogenic fungi, and may be used in agricultural and medicinal scales after being further studied. Vimal et al. (2009) conducted a study in whichthe aimwas to isolate and identify the actinomycetes having antagonistic activity. Actinomycetes strains isolated from marine sediment samples were collected at the Pondicherry coast of India which showed antibacterial activity against some selected microbial pathogens. The cultural conditions and nutritional requirements for maximum growth and yield of secondary metabolites is been optimized under the shake - flask conditions. The growth and yield of the secondary metabolites was maximum when grown in ISP2 medium supplemented with sea water, pH 7.4, and incubation temperature of 28ºC, salt tolerance is 2% and incubation time is 4-7 days. Basing on the morphological, physiological,phylogenetic and biochemical characterization the strains were identified as Nocardiopssp. VIT SVK5 (FJ973467). The petroleum ether extract (1000g/ml) obtained from the isolate showed significant antibacterial activity against Gram negative bacteria - Escherichia coli(20mm), Pseudomonas aeruginosa(18 mm) and Klebsiella pneumonia (15mm) and Gram positive bacteria - Enterococcus faecalis(20mm), Bacillus cereus (13 mm) and Staphylococcusaureus(6mm) when compared with streptomycin (25 μg /disc). The ethyl acetate extract (1000 μg/ml) showed antifungal activity against Aspergillusfumigatus( 23mm), Aspergillusflavus(15 mm) and Aspergillusniger(12mm) was then compared with amphotericin-B (25 μg/disc). The chloroform extract (1000μg/ml) was very much effective against yeasts, Candidacruzi(18mm), Candida tropicans(15 mm) and Candida albicans(14mm) was when compared to streptomycin (25μg/disc). It was then concluded that the isolated strain had broad spectrum of antagonistic activity against gram positive and gram negative bacteria and Aspergillussp. Screening of six marine sediment samples studied by Dasari et al. (2011) near NTPC of the Visakhapatnam (India) Coast of Bay of Bengal resulted in the isolation of 72 isolates of actinomycetes. Among all these, Amycolatopsis Alba var. nov. DVR D4 had showed broad spectrum of antibacterial activity against Gram-positive bacteria and Gram-negative bacteria; and these produced antibacterial metabolite extracellulary under submerged fermentation conditions. The chemical and physical process parameters affecting the production of the antibiotic were optimized. The maximum antibiotic activity was observed with the optimized production medium containing D-glucose, 2.0 %w/v; malt extract, 4.0 %w/v; yeast extract, 0.4 %w/v; dipotassium hydrogen phosphate, 0.5 %w/v; sodium chloride, 0.25 %w/v; zinc sulphate, 0.004 %w/v; calcium carbonate, 0.04 %w/v; with inoculums volume of 5.0 %v/v at 6.0 pH, incubated at 28°C temperature at 220 rpm and for 96 hrs. The work was carried out by Houssamet al. (2011) in the course of a screening program for specifying the bioactive substances demonstrated inhibitory affects against microbial pathogenic from actinomycetes strains. Eighty eight actinomycete strains isolated from twelve soil samples collected from different localities in Egypt. A single actinomycete culture AZ-146 from eight cultures was found to be exhibiting to produce wide spectrum antimicrobial activities. A study on induced antibiotic production by E.coli, B.substilis, pseudomonas and klebsiellawith Streptomyces paves way for the discovery of pathogen targeted/ specific antibioticproduction. As we know that actinomycetes strain streptomyces itself can produce antibiotics butwhen it is induced with the human pathogens, the cell, it produces antibiotics the secondary metabolities in large amount in same time so in less time more production of antibiotics. This may further become very useful for mankind. So lot of work has been recorded which deals with the production of antibiotic described above and not much work is done on the current topic cellinduced production. So some of the following are. Cross species signal transfer mediated induction of antibiotic production by the soil actinomycetes against human bacterial pathogens was investigated by Musthafa et al. (2010). Soil actinomycetes isolated and analyzed for their efficacy in antibiotic production against common clinical pathogens viz. Staphylococcus aureus, Pseudomonas fluorescens, Escherichia coli, Bacillus subtilis and Salmonella typhi. Out of 36 actinomycetes isolates that were analyzed, four isolates exhibited significant antibacterial activity against S. aureuscells. Cell free extract of SA25 that was cultivated in the presence of heat killed S.aureuscells were then subjected to solvent extraction and was partially purified. In TLC band Antibacterial compound of the strain SA25 was identified as Streptomyces sp. Actinomycetes are known to be producing antibiotics and this property has been exploited for years for the commercial production of antibiotics, but exposure of actinomycetes(Streptomyces) to bacterial pathogens is expected to pave for the production of novel antibiotics with high degree of specificity and huge production. It can be looked upon as an alternative way to overcome the problem of drug resistance and reemergence of the resistant pathogens. The study was done by Selvinet al. (2009) on sponge-associated actinomycetes which were isolated from the marine sponge Dendrillanigraand collected from the southwest coast of India. A total of Elevenactinomycetes were isolated depending upon the heterogeneity and stability in the subculturing. Out of these, NocardiopsisdassonvilleiMAD08 showed 100% activity against the multidrug resistant pathogens were tested. The culture conditions for N. dassonvilleiMAD08 was optimized under submerged fermentation conditions for enhanced antimicrobial production. The unique and significant feature of MAD08 includes extracellular amylase, cellulase, protease and lipase production. These enzymes ultimately increase the scope of optimization using broad range of raw materials which might be efficiently utilized. The extract of the cell free supernatant with ethyl acetate yielded bioactive crude extract that displayed activity against a panel of pathogens tested. Analysis of this active thin layer chromatography fraction by Fourier transform infrared and gas chromatography-mass spectrometry evidenced showed 11 compounds with antimicrobial activity. The culture supernatant ammonium sulfate precipitation was at 80% saturation which yielded an anticandidal protein of molecular weight 87.12 kDa. This was the first strain that produces both organic solvent and water soluble antimicrobial compounds. The active extract was non-hemolytic as well as it showed surface active property envisaging its probable role in the inhibition to the attachment of pathogens to host tissues, thus, blocking the host–pathogen interaction at an earlier stage of pathogenesis. Aminoglycoside Aminoglycosides are potent bactericidal antibiotics that act by creating fissures in the outer membrane of the bacterial cell. They are particularly active against aerobic, gram-negative bacteria and act synergistically against certain gram-positive organisms. Gentamicin is the most commonly used aminoglycoside, but amikacin may be particularly effective against resistant organisms. Aminoglycosides are used in the treatment of severe infections of the abdomen and urinary tract, as well as bacteremia and endocarditis. They are also used for prophylaxis, especially against endocarditis. Resistance is rare but increasing in frequency. Avoiding prolonged use, volume depletion and concomitant administration of other potentially nephrotoxic agents decreases the risk of toxicity. Single daily dosing of aminoglycosides is possible because of their rapid concentration-dependent killing and post-antibiotic effect and has the potential for decreased toxicity. Single daily dosing of aminoglycosides appears to be safe, efficacious and cost effective. In certain clinical situations, such as patients with endocarditis or pediatric patients, traditional multiple dosing is still usually recommended. The first aminoglycoside, streptomycin, was isolated from Streptomyces griseus in 1943. Neomycin, isolated from Streptomyces fradiae, had better activity than streptomycin against aerobic gram-negative bacilli but, because of its formidable toxicity, could not safely be used systemically. Gentamicin, isolated from Micromonospora in 1963, was a breakthrough in the treatment of gram-negative bacillary infections, including those caused by Pseudomonas aeruginosa. Other aminoglycosides were subsequently developed, including amikacin (Amikin), netilmicin (Netromycin) and tobramycin (Nebcin), which are all currently available for systemic use in the United States. Stretomycin Streptomycin belongs to a group of compounds, known as antibiotics, which are produced by microorganisms and which possess the property of inhibiting the growth and even of destroying other microorganisms. Antibiotics vary greatly in their chemical nature, mode of action upon different organisms, and effect upon the animal body. The selective action of antibiotics upon bacteria and other microorganisms is known as the antibiotic spectrum. Streptomycin is an antibiotic drug and a water-soluble aminoglycoside. It was found to be soluble in water and insoluble in organic solvents, such as ether, chloroform, and acetone. The chloride was completely soluble in methanol, less soluble in ethanol, and virtually insoluble in butyl alcohol, acetic acid, and pyridine. The sulfate was only slightly soluble in methanol and virtually insoluble in the other solvents. It inhibits both gram positive and gram negative bacteria. It is the first of a class of drugs called aminoglycosides to be discovered, and was the first antibiotic remedy for tuberculosis.It is active against a large number of bacteria found among the gram-negative, gram-positive, and acid-fast groups and among the spirochaetes; it has relatively little activity against anaerobic bacteria, fungi, protozoa, and viruses. No absolute value can be given for the sensitivity of an organism to streptomycin; this depends not only upon the particular species. Streptomycin belongs to the glucosides in which a diguanido-group is linked to a nitrogen-containing disaccharide-like compound. It is a strong base, with three basic functional groups. The molecular weight determination on the trihydrochloride in water gave about 800 for the free base after the necessary corrections for the chloride ion. As a drug, it is used in a variety of pharmaceutical applications. It is applied as a potential control treatment for walnut blight and has an effect on arrhythmias induced by an increase of ventricular wall stress in the rat heart.Streptomycin stops bacterial growth by damaging cell membranes and inhibiting protein synthesis. Specifically, it binds to the 16S rRNA of the bacterial ribosome, which prevents the release of the growing protein. Streptomycin proved to be highly effective in the treatment of a large number of infectious diseases brought about in experimental animals by various bacteria. This was true of Salmonella schottmülleri, Shigellagallinarum, Brucellaabortus, Pseudomonas aeruginosa, Klebsiella or Friedländer’s bacillus, Diplococcuspneumoniae, and a number of organisms commonly found in urinary tract infections. Streptomycin is also used as a pesticide, to combat the growth of bacteria, fungi, and algae. Streptomycin controls bacterial and fungal diseases of certain fruit, vegetables, seed, and ornamental crops, and controls algae in ornamental ponds and aquaria. A major use is in the control of fireblight on apple and pear trees. As in medical applications, extensive use can be associated with the development of resistant strains. Streptomycin and Rifampicin are potent antibiotic drugs. Streptomycin is broad spectrum drug effective against both gram positive and gram negative bacteria. However, its major use is as an anti-TB drug. Rifampicin also is used extensively as an anti-TB drug but it is also used in treatment of leprosy. As both the diseases are rampant in India, their importance cannot be over emphasized. Streptomycin drug is refined from the fermentation of the actinomycete Streptomyces griseus. Spores of this actinomycete are inoculated into a medium to establish a culture with a high mycelial biomass for introduction into an inoculum tank, with subsequent use of the mycelial inoculum to initiate the fermentation process in the production tank. During its synthesis, proteins are needed to further enhance the efficiency of the process. Such proteins are called streptomycin. Moreover, enhancement of streptomycin production is also made possible by special combination between myo-inositol and arginine Isolated streptomyces strainsfrom soil were found to possess various numbers of genes from the streptomycin biosynthesis cluster. The biosynthesis of streptomycin in Streptomyces griseus has been studied by adding D-[3,4-14C2]glucose or D-t1,3-14C2]glucose to the growth medium and degrading the streptomycin produced. Today, India is a major producer of streptomycin and production has almost stopped in all the major developed countries. Indigenous demand is fully met from indigenous supply. Estimated demand matrix may be generated for each drug especially for streptomycin and rifampicin.Consumption has stagnated and is likely to decline in years to come. There is a considerable export market. However, Indian price of about Rs.l 100 per kg.is way above the international price. More potent drugs like rifampicin which are now available, are likely reduce the demand for streptomycin. A commercial source of streptomycin has been used to induce seedlessness in the seeded grape cultivar Rubi (Italia Red).A commercial source of streptomycin has been used to induce seedlessness in the seeded grape cultivar Rubi (Italia Red). Tetracycline Tetracycline, a protein synthesis inhibitor, is an antibiotic produced by the Streptomyces genus of Actinobacteria. It is indicated for use against many bacterial infections which include chlorea and It is commonly used to treat acne and rosacea.Tetracyclines were discovered in the 1940s and exhibited activity against a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites. They are inexpensive antibiotics, which have been used extensively in the prophlylaxis and therapy of human and animal infections and also at subtherapeutic levels in animal feed as growth promoters. The first tetracycline-resistant bacterium, Shigelladysenteriae, was isolated in 1953. Tetracycline resistance now occurs in an increasing number of pathogenic, opportunistic, and commensal bacteria. The presence of tetracycline-resistant pathogens limits the use of these agents in treatment of disease. Tetracycline resistance is often due to the acquisition of new genes, which code for energy-dependent efflux of tetracyclines or for a protein that protects bacterial ribosomes from the action of tetracyclines. The production of tetracycline by fermentation was disclosed by Minieri et al. (1953). In this fermentation the composition of the medium and the strain of streptomycete are both important factors, since Streptomyces aureofaciens is capable of producing at least two antibiotic substances. In the presence of chloride, which is incorporated within the chlortetracycline molecule, the antibiotic formed was predominately chlortetracycline. In media low in chloride tetracycline predominates and the chlortetracycline fraction diminishes since 1 ppm of available chloride ion can produce at most 14 ,ug/ml chlortetracycline. The simultaneous production of two or more antibiotics in a fermentation is well known, and the substances formed may be either closely related on a chemical or a biological basis or widely separated. Typical examples of closely related compounds produced simultaneously by the same organism are the penicillins, streptomycins, polymyxins, bacitracins, cephalosporins, nisins, neomycins,rhodomycins, and candicidins. Works Cited Aminoglycosides: A Practical Review - November 15, 1998 - American Family Physician. 2013. Aminoglycosides: A Practical Review - November 15, 1998 - American Family Physician. [ONLINE] Available at: http://www.aafp.org/afp/1998/1115/p1811.html. [Accessed 17 March 2013]. 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In Antibiotics annual 1953-1954. Medical Encyclopedia, Inc., New York, New York, pp. 81-87. Read More