Microbial Products A. Primary Metabolites: log phase, use nutrients fast, produce PM B. Secondary Metabolites: depletion of nutrients, growth retards, produce SM Primary Metabolites: Vitamins Vitamins: cannot be synthesized by higher organisms But microorganisms are capable of synthesizing (gut) Studies reveal vitamin deficiencies Thiamine Reported beneficial health effects Riboflavin Growing vitamin market demand (cost Pyridoxine effective) Folic acid Genetically engineered MO as alternatives to Pantothenic acid chemical synthesis Biotin Vitamin B12 Ascorbic acid b- carotene (provitamin A) Ergosterol (vitamin D) Vitamins Fat soluble Carotenoids b-carotene (provitamin A) Astaxanthin Poly unsaturated Fatty acids (PUFA; vitamin F) Docosahexaenoic acid (DHA) Arachidonic acid (ARA) Ergosterol (vitamin D) Water soluble Riboflavin (vitamin B2) Cobalamin (vitamin B12) L-Ascorbic acid (Vitamin C) R-Pantothenic acid (vitamin B5) D-Biotin (vitamin H or B7) Vitamin B1 (Thiamine) Vitamin B6 (pyridoxol) Folic acid Vitamin B12 or Cyanocobalamin • Water soluble vitamin ; complex sructure • Has role in functioning of brain and nervous system, formation of blood • Contains rare element cobalt • Deficiency causes pernicious anemia which is an causes low Hb, less RBCs • Pernicious anemia: autoimmune disorder, parietal cells (stomach) responsible for secreting intrinsic factor are destroyed. Intrinsic factor is crucial for the normal absorption of B12, so a lack of intrinsic factor, as seen in pernicious anemia, causes a deficiency of Vitamin B12 • dietary reference intake for an adult ranges from 2 to 3 µg per day • used in treating cyanide poisoning, prevents brain atrophy in Alzheimer’s patients • COMMON INGREDIENT IN ENERGY DRINKS C63 H88 CoN14 O14P Pyrrole nitrogen 4 Pyrrole units cobinamide • Corrin ring • Deep red colour due to corrin ring • Central Co atom • Coordination state 6 • 4 of 6 coord sites have pyrrole ring • 5 has dimethylbenzimidazole group • 6 is center of reactivity, variab;e • CN, OH, Me, 5-deoxyadenosyl for 4 types of B12 nucleotide 6 2 1 3 4 5 5,6-dimethyl benzimindazole Commercial production Chemical syn not feasible Genera known to produce vit B12 Most commonly used for industrial production are 20mg/L Streptomyces griesus Pseudomonas denitrificans (aerobic) Salmonella typhimuriu (anaerobic) Propionibacterium shermanii GRAS by FDA (anaerobic) (Generally Regarded As Safe) Sanofi-Aventis (FRENCH) use genetically engineered versions to produce vit B12 under specialized conditions from Propionibacterium since they have no endotoxins or exotoxins P. denitrificans also used after strain modification; mutant more efficient than wild type Commercial production • Produced in continuous culture with 2 fermenters in series Addition of 5,6dimethylbenzimidazol (0.1%) Glucose Corn steep Betaine (5%) Cobalt (5ppm) pH 7.5 + Propionibacterium freudenreichii Anaerobic 70h Aerobic 50h Cobinamide production and accumulation KCN added CYNACOBALAMIN 80% purity Used as feed additive Betaine: sugar beet molasses Filtrate Nucleotide synthesized Combined with cobinamide To yield 2ppm of cobalamin Acidification of culture To 2-3pH/ 100oC Filter to remove cell debris Commercial production ANAEROBIC PHASE AEROBIC PHASE 2-4 DAYS 5-deoxyadenosylcobinamide produced 5,6-dimethylbenzimidazole is added and gets incorporated to form 5’-deoxyadenosylcobalamin During the 7-day fermentation run, adenosylcobalamin is predominantly secreted from the biomass and accumulates in the fermentation broth in milligram amounts. The down- stream steps comprise filtration, cyanide treatment, chromatography, extraction, and crystallization yielding vitamin B12 in high purity. If to be used for treatment further purification (95-98% Purity) Commercial production Pseudomonas denitrificans: strain improvements resulted in increase in yeild From 0.6mg/L to 60mg/L Glucose : common carbon Alcohols (methanol, ethanol, isopropanol) Hydrocarbons(alkanes, decane, hexadecane) With methanol 42mg/L was obtained using Methanosarcina barkeri Riboflavin (Rf) or Vitamin B2 • • • • • Water soluble Essential for growth and reproduction; key role in energy metabolism, ketone bodies, fats, CHO and protein metabolism Deficiency leads to cheliosis (fissures around mouth), glossitis (purple tounge) and dermatitis Required in coenzymes FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide) Used as an orange-red food colour additive, designated in Europe as E101 7,8-dimethyl-10- (D-19-ribityl) isoalloxazine Participates in O-R reactions Flavin is ring moiety with yellow colour to oxidized form Isoalloxazine ring Isoalloxazine ring H H Ribitol FAD E101 FMN E101a genes encoding the riboflavin biosynthetic enzymes are well conserved among bacteria and fungi INDUSTRIAL USE Processed food is often fortified by the use of riboflavin as a colorant or vitamin supplement. The main application (70%) of commercial riboflavin is in animal feed, since productive livestock, especially poultry and pigs, show growth retardation and diarrhea in case of riboflavin deficiency. According to a report by SRIC, a consulting company in Menlo Park (California), in 2005 the need for industrially produced riboflavin was estimated at 6500–7000 tons per year. Commercial production Glucose 1/3rd production by direct fermentation Acetone butanol fermentation Clostridium acetobutylicum C. butylicum riboflavin as by product 50% by biotransformation using Bacillus pumulis D-ribose 20% production by Chemical synthesis Riboflavin Ashbya gossypii Candida famata Bacillus subtillis (genetically modified) Commercial production Phase I use of glucose, accumulation of pyr, pH acidic, growth stops, no Riboflv Phase II decr pyr, incr in ammonia, alkalinity incr, prod of Riboflv in form of FAD and FMN Phase III autolysis, cell disruption, release of free FAD, FMN and riboflv Carbon sources: glucose, acetate, methanol, aliphatic hydrocarbons Major riboflavin producers are DSM Nutritional Products (Switzerland) and Hubei Guangji (Hubei Province, China), both using genetically engineered B. subtilis production strains, and BASF (first in Germany but now in South Korea), employing genetically engineered A. gossypii. Ascorbic acid or Vitamin C • • Used in collagen biosynthesis, protects against nitrosamines, free radicals Deficiency causes scurvy Precursor for its chemical synthesis can be obtained by biological methods feed applications of L-ascorbic acid account for only 10%, whereas the main uses are in the pharmaceutical industry (50%), food (25%), and beverages (15%). Pharmaceutical applications include stimulation of collagen synthesis (especially cosmetic products) and high antioxidant capacity, used for the reported health benefits in the prevention of flu, heart diseases, and cancer, as well as an antidote for poisoning. The food and beverage industry predominantly exploits the antioxidant capacity of L-ascorbic acid to extend durability, prevent discoloration, and to protect flavor and nutrient contents of their products. Submerged bioreactor fermentation Erwinia sp. Acetobacter sp. Gluconobacter sp. 2,5-diketogluconic acid 2,5-diketogluconic acid reductase D-glucose (200g) Glucuronic acid Corynebacterium sp. 2-keto L-gluconic acid Bacillus megaterium L-ASCORBIC ACID Cloning of gene 2,5-diketogluconic acid Reductase of Corynebacterium into Erwinia herbicola D-sorbitol sorbitol dehydrogenase Acetobacter xylinum, A,suboxydans L-Sorbose chemical oxidation 2 keto L gulonic acid Enol form of 2 keto L gulonic acid Reichstein Grussner synthesis Gluconolactone acid treatment L-ASCORBIC ACID (100g) L-Gluconolactone L-Gluconolactone dehydrogenase L-ASCORBIC ACID b- carotene or provitamin A Provitamin A -----> Vitamin A (intestine) • • • Fat soluble Deficiency leads to night blindness Best source is liver and whole milk also coloured fruits and vegetables • • • Isoprene derivatives Tetraterpenoids with eight isoprene residues 400 naturally occurring carotenoids: b-carotene, a-carotene, d-carotene, lycopene, zeaxanthin Carotenoids Used as food colorants and animal feed supplements for poultry and aquaculture, carotenoids play an increasing role in cosmetic and pharmaceutical applications due to their antioxidant properties. The pigments are often regarded as the driving force of the nutraceutical boom, since they not only exhibit significant anticarcinogenic activities but also promote ocular health, can improve immune response, and prevent chronic degenerative diseases. Commercial production Microbial fermentation Submerged Fermentation process Blakeslea trispora (high yeild; 7g/L) Phycomyces blakesleeanus Choanephora cucurbitarum Corn starch, soyabean meal, b-ionone, antioxidants stimulators Trisporic acid: act as microbial sex hormone, improves yield b-Ionone: incr b-carotene syn by incr enzyme activity Purified deodorized kerosene increases solubility of hydrophobic substrates Recovery: b- carotene rich mycelium used as feed additive Mycelium is dehydrated by methanol, extracted in methylene chloride and crystallized which is 70-85% pure DSM Nutritional Products (Switzerland) and BASF (Germany) dominate the market with their chemical synthesis processes, but Chinese competitors are catching up. Halophilic green microalgae Dunaliella salina. It accumulates the pigments in oil glo- bules in the chloroplast interthylakoid spaces, protecting them against photoinhibition and photodestruction. Excessive pigment formation in D. salina is achieved by numerous stress factors like high temperature, lack of nitrogen and phosphate but excess of carbon, high light intensity, and high salt concentration, the latter two having the highest impact. Dried D. salina biomass for sale contains 10–16% carotenoids, mainly b-carotene. In addition crystalline material obtained after extraction with edible oil is also sold. Primary Metabolites: Organic Acids Organic acids are produced by through metabolisms of carbohydrates. They accumulate in the broth of the fermenter from where they are separated and purified. Glycolysis Krebs cycle I. Terminal end products (pyruvate, alcohol) lactic acid Propionic acid II. Incomplete oxidation of sugars (glucose) citric acid Itaconic acid Gluconic acid III. Dehydrogenation of alcohol with O2 acetic acid Manufactured on large scale as pure products or as salts CITRIC ACID: industrial uses Flavoring agent In food and beverages Jams, candies, deserts, frozen fruits, soft drinks, wine Antioxidants preservative and Chemical industry Antifoam Treatment of textiles Metal industry, pure metals +citrate (chelating agent) Acidifyer Flavoring Chelating agent Primary metabolite Present in all organisms Agent for stabilization of Fats, oil or ascorbic acid Stabilizer for cheese preparation Pharmaceutical industry Trisodium citrate (blood preservative) Preservation of ointments and cosmetics Source of iron Detergent cleaning industry Replace polyphosphates Commercial Production Strains that can tolerate high sugar and low pH with reduced synthesis of undesirable by products (oxalic acid, isocitric acid, gluconic acid) Glucose Glucose Pyruvate Aspergillus niger A. clavatus Pencillium luteum MEDIUM CYTOPLASM Pyruvate CO2 Pyr carboxylase OXA Malate Pyruvate Pyr Dehydrogenase Acetyl CoA MITOCHONDRIA Malate CO2 OXA Fumarate Succinyl CoA Citrate synthase citric acid a-KG 100g sucrose --- 112g any citric acid or 123g citric acid-1hydrate Factors for regulation CARBOHYDRATE SOURCE: sugar should be 12-25% Molasses (sugar cane or sugar beet) Starch (potato) Date syrup Cotton waste Banana extract Sweet potato pulp Brewery waste Pineapple waste High sugar conc incr uptake and production of citric acid TRACE METALS: Mn2+, Fe3+, Zn2+ incr yield Mn2+ incr glycolysis Fe3+ is a cofator for enzymes like aconitase pH: incr yield when pH below 2.5, production of oxalic acid and gluconic acid is suppressed and risk of contamination is minimal DISSOLVED O2: high O2, sparging or incr aeration can affect if interrupted NITROGEN SOURCE: addition of ammonium stimulates overproduction, molasses is good source of nitrogen Citric acid production Surface fermentation Solid liquid submerged fermentation Stirred Bioreactor Airlift bioreactor N alkanes (C9-C23) can also be used to produce citric acid; can result in excess production of isocitric acid ACETIC ACID: industrial uses ACETIC ACID Incomplete oxidation of ethanol Vinegar is prepared from alcoholic liquids since ceturies NAD+ NADH +H+ NADP+ NADP +H+ CH3 CH2OH---- CH3CHO-------- CH3CH(OH)2 ------- Ethanol acetaldehyde acetaldehyde hydrate Alcohol dehydrogenase CH3COOH acetic acid Acetaldehyde dehydrogenase Gluconobacter, Acetobacter with acid tolerant A. aceti One molecule of ethanol one molecule of acetic acid is produced 12% acetic acid from 12% alcohol Clostridium thermoaceticum It is an obligate anaerobe, Grampositive, spore-forming, rod-shaped, thermophilic organism with an optimum growth temperature of 55– 60 o C and optimum pH of 6.6– 6.8. VINEGAR: 4% by volume acetic acid with alcohol, salts, sugars and esters flauoring agent in sauces and ketchups, preservative also Wine, malt, whey (surface or submerged fermentation process) Surface: trickling generator; fermentale material sprayed over surface, trickle thro shavings contaning acetic acid producing bacteria; 30oC (upper) and 35oC (lower). Produced in 3 days. Submerged: stainless steel, aerated using suction pump, production is 10X higher Clostridium thermoaceticum (from horse manure) is also able to utilize fivecarbon sugars: 2C5H10O5 --- 5CH3COOH A variety of substrates, including fructose, xylose, lactate, formate, and pyruvate, have been used as carbon sources in an effort to lower substrate costs. This factor is also important if cellulosic renewable resources are to be used as raw materials. Typical acidogenic bacteria are Clostridium aceticum, C. thermoaceticum, Clostridium formicoaceticum, and Acetobacterium woodii. Many can also reduce carbon dioxide and other one-carbon compounds to acetate. 1mol 2moles 1mol 2moles 1mol CODH These enzymes are metalloproteins; for example, CODH contains nickel, iron, and sulfur; FDH contains iron, selenium, tungsten, and a small quantity of molybdenum; and the corrinoid enzyme (vitamin B12 compound) contains cobalt. C. thermoaceticum does not have any specific amino acid requirement; nicotinic acid is the sole essential vitamin LACTIC ACID: industrial uses Technical grade 20-50% >90% Intestinal treatment (metal ion lactates) Food additive (sour flour and dough) Ester manufacture Textile industry Glucose G3P G3P dehydrogenase Pharmaceutical grade Food grade >80% NAD+ Lactic acid NADH +H+ 1,3-biphosphoglycerate Pyruvate LDH (Lactate dehydrogenase) LACTIC ACID 2 isomeric forms L(+) and D(-) and as racemic mixture DL-lactic acid First isolated from milk Toady produced microbial Heterofermentation Homofermentation Other than lactate products only lactate as product Lactobacillus L. delbrueckii L. leichmanni Mostly one isomer is produced Glucose L. bulgaricus L.helvetii Whey (lactose) L.lactis ------L.amylophilus -------L.pentosus ------ Maltose Starch Sulfite waste liquor LACTIC ACID: production process 1mol of glucose gives 2 moles of lactic acid; L lactic acid is predominantly produced Fermentation broth (12-15% glucose, N2, PO4, salts micronutrients) pH 5.5-6.5/temp 45-50oC/75h Heat to dissolve Ca lactate Addition of H2SO4 (removal of Ca SO4) Filter and concentrate Addtion of Hexacyanoferrant (removes heavy metal) Purification (Ion exchange) Concentration Lactic acid GLUCONIC ACID: Applications 1. Used in stainless steel manufacturing, leather (can remove rust and calcareous deposits) 2. 3. 4. 5. 6. Food additive for breverages Used in Ca and Fe therapy Na gluconate used in sequestering agent in detergets Desizing polyester or polyamide fabric Manufacture of frost and cracking resistant concrete Bacteria: Gluconobacter, Acetobacter, Pseudomonas, Vibrio Fungi: Aspergillus, Penicillium, Gliocladium intracellular PQQH2 PQQ Glucose dehydrogenase D-gluconolactone D-Glucose Extracellular Inducible Glucose oxidase FAD H2O2 extracellular Bacteria H2O Lactonase Gluconic Acid fungi FADH2 Catalase Fungi O2 High conc of glucose and pH above 4 H2O2 antagonist for other micro-organisms Submerged fermentation process Use glucose from corn H 4.5-6.5 28-30oC for 24h Incr supply of O2 enhances yield PQQ: pyrroliquinoline quinone coenzyme ITACNIC ACID: Applications Aspergillus itoconicus and A.terreus 1. Used in plastic industry, paper industry 2. Manufacturing of adhesives Cis-aconitic acid undergoes decarboxylation Itaconic acid Oxidase Itaconic acid Itatartaric acid (-) By Ca to incr yield SECONDARY METABOLITES ANTIBIOTICS BROAD SPECTRUM Control growth of wide range of unrelated organisms Tet, Cm NARROW SPECTRUM Control growth selected number organisms Pen, Str Streptomyces,eg. Tetracyclin, actinomycin D, of of ANTIBIOTICS: applications 1. Antimicrobial agents for chemotherapy 2. Antitumour antibiotics eg. Actinomycin D and mitomycin D 3. Food preservative antibiotics eg in canning (chlortetracycline) or fish or meat preservation (pimarcin, nisin) 4. Antibiotics in animal feed and veterinary medicine eg enduracidin, tylosin and hygromycin B, theostrepton, salinomycin 5. Control of plant diseases eg blasticidin, teranactin, polyoxin 6. Molecular biology MODE OF ACTION OF ANTIBIOTICS DNA GYRASE RNA ELONGATION CELL WALL SYNTHESIS DNA DIRECTED RNA POLYMERASE DNA THF RIBOSOMES DHF RNA PROTEIN SYNTHESIS (50S INHIBITORS) PROTEIN SYNTHESIS (30S INHIBITORS) PROTEIN SYNTHESIS (tRNA) CYTOPLASMIC MEMBRANE STRUCTURE AND FUNCTION PABA LIPID BIOSYNTHESIS SYTHETIC ANTIBIOTICS Selective toxicity: concept, Paul Ehrlich 1. GROWTH FACTOR ANALOGS: structurally similar to a growth factor required in a micro-organism; small differences of analogs in authentic growth factor prevent analog to function in the cell. A. SULFA DRUGS: specifically inhibit bacteria (streptococcal infections) eg. SULFANILAMIDE: is an analog of PABA (p-aminobenzoic acid) which is part of folic acid and nucleic acid precursor. Combination: sulfamethoxazole and trimethoprim; disadvantages and advantages B. ISONIAZID: important growth factor with narrow spectrum only against Mycobacterium. It interferes with synthesis of mycolic acids, a cell wall component. It is an analog of nicotinamide (vitamin). Single most effective drug against tuberculosis. 2. NUCLEIC ACID BASE ANALOGS URACIL PHENYLALANINE THYMINE 5-FLOUROURACIL (Uracil analog) p-FLOUROPHENYLALANINE 5-BROMOURACIL (thymine analog) Addition of F or Br does not alter the shape but changes chemical properties such that the compound does not function in the cell metabolism, thereby blocking the nucleic acid synthesis. These analogs are used in treatment of viral and fungal infections and many of these occur as mutagens. 3. QUINOLONES: Antibacterial compounds interfere with bacterial DNA gyrase, prevent supercoiling (packaging of DNA) eg Flouroquinolones like ciprofloxin (UTI, anthrax). B. anthracis maybe resistant to pencillin. These are effective in both G+ve and G-ve bacteria since DNA gyrase is present in all. Also used in beef and poultry for prevention and treatment of respiratory diseases. New generation Flouroquinolnes Ouinolones NATURALLY OCCURING ANTIBIOTICS FROM BACTERIA, FUNGI LESS THAN 1% OF 1000S OF ANTIBIOTICS ARE USEFUL BECAUSE OF TOXICITY OR LACK OF UPTAKE BY HOST CELLS Natural antibiotics can be artificially modified to enhance their efficacy then they are semi-synthetic antibiotics Broad spectrum antibiotics: effective against both gram +ve and gram-ve Narrow may also be beneficial to target specific group of bacteria eg. Vancomycin: narrow spectrum effective for gram positive pencillin resistant Staphylococcus, Bacillus, Clostridium Targets for antibiotics maybe ribosomes (Cm and Str for Bacteria and Cyclohexamide for eukarya), Cell wall, cytoplasmic membrane, lipid biosynthesis, enzymes, DNA replication and transcription elements Protein synthesis, Transcription (RNA poly, RNA elongation etc) Produced By Fungi B-LACTAMS (b-lactam ring) Penicillin Cephalosporins Produced by Prokaryotes AMINOGLYCOSIDES (amino sugars with glycosidic linkage) MACROLIDES (lactone ring bonded to sugars) TETRACYLINES (Streptomyces) PEPTIDE ANTIBIOTICS (Daptomycin, (Streptomyces) PLATENSIMYSIN (Streptomyces) Beta Lactam Antibiotics 1. 2. 3. 4. PENICILLINS, CEPHALOSPORINS, MONOBACTAMS AND CARBAPENEMS PENCILLIN--------b-LACTAM ANTIBIOTIC Alexander Fleming Pencillin G and V (natural) Penicillium chrysogenum Pencillin G first clinically useful antibiotic For Gram positive bacteria Used for Pneumococcal Streptococcal infections 6-AMINOPENICILLIANIC ACID Ampicillin, carbencillin Slight modification in N-acyl groups results in semi synthetic penicillin which is able to act on gram negative bacteria (goes past outer membrane) to act on cell wall MANY BACTERIA HAVE BETA LACTAMASE HENCE THOSE BACTERIA ARE PENCILLIN RESISTANT EG. Oxacillin and Methicillin beta lactamase resistant semi synthetic antibiotics MECHANISM OF ACTION • • • • • • • Pencillins block cell wall synthesis: transpeptidation (cross linking 2 glycan peptide chains) Transpeptidases bind to pencillin hence they are called PENCILLIN BINDING PROTEINS (PBP) Newly synthesized bacterial wall is no longer cross linked and has poor strength PBP also stimulates release of AUTOLYSINS (ENZYMES TO DIGEST CELL WALL) Osmotic pressure differences cause lysis VANCOMYCIN: does not bind PBPs but D-alanyl- Dalanine peptide to block transpeptidation BECAUSE OF SELECTIVE PROCESS B-LACTAMS DO NOT AFFTECT HOST CELLS AND MECHANISM IS UNIQUE TO BACTERIA MECHANISM OF ACTION Natural penicillin: i.e. V and G are effective against several gram positive bacteria They are effective against b-lactamase producing MO (enz which can hydrolyze penicillins) Eg. Staphylococcus aureus Production of penicillin is used: 45% (human), 15% (animal health) and 45% for production of semi synthetic penicillin P. notatum, P.chrysogenum and its mutant strain which is a high yeilding strain (Q176) Genetically engineered strains for improved pencillin production are being used now UDP deriv of NAM and NAG are synthesized Sequentially aa are added to UDPNAM to form NAM -pentapeptide ATP is used, no tRNA or ribosomes involved in peptide bond formation UDP tansfers NAG to bactoprenolNAM peptapeptide. For pentaglycine use special glycyltRNA moc but not ribosomes Transfer of UDP-NAMpentapeptideto bactoprenol PO4 LIPID I Bactoprenol carrier moves back across membrane by losing one PO4 for a new cycle Transport of completed NAMNAG-pepntapeptide across membrane LIPID II Attached to growing end of PG chain and incr by one repeat unit Bactoprenol is a 55 carbon alcohol and linked to NAM by pyrophosphate In S. aureus pepntapeptide has L-lys and in E. coli DAP UDP glucose Final step is TRANSPEPTIDATION which creates peptide cross links between PG chains. The enzyme removes terminal D-alanine as cross link is formed The b-lactam group of antibiotics includes an enormous diversity of natural and semi-synthetic compounds that inhibit several enzymes associated with the final step of peptidoglycan synthesis. All of this enormous family are derived from a b-lactam structure: a four-membered ring in which the b-lactam bond resembles a peptide bond. The multitude of chemical modifications based on this four-membered ring permits the astonishing array of antibacterial and pharmacological properties within this valuable family of antibiotics. Clinically useful families of b-lactam compounds include the penicillins, cephalosporins, monobactams and carbapenems. Many new variants on the b-lactam theme are currently being explored. Certain b-lactams have limited use directly as therapeutic agents, but may be used in combination with other b-lactams to act as b-lactamase inhibitors. Co-amoxyclav, for example is a combination of amoxycillin and the b- lactamase inhibitor clavulanic acid. During cross-linking of the peptidoglycan polymer, one D-alanine residue is cleaved from the peptidoglycan precursor and this reaction is prevented by b-lactam drugs. More recent studies have shown that the activity of this class of drugs is more complicated and involves other processes as well as preventing cross-linking of peptidoglycan. B-lactamase An increasing number of bacteria are penicillin resistant. Penicillinase-resistant penicillins such as methicillin, nafcillin, and oxacillin are frequently employed against these bacterial pathogens. Although penicillins are the least toxic of the antibiotics, about 1 to 5% of the adults in the United States are allergic to them. Occasionally a person will die of a violent allergic re- sponse; therefore patients should be questioned about penicillin allergies before treatment is begun. MRSA VRSA CEPHALOSPORINS Cephalosporium: Cephalosporin C B-lactam ring Dihydrothiazine ring (6 member) cefatrioxone Same mode of action with broader spectrum than penicillins Resistant to b-lactamases Hence used to treat infections which are penicillin resistant Used to treat Nesseria gonorrhea (STD) Most cephalosporins (including cephalothin, cefoxitin, ceftri- axone, and cefoperazone) are administered parenterally. Cefoperazone is resistant to destruction by b-lactamases and effective against many gram-negative bacteria, including Pseudomonas aeruginosa. Cephalexine and cefixime are given orally rather than by injection. 7-ACA: 7- aminocephalosporanic acid nucleus structure in all cephalosporins G+ = G- G+ > G- R1 R2 G+ < G- TETRACYCLINES • • • • Broad spectrum Effective for G+ and G- (mycoplasmas, rickettesia, chlamydia) Used for combatting stomach ulcer (Helicobacter pylori) Inhibit protein synthesis by blocking binding of amino acyl tRNA to ribosome (A site) BASIC STRUCTURE • • Napthacene ring Chlortetracycline and oxytetracycline are most commonly used in human and veterinary diseases and for preservation of meat, fish and poultry Three members of the tetracycline family. Tetracycline lacks both of the groups that are shaded. Chlortetracycline (aureomycin) differs from tetracycline in having a chlorine atom (blue); doxycycline consists of tetracycline with an extra hydroxyl (purple). TETRACYCLINES Str. aureus. S.flavus S. rimosus, S. antibioticus Streptomyces aureofaciens 20 diff species producing mix of tet Genetic modification Polyketide synthesis Antibiotics synthesized by successive condensation of small carboxylic acids Like acetate, butyrate, propionate, malonate High doses of tetracycline may result in nausea, diarrhea, yellowing of teeth in children, and damage to the liver and kidneys. AMINOGLYCOSIDES • • Oligosaccharide antibiotics Structurally all contain a cyclohexane ring and amino sugars bound by glycosidic linkages Bind to the 30S small ribosomal subunit and interfere with protein synthesis in at least two ways. They directly inhibit protein synthesis and also cause misreading of the genetic message carried by mRNA…prolonged use can cause kidney damage and hearing loss Streptomycin, kanamycin, neomycin, and tobramycin are synthesized by Streptomyces, whereas gentamicin comes from a related bacterium, Micromonospora purpurea. Known as reserve antibiotics as they develop resistance quickly AMINOGLYCOSIDES producing organisms Streptomycin Streptomyces griesus Neomycin B and C S.fradiae Kanamycin A, B and C S.kanamyceticus Hygromycin B S.hygroscopicus Gentamycin Micromonospora purpurea Sisimicin M.inyoensis MACROLIDES Antibiotics with a large lactone ring (macrocyclic lactone ring) Which consists of 12-, 14- and 16-membered lactone rings with 1-3 sugars linked by glycosidic bond Effective agaist penicillin resistant MO, G+ org, inhibitb y binding to 50S ribosome Clarithromycin (Erythromycin derv) Used to treat stomach ulcers Erythromycin : Streptomyces erythreus 14-membred connected to 2 sugars Genetic modifications by polyketide synthesis MACROLIDES Polyene macrolides: lactone rings in range of 26-28 Eg. Nystatin, amphotericin Actinomycetes are most common organisms which produce them Erythromycin is a relatively broad-spectrum antibiotic effective against grampositive bacteria, mycoplasmas, and a few gram-negative bacteria. It is used with patients allergic to penicillins and in the treatment of whooping cough, diphtheria, diarrhea caused by Campylobacter, and pneumonia from Legionella or Mycoplasma infections. Newer macrolides are now in use. Clindamycin is effective against a variety of bacteria including staphylococci and anaerobes such as Bacteroides. Azithromycin is particularly effective against Chlamydia trachomatis. AROMATIC ANTIBIOTICS Aromatic rings in structure Chloroamphenicol, griesofluvin, novobiocin CHLORAMPHENICOL Broad spectrum antibiotic against G+ and G- bacteria, rickettesia, chlamydia, actinomycetes chloramphenicol binds to 23S rRNA on the 50S ribosomal subunit. It inhibits the peptidyl transferase and is bacteriostatic. Streptomyces venezuelae and S.omiyanesis This antibiotic has a very broad spectrum of activity but unfortunately is quite toxic. One may see allergic responses or neurotoxic reactions. The most common side effect is a temporary or permanent depression of bone marrow function, leading to aplastic anemia and a decreased number of blood leukocytes. Chloramphenicol is used only in life-threatening situations when no other drug is adequate. GRIESOFULVIN Penicillium patulum Maybe attacks chitin biosynthesis hence acts as anti fungal antibiotic Following a 40-year hiatus in discovering new classes of antibacterial compounds, three new classes of antibacterial antibiotics have been brought into clinical use: Cyclic lipopeptides (Daptomycin), Glycylcyclines (tigecycline) and Oxazolidinones (Linezolid) PEPTIDE ANTIBIOTICS Daptomycin : Streptomyces roseosporus used to treat MDR infections Tigecycline: Tygacil® marketed by Wyeth used to treat MDR strains of Staphylococcus aureus and Acineotobacter baumanii. Mechanism similar to tetracycline. Also shows suceptibility to NDML (New Delhi metallo-b-lactamase multidrug resistant Enterobacteriaceae) NDML is an enzyme which makes bacteria resistant to broad range of b-lactam antibiotics. This includes antibiotics of carbapenems for treatment of antibiotics resistant infections. Termed as “SUPERBUGS” Such bacteria susceptible to polymixins and tigecyclines MECHANISM OF DRUG RESISTANCE Plasmids R-Plasmids Superinfection: Clostridium difficile, Candida albicans Transformation, conjugation, transduction, ABC transporters Phage therapy There has been some recent progress in developing new antibiotics that are effective against drug-resistant pathogens. Two new drugs are fairly effective against vancomycin-resistant enterococci. Synercid is a mixture of the streptogramin antibiotics quinupristin and dalfopristin that inhibits protein synthesis. A second drug, linezolid (Zyvox), is the first drug in a new family of antibiotics, the oxazolidinones. It inhibits protein synthesis and is active against both vancomycinresistant enterococci and methicillin-resistant Staphylococcus aureus.