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Microbial Metabolism Biochemical tests Zivuku.M Content • Microbial metabolism (anabolism and catabolism) • Microbial biochemical tests (carbohydrates fermentation tests, Methyl red test, VP test, Indole test, Citrate test) • Microbial products (pyrogen's, antibiotics, vitamins, bacteriocins) Microbial Metabolism Metabolism refers to all the biochemical reactions that occur in a cell or organism. 1) pathways for the interconversion of focal metabolites 2) assimilatory pathways for the formation of focal metabolites 3) biosynthetic sequences for the conversion of focal metabolites to end products 4) pathways that yield metabolic energy for growth and maintenance. Microbial Metabolism The key concepts of glycolysis, Krebs Cycle, oxidative phosphorylation have been or will be discussed in biochemistry. Concentrates here are put on the products of bacterial metabolism with medical importance (e.g. bacterial pathogenesis). for laboratory diagnosis or for • Medical Important Metabolic Products 1. anabolic processes (synthesis or build up) 2. catabolic processes (decomposition or break down) Catabolic Products and Biochemical Testing a. Carbohydrate Fermentation Tests Medium: Carbohydrate fermentation broth with a Durham tube (a small inverted vial filled with the carbohydrate fermentation broth). If gas is produced during fermentation of the sugar, it is trapped at the top of the Durham tube and appears as a bubble. pH indicator: phenol red a. Carbohydrate Fermentation Tests Principle: Because the type of enzyme(s) produced by bacteria is genetically controlled, the fermented pattern of sugars may be unique to a species, but may be different between different species. Fermentation products are usually acid (lactic acid, acetic acid etc.), neutral (ethanol etc.) or gases (carbon dioxide, hyrogen, etc). a. Carbohydrate Fermentation Tests Result: positive yellow color yellow color with gas bubble negative red color, no gas bubble b. Methyl Red (MR) Test Medium: contains glucose and peptone Indicator: methyl Red All enterics oxidize glucose for energy; however the end products vary depending on bacterial enzymes. b. Methyl Red (MR) Test For bacteria (e.g. E. coli ) that produces acids, causing the pH to drop below 4.4. When the pH indicator methyl red is added to this acidic broth it will be cherry red (a positive MR test). For bacteria (e.g. Klebsiella and Enterobacter) that produce more neutral products from glucose (e.g. ethyl alcohol, acetyl methyl carbinol). In this neutral pH the growth of the bacteria is not inhibited. The bacteria thus begin to attack the peptone in the broth, causing the pH to rise above 6.2. At this pH, methyl red indicator is a yellow color (a negative MR test). Result: c. Voges-Proskauer (VP) Test Medium: contains glucose and peptone Indicator: Barritt's A (alpha-napthol) and Barritt's B (potassium hydroxide) When these reagents are added to a broth in which acetyl methyl carbinol is present, they turn a pink-burgundy color (a positive VP test). This color may take 20 to 30 minutes to develop. E. coli does not produce acetyl methyl carbinol, but Enterobacter and Klebsiella do. c. Voges-Proskauer (VP) Test d. Citrate Utilization Test The citrate test utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate as its sole carbon and energy source. Growth of bacteria in the media leads to development of a Prussian blue color (positive citrate). e. Indole Test The test organism is inoculated into tryptone broth, a rich source of the amino acid tryptophan. Indole positive bacteria such as E. coli produce tryptophanase, an enzyme that cleaves tryptophan, producing indole and other products. When Kovac's reagent is added to a broth with indole in it, a dark pink color develops. Indol test Methyl test VP test Citrate utilization test I M Vi C test E. coli C. perfringens I M Vi C + — + — — + — + • f. Hydrogen Sulfide (H2S ) formation To determine the ability of a bacterium to produce hydrogen sulfide (H2S) by enzymatic reaction on amino acids such as cysteine, cystine and methionine. The hydrogen sulfide combines with ferrous sulfide (Fe2S) in the triple sugar iron (TSI) agar to form a black to dark insoluble precipitate. • g. Urease Test Medium: urea agar slant Indicator: phenol red Principle: The hydrolysis of urea by urease produces ammonia and carbon dioxide. The formation of ammonia alkalinizes the medium, and the pH is detected by the color change from light orange to pink-red. Positive result: pink-red color Negative result: light orange What are the medically important anabolic products of bacteria? Synthetic Products • Pyrogens • Toxins and Invasive Enzymes • Antibiotics • Vitamins • Bacteriocins • Pigments • Pyrogens • the products of many bacteria, especially gram-negative bacteria, which resulting in fever when injected into animals or humans. • They are polysaccharides in cell wall (G+ bacteria) or LPS (G- bacteria). • They are highly resistant to high heat (not being destroyed heating at 121C for 15-20 min), but can be destroyed by heating to 250 C for 30 min. • They can be removed from most fluid materials by adsorption using special materials. • Toxins and Invasive Enzymes • According to difference on the chemical composition, toxicity, bioactivity, antigenicity and so on, bacterial toxins can be divided into exotoxin (polypeptide or protein) and endotoxin (LPS). • Invasive enzymes secreted by bacteria can help bacteria to invade host. So they contributed to the pathogenesis of bacteria. • Antibiotics • The substance produced by many fungi and a small number of bacteria that selectively kill or inhibit other organisms. • Vitamins • A few bacteria produce various vitamins, e.g. some certain bacteria growing in the intestine produce vitamin K. This action is thought to be beneficial to the host. • Bacteriocins • Substances produced by specific strains of bacteria that are lethal against other strains of the same or related species. • They are protein or lipopolysaccharide-protein complexes. • pigments • Produced by a small number of bacteria with characteristic colors. I) water soluble Pseudomonas aeruginosa can produce a green water soluble pigment, so that the color is distributed throughout the culture. II) liposoluble Staphylococcus aureus can produce a golden yellow liposoluble pigment and this color only show in their colonies. • Help to identify some of bacteria. Pseudomonas aeruginosa produces a blue-green pigment, which diffuses into the medium giving the plate a characteristic color. Staphylococcus aureus produces a golden yellow pigment and give the colonies this color. The medical significance of these products Products Medical significance pyrogen toxin Pathogenicity of bacteria Invasive enzyme Antibiotic Vitamin Treatment of infectious diseases Bacteriocin Identification of bacteria pigment Antibiotics and Antibiotic Resistance Antibiotics Antibiotics are powerful medicines that fight bacterial infection Literal translation • anti – against • biotic – living things How antibiotics work Antibiotics can be either • Broad Spectrum – Kill a wide range of bacteria e.g. Penicillin • Narrow Spectrum – Kill a specific type or group of bacteria e.g. Isoniazid, Rifampicintablets Antibiotics work in one of two ways • Bactericidal – Kills the bacteria • Bacteriostatic – Prevents the bacteria from dividing Miracle Cure? – Before the 1930s there were no treatments for bacterial infections – Following the discovery of penicillin industry started searching for more antibiotics in nature – Streptomycin was the first drug to have an effect on tuberculosis – a condition previously untreatable – Surgeons could attempt more dangerous operations Miracle Cure? Overuse of antibiotics can damage our normal/good bacteria. – Many antibiotics prescribed by the doctor are broad spectrum – These kill the body’s good bacteria as well as the bad – With the good bacteria gone there is more room for bad microbes to invade! Miracle Cure? Antibiotics resistance Many bacteria have developed the ability to become resistant to antibiotics. These bacteria are now a major threat in our hospitals. Antibiotic resistant bacteria aureus (MRSA) include Methicillin Resistant Staphylococcus Antibiotic Resistance The Causes – Overuse • Antibiotics used to treat infections when they are not needed or not effective i.e. for the flu – Misuse • Not completing a prescribed course • Using antibiotics not prescribed for you Bacterialcidal Mechanism Cell Membrane Cell Wall Mitochondria DNA Ribosomal Complex Bacterialcidal Mechanism • Damaging or inhibiting synthesis of the bacterial cell wall (penicillins, cephalosporins, monobactams, carbapenems, bacitracin, vancomycin, cycloserine, fosfomycin) • Damaging or inhibiting synthesis of the cell membrane (polymyxins) Bacterialcidal Mechanism • Metabolizing or inhibiting DNA synthesis of nucleic acids (rifampin, nitrofurantoins, nitromidazoles) • Modifying ribosomal energy metabolism (sulfonamides, trimethroprim, dapsone, isoniazid) • Inhibiting ribosomal protein biosynthesis (aminoglycosides, tetracyclines, chloramphenicol, erythromycin, clindamycin, spectinomycin, mupirocin, fusidic acid) Acquired Bacterial Resistance Virus Plasmid Acquired Bacterial Resistance • Receiving a plasmid bearing a resistance gene from another bacterium directly • Receiving a resistance gene from other bacterium by viral transfection • Chromosomal mutation • DNA scavenged from dead bacteria Mechanisms of resistance Imipenem resistant Pseudomonas aeruginosae Streptococcus pneumoniae resistance to penicillins MRSA penicillin binding protein PBP2A Tetracycline Penicillins, Cephalosporins Mechanisms of resistance • Antibiotic modification: some bacteria have enzymes that cleave or modify antibiotics: e.g. β-lactamase inactivates penicillin • Denied access: membrane becomes impermeable for antibiotic: e.g. imipenem • Pumping out the antibiotic faster than it gets in: e.g. tetracyclines • Altered target site: antibiotic cannot bind to its intended target because the target itself has been modified • Production of alternative target (typically enzyme): e.g. Alternative penicillin binding protein (PBP2a) in MRSA Loss of Antibiotic Resistance • Antibiotic resistant bacteria are at a selective disadvantage - they must expend energy and resources to manufacture proteins that confer resistance • The prevalence of resistant bacteria declines after antibiotics are withdrawn How antibiotic resistance can be prevented – Antibiotics should be the last line of defence NOT the first • Most common infections will get better by themselves through time, bed rest, liquid intake and healthy living. – Only take antibiotics prescribed by a doctor – If prescribed antibiotics, finish the course. – Do not use other peoples or leftover antibiotics • they be specific for some other infection Summary 1) Definitions: pyrogen, antibiotic and acteriocin 2) Bacteria growth curve, especially the characteristics and application of log phase and maximum stationary phase. 3) The medically important antibiotic products of bacteria 4) The requirements of bacterial growth 5) The mode of bacterial reproduction THANK YOU HAVE A BLESSED DAY