Medical Microbiology B.Sc (H) Biomedical Science PDF

Medical Microbiology B.Sc (H) Biomedical Science Dr Satendra Singh BMS, ANDC, DU \ Microbiology often has been defined as the study of organisms and agents too small to be seen clearly by the unaided eye—that is, the study of microorganisms 1. Medical microb...

Medical Microbiology B.Sc (H) Biomedical Science Dr Satendra Singh BMS, ANDC, DU \ Microbiology often has been defined as the study of organisms and agents too small to be seen clearly by the unaided eye—that is, the study of microorganisms 1. Medical microbiologists identify the agents causing infectious diseases and plan measures for their control and elimination. 2. Frequently they are involved in tracking down new, unidentified pathogens such as the agent that causes variant Creutzfeldt-Jakob disease, (the human version of “mad cow disease”) the hantavirus, the West Nile virus, and the virus responsible for SARS. 3. These microbiologists also study the ways in which microorganisms cause disease 1. It is estimated that microbes contain 50% of the biological carbon and 90% of the biological nitrogen on Earth—they greatly exceed every other group of organisms on the planet. 2. Microbes Found everywhere. 3. They are major contributors to the functioning of the biosphere, being indispensable for the cycling of the elements essential for life. 4. They also are a source of nutrients at the base of all ecological food chains and webs. 5. Photosynthesis, Fermented food, antibiotics, 6. Those microbes that inhabit humans also play important roles, including helping the body digest food and producing vitamins B and K. 7. Microbial diseases-- The plague, AIDS 8. they are necessary for the production of bread, cheese, beer, antibiotics, vaccines, vitamins, enzymes, and many other important products. Indeed, modern biotechnology rests upon a microbiological foundation. Procaryotic cells [Greek pro, before, and karyon, nut or kernel; AYTGCCCTTCCG Eucaryotic cells [Greek, eu, true 1. The comparison of ribosomal RNA (rRNA), begun by Carl Woese in the 1970s, was instrumental in demonstrating that there are two very different groups of procaryotic organisms: Bacteria and Archaea, which had been classified together as Monera in the five-kingdom system. 2. Many Microbiologists believe that organisms should be divided among three domains: Bacteria (the true bacteria or eubacteria), Archaea, and Eucarya (all eucaryotic organisms) 1. Bacteria are procaryotes that are usually single- celled organisms 2. Archaea are procaryotes that are distinguished from Bacteria by many features, most notably their unique ribosomal RNA sequences. 3. They also lack peptidoglycan in their cell walls and have unique membrane lipids. 4. Protists are generally larger than procaryotes and include unicellular algae, protozoa, slime molds, and water molds 5. Fungi and Viruses THE DISCOVERY OF MICROORGANISMS 1. Roman philosopher Lucretius (about 98–55 B.C.) and the physician Girolamo Fracastoro (1478–1553) suggested that disease was caused by invisible living creatures. 2. In 1665, the first drawing of a microorganism was published in Robert Hooke’s Micrographia. 3. However, the first person to publish extensive, accurate observations of microorganisms was the amateur microscopist Antony van Leeuwenhoek (1632–1723) of Delft, The Netherlands 1. From earliest times, people had believed in spontaneous generation—that living organisms could develop from nonliving matter. 2. Even Aristotle (384–322 B.C.) thought some of the simpler invertebrates could arise by spontaneous generation. 3. This view finally was challenged by the Italian physician Francesco Redi (1626–1697), who carried out a series of experiments on decaying meat and its ability to produce maggots spontaneously. 4. Redi placed meat in three containers. One was uncovered, a second was covered with paper, and the third was covered with a fine gauze that would exclude flies. 5. Flies laid their eggs on the uncovered meat and maggots developed. The other two pieces of meat did not produce maggots spontaneously. 6. However, flies were attracted to the gauze-covered container and laid their eggs on the gauze; these eggs produced maggots. Thus the generation of maggots by decaying meat resulted from the presence of fly eggs, and meat did not spontaneously generate maggots as previously believed. Louis Pasteur (1822–1895) Rejected Spontaneous generation THE GOLDEN AGE OFMICROBIOLOGY 1. Pasteur’s work with swan neck flasks ushered (Guide) in the Golden Age of Microbiology. 2. Within 60 years (1857–1914), a number of disease-causing microbes were discovered, great strides (Steps) in understanding microbial metabolism were made, and techniques for isolating and characterizing microbes were improved. 3. Indirect evidence for the germ theory of disease came from the work of the English surgeon Joseph Lister (1827–1912) on the prevention of wound infections Koch’s Postulates 1. The first direct demonstration of the role of bacteria in causing disease came from the study of anthrax by the German physician Robert Koch (1843–1910). 2. Koch used the criteria proposed by his former teacher, Jacob Henle (1809–1885), to establish the relationship between Bacillus anthracis and anthrax, and published his findings in 1876 Robert Koch. Koch (1843–1910) examining a specimen in his laboratory 1. Koch injected healthy mice with material from diseased animals, and the mice became ill. 2. After transferring anthrax by inoculation through a series of 20 mice, he incubated a piece of spleen containing the anthrax bacillus in beef serum. 3. The bacilli grew, reproduced, and produced endospores. When the isolated bacilli or their spores were injected into mice, anthrax developed. 4. His criteria for proving the causal relationship between a microorganism and a specific disease are known as Koch’s postulates. 5. In 1884, he reported that tuberculosis was caused by a rod- shaped bacterium, Mycobacterium tuberculosis; he was awarded the Nobel Prize in Physiology or Medicine in 1905 for his work. 1. Fannie Eilshemius Hesse, one of Koch’s assistant, suggested the use of agar as a solidifying agent—she had been using it successfully to make jellies for some time. 2. Agar was not attacked by most bacteria and did not melt until reaching a temperature of 100°C. 3. Furthermore, once melted, it did not solidify until it reached a temperature of 50°C. 4. Another important tool developed in Koch’s laboratory was a container for holding solidified media—the petri dish (plate), named after Richard Petri, who devised it. 5. These developments directly stimulated progress in all areas of bacteriology 1. Viral pathogens were also studied during this time. The discovery of viruses and their role in disease was made possible when Charles Chamberland (1851–1908), one of Pasteur’s associates, constructed a porcelain bacterial filter in 1884. 2. Dimitri Ivanowski and Martinus Beijerinck (pronounced “by-a-rink”) used the filter to study tobacco mosaic disease. 3. Pasteur and Roux discovered- attenuation and they called attenuated culture a vaccine. 4. Shortly after this, Pasteur and Chamberland developed an attenuated anthrax vaccine in two ways: by treating cultures with potassium bichromate and by incubating the bacteria at 42 to 43°C. Unit II: Bacterial Cells - fine structure and function (5 Lectures) Size, shape and arrangement of bacterial cells. Cell membrane, cytoplasmic matrix, inclusion bodies (eg magnetosomes), nucleoid, Ultrastructure of Gram +ve and Gram –ve bacterial cell wall, Pili, Capsule, Flagella, motility and endospores. AN OVERVIEW OF PROCARYOTIC CELL STRUCTURE Shape, Arrangement, and Size 1. Most commonly encountered procaryotes have one of two shapes--- Cocci (s., coccus) are roughly spherical cells. 2. They can exist as individual cells, but also are associated in characteristic arrangements that are frequently useful in their identification. 3. Diplococci (s., diplococcus) arise when cocci divide and remain together to form pairs. 4. Long chains of cocci result when cells adhere after repeated divisions in one plane; this pattern is seen in the genera Streptococcus, Enterococcus, and Lactococcus. 5. Staphylococcus divides in random planes to generate irregular grapelike clumps. 6. Divisions in two or three planes can produce symmetrical clusters of cocci. Members of the genus Micrococcus often divide in two planes to form square groups of four cells called tetrads 1. The other common shape is that of a rod, sometimes called a bacillus (pl., bacilli). Bacillus megaterium is a typical example of a bacterium with a rod shape. 2. Vibrios most closely resemble rods, as they are comma- shaped. 3. Spiral-shaped procaryotes can be either classified as spirilla, which usually have tufts of flagella at one or both ends of the cell or spirochetes. Spirochetes are more flexible and have a unique, internal flagellar arrangement. 4. Actinomycetes typically form long filaments called hyphae that may branch to produce a network called a mycelium   Size  Escherichia coli is a rod of about average size, 1.1 to 1.5 um wide by 2.0 to 6.0 um long  Nanobacteria range from around 0.2 um to less than 0.05 um in diameter 1. Spirochaetes, which can reach 500 um in length, and the photosynthetic bacterium Oscillatoria, which is about 7 um in diameter (the same diameter as a red blood cell) 2. Huge bacterium in intestine of brown surgeon fish, Acanthurus nigrofuscus. Epulopiscium fishelsoni grows as large as 80x600 um 1. The preceding techniques require the use of special culture dishes named petri dishes or plates after their inventor Julius Richard Petri, a member of Robert Koch’s laboratory. 2. Petri developed these dishes around 1887 and they immediately replaced agar-coated glass plates. 3. In nature, microorganisms often grow on surfaces in biofilms—slime-encased aggregations of microbes. However, sometimes they form discrete colonies. 4. Nutrient diffusion and availability, bacterial chemotaxis, and the presence of liquid on the surface all appear to play a role in pattern formation 1. Generally the most rapid cell growth occurs at the colony edge. Growth is much slower in the center, and cell autolysis takes place in the older central portions of some colonies. 2. These differences in growth are due to gradients of oxygen, nutrients, and toxic products within the colony. 3. At the colony edge, oxygen and nutrients are plentiful. The colony center is much thicker than the edge. 4. Consequently oxygen and nutrients do not diffuse readily into the center, toxic metabolic products cannot be quickly eliminated, and growth in the colony center is slowed or stopped. 5. Because of these environmental variations within a colony, cells on the periphery can be growing at maximum rates while cells in the center are dying. Procaryotic Cell Organization PROCARYOTIC CELLMEMBRANES  Retain cytosol  Selective barrier  Essential for survival The procaryotic plasma membrane also is the location of a variety of crucial metabolic processes: respiration, photosynthesis, and the synthesis of lipids and cell wall constituents. Finally, the membrane contains special receptor molecules that help procaryotes detect and respond to chemicals in their surroundings. Membrane chemistry can be used to identify particular bacterial species The Fluid Mosaic Model of Membrane Structure The most widely accepted model for membrane structure is the fluid mosaic model of Singer and Nicholson, which proposes that membranes are lipid bilayers within which proteins float CM 5-10 nm thick Membrane Proteins  Peripheral Proteins: Make up about 20 to 30% of total membrane protein, water soluble  Integral proteins: about 70 to 80% of membrane proteins, insoluble Membrane chemistry Lipid: Amphipathic  Bacterial Membranes Lipids  Cholesterol found in eukaryotic cells are absent in bacterial cell  Many bacterial membranes contain sterol-like molecules called hopanoids.  Hopanoids are synthesized from the same precursors as steroids, and like the sterols in eucaryotic membranes, they probably stabilize the membrane It has been estimated that the total mass of hopanoids in sediments is around 10–11-12 tons—about as much as the total mass of organic carbon in all living organisms (10- 12 tons)—and there is evidence that hopanoids have contributed significantly to the formation of petroleum. Mesosomes Archaeal Membranes One of the most distinctive features of the Archaea is the nature of their membrane lipids. They differ from both Bacteria and Eucarya in having branched chain hydrocarbons attached to glycerol by ether links rather than fatty acids connected by ester links  Sometimes two glycerol groups are linked to form an extremely long tetraether. Usually the diether hydrocarbon chains are 20 carbons in length, and the tetraether chains are 40 carbons.  Cells can adjust the overall length of the tetraethers by cyclizing the chains to form pentacyclic rings  Phosphate-, sulfur- and sugar-containing groups can be attached to the third carbons of the diethers and tetraethers, making them polar lipids.  These predominate in the membrane, and 70 to 93% of the membrane lipids are polar. The remaining lipids are nonpolar and are usually derivatives of squalene Extreme thermophiles such as Thermoplasma and Sulfolobus, which grow best at temperatures over 85°C, are almost completely tetraether monolayers. Archaea that live in moderately hot environments have a mixed membrane containing some regions with monolayers and some with bilayers. THE BACTERIAL CELLWALL 1. it helps determine the shape of the cell; 2. it helps protect the cell from osmotic lysis; 3. it can protect the cell from toxic substances; 4. and in pathogens, it can contribute to pathogenicity. 5. The procaryotic cell wall also is the site of action of several antibiotics Overview of Bacterial Cell Wall Structure Periplasmic space and periplasm. Gram stain in 1884 Peptidoglycan Structure The gram-positive cell wall consists of a single 20 to 80 nm thick homogeneous layer of peptidoglycan (murein) lying outside the plasma membrane. In contrast, gram-negative cell wall is quite complex. It has a 2 to 7 nm peptidoglycan layer covered by a 7 to 8 nm thick outer membrane Peptidoglycan Structure 1. The polymer contains two sugar derivatives, N- acetylglucosamine and N-acetylmuramic 2. acid (the lactyl ether of N-acetylglucosamine), and several different amino acids. 3. Three of these amino acids are not found in proteins: D- glutamic acid, D-alanine, and mesodiaminopimelic acid. 4. The presence of D-amino acids protects against degradation by most peptidases, which recognize only the L-isomers of amino acid residues. 1. Gram-Positive Cell Walls Consist Primarily of Peptidoglycan Most bacteria that stain Gram positive belong to the phyla Firmicutes and Actinobacteria, and most of these bacteria have thick cell walls composed of peptidoglycan and large amounts of other polymers such as teichoic acids (figure 3.22). 2. Teichoic acids are polymers of glycerol or ribitol joined by phosphate groups (figure 3.23). Some teichoic acids are covalently linked to peptidoglycan and are referred to as wall teichoic acids. 3. Others are covalently connected to the plasma membrane; they are called lipoteichoic acids. Wall teichoic acids extend beyond the surface of the peptidoglycan. 4. They are negatively charged and help give the cell wall its negative charge. Teichoic acids are not present in other bacteria. Gram-Positive Cell Wall 1. Teichoic acids have several important functions. They help create and maintain the structure of the cell envelope by anchoring the wall to the plasma membrane. 2. They are important during cell division, and they protect the cell from harmful substances in the environment (e.g., antibiotics and host defense molecules). 3. In addition, they function in ion uptake and are involved in binding pathogenic species to host tissues, thus initiating the infectious disease process. Gram-Positive Cell Wall glycerol and ribitol groups Exoenzymes In addition, gram-positive cell walls usually contain large amounts of teichoic acids, polymers of glycerol or ribitol joined by phosphate groups. Teichoic acids appear to extend to the surface of the peptidoglycan, and, because they are negatively charged, help give the gram-positive cell wall its negative charge. Teichoic acids are not present in gram-negative bacteria Comparison of Gram positive and Gram negative bacterial cell wall 1. The periplasm has relatively few proteins; this is probably because the peptidoglycan sacculus is porous and many proteins translocated across the plasma membrane pass through the sacculus. 2. Some secreted proteins are enzymes called exoenzymes. Exoenzymes often serve to degrade polymers such as proteins and polysaccharides that would otherwise be too large for transport across the plasma membrane; the degradation products, the monomer building blocks, are then taken up by the cell. 3. Those proteins that remain in the periplasmic space are usually attached to the plasma membrane. 1. Membrane-bound enzymes called sortases catalyze the formation of covalent bonds and join many membrane proteins to the peptidoglycan. 2. Many covalently attached proteins have roles in virulence. For example, the M protein of pathogenic streptococci aids in adhesion to host tissues and interferes with host defenses. Gram-Negative Cell Wall The peptidoglycan layer is very thin in G –ve bacterial cell wall (2 to 7 nm, depending on the bacterium) and sits within the periplasmic space. The periplasmic space is usually 30 to 70 nm wide. Some studies indicate that it may constitute about 20 to 40% of the total cell volume. Thus it is much larger than that observed in typical Gram-positive cells. 1. The outer membrane lies outside the thin peptidoglycan layer and is linked to the cell in two ways. 2. The first is by Braun’s lipoprotein, the most abundant protein in the outer membrane. This small lipoprotein is covalently joined to the underlying peptidoglycan, and is embedded in the outer membrane by its hydrophobic end. 3. The outer membrane and peptidoglycan are so firmly linked by this lipoprotein that they can be 4. isolated as one unit. 5. The second linking mechanism involves the many adhesion sites joining the outer membrane and the plasma membrane. 6. The two membranes appear to be in direct contact at these sites. In E. coli, 20 to 100 nm areas of contact between the two membranes can be seen. 7. Adhesion sites may be regions of direct contact or possibly true membrane fusions. It has been proposed that substances can move directly into the cell through these adhesion sites, rather than traveling through the periplasm. 1. Possibly the most unusual constituents of the outer membrane are its lipopolysaccharides (LPSs). 2. These large, complex molecules contain both lipid and carbohydrate, and consist of three parts: (1) lipid A, (2) the core polysaccharide, and (3) the O side chain. 3. The lipid A region contains two glucosamine sugar derivatives, each with three fatty acids and phosphate or pyrophosphate attached. The fatty acids attach the lipid A to the outer membrane, while the remainder of the LPS molecule projects from the surface. 4. The core polysaccharide is joined to lipid A. In Salmonella it is constructed of 10 sugars, many of them unusual in structure. 5. The O side chain or O antigen is a polysaccharide chain extending outward from the core. It has several peculiar sugars and varies in composition between bacterial strains Functions of LPS 1. It contributes to the negative charge on the bacterial surface because the core polysaccharide usually contains charged sugars and phosphate ( figure 3.25). (2) It helps stabilize outer membrane structure because lipid A is a major constituent of the exterior leaflet of the outer membrane. 2. It helps create a permeability barrier. The geometry of LPS (figure 3.25b) and interactions between neighboring LPS molecules are thought to restrict the entry of bile salts, antibiotics, detergents, and other toxic substances that might kill or injure the bacterium. 3. LPS helps protect pathogenic bacteria from host defenses. The O side chain of LPS is also called the O antigen because it elicits an immune response by an infected host. 1. This response involves the production of antibodies that bind the strain-specific form of LPS that elicited the response. 2. For example, microbiologists refer to specific strains of Gram-negative bacteria using the O antigen, such as E. coli O157; here the O side chain is the antigenic type number 157. 3. Unfortunately, many bacteria can rapidly change the antigenic nature of their O side chains, thus thwarting host defenses. Importantly, the lipid A portion of LPS can act as a toxin and is called endotoxin; 1. Despite the role of LPS in creating a permeability barrier, the outer membrane is more permeable than the plasma membrane and permits the passage of small molecules like glucose and other monosaccharides. 2. This is due to the presence of porin proteins. Most porin proteins cluster together to form a trimer in the outer membrane. 3. Each porin protein spans the outer membrane and is more or less tube-shaped; its narrow channel allows passage of molecules smaller than about 600 to 700 daltons.