01 - Brief history of microbiology - Adan Abu Rashid, Lucia Baglieri.pdf

11.10.21 Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri MOD-Microbiology-prof.Garlanda Microbes: history, structure, classification Part 1- A brief History of Microbiology: Antoni van Leeuwenhoek: • • • discovered bacteria first to made magnifying glass lenses first to desc...

11.10.21 Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri MOD-Microbiology-prof.Garlanda Microbes: history, structure, classification Part 1- A brief History of Microbiology: Antoni van Leeuwenhoek: • • • discovered bacteria first to made magnifying glass lenses first to describe small creatures living in water as animacules now known as protozoa and bacteria Carolus Linnaeus: • • • developed taxonomic system divided animals and plants the classification of microorganisms included: bacteria, archea, fungi, algae and parasites, but did not include viruses The Golden age of Microbiology (late1800s): Until the 17th century Aristotle’s theory of spontaneous generation (= abiogenesis) was accepted, which indicated that living things can arise from non-living matter. Francesco Redi’s experiment (late 1600s): demonstrated that flies which apparently originated from meat did not originate from spontaneous generation, but rather they came from eggs that were laid when the flask was unsealed. As a result of his experiment, scientists started to doubt Aristotle's theory. Lazzaro Spallanzani (1799): showed that when heating and sealing vials, microbes did not grow. His theory was criticized because heating destroyed the “living force”, and sealing did not allow enough air for organisms to thrive. Louis Pasteur (1822-1895): his studies on fermentation (yeast cells produce alcohol → pasteurization, immunization) led him to the development of the germ theory of diseases: microorganisms are responsible for diseases. He demonstrated that when boiling solution in a flask while the neck of the flask is bent, you prevent the entry of microbes, while allowing air to enter (dust and some microbes will remain in the bended neck of the flask). 11.10.21 Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri Buchner: demonstrated that a process of fermentation can be performed through enzymes, and it does not require living cells. At the end of 19th century, the term “microorganisms” was presented Ivanowsky & Beijerinck: showed that some diseases may be caused by microorganism that can pass through filters (viruses). Robert Koch: he is considered the father of etiology, and studied the causation of diseases. He is the person who was able to analyze colonies of microorganisms and describe them. He proved for the first time that anthrax was caused by a bacterium. He also discovered that tuberculosis is caused by Mycobacterium tuberculosis, for which he earned the Nobel prize in 1905. Robert Koch's postulates: • • • suspected causative agent MUST be found in every case of disease and absent from healthy host. agents must be isolated and grown outside the host because we have to demonstrate that it is the causative agent, although now we know that not all microorganisms can be grown outside a host. when an agent is introduced to healthy person, the host will get the disease, and the same agent must be found in the diseased experimental host Koch's Advances: • • • • • collect specimens from diseases individuals smearing onto solid surfaces such as petri dishes distinct colonies of a single cell with different morphology staining techniques for bacteria techniques to count bacteria by originating colonies Hans Gram: introduced the gram staining technique, which is still used, and presented the gram positive/negative classification. With this technique, depending on whether microbes are Gram+ or Gram-, they acquire a different dye, therefore it is possible to discriminate between them. Also, the importance of hygiene to prevent diseases was introduced: Ignaz Semmelweis: handwashing to prevent puerperal fever after childbirth. He observed that women often underwent fever after parturition. He understood that students and doctors were “moving” microbes from one patient to the other, and insisted on the importance of handwashing, but without being believed. Joseph Lister: founder of antiseptic surgery Florence Nightingale: insisted on hygiene standards in hospitals & nursing education 11.10.21 Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri John Snow: studied the cholera epidemic in London. He introduced the concepts of infection control and epidemiology Edward Jenner: father of vaccination and immunology, discovered vaccine that protected humans from cowpox (he observed that there was a virus originating in bovines, which could be used to protect humans from cowpox). Antibiotics work by targeting the specificity of bacteria. Instead, viruses replicate by using the enzymes and the replication machinery of the host. Therefore, viruses cannot be targeted by antibiotics. Ehrlich’s "magic bullets": studied and proved the we need specific chemicals for killing pathogens (field of chemotherapy) From 2nd half of the 1800 (golden age) it was accepted that life comes from other life, and microorganisms can cause diseases. The modern age of microbiology: Development of disciplines based on microbiology • • • • • Basic chemical reactions of life: microbes as models. Biochemistry: studies the basic biochemical reactions (shared by all living organisms) to answer questions about metabolism, using microbes as a model system. How do genes work? Microbial genetics: study of microbial genes as a model and a tool, especially for recombinant DNA technology, molecular biology and gene therapy (inserting missing genes or repairing defective ones in humans The role of microbes in the environment: microbial colonies are essentials in diseases. The defense against diseases. Immunology: microorganisms can cause diseases, but the body can defend itself. How to control or eradicate diseases (malaria, Tb, polio), how to reduce microbes' resistance to antibiotics, how to reduce emerging (covid19) and reemerging diseases. 11.10.21 fields of microbiology: Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri 11.10.21 Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri Part 2- Cell Structure and Function: Overview of Prokaryotic and Eukaryotic cells Prokaryotes includes bacteria and archaea domains. Thet lack a nucleus, so the genomic material is confined to a specific part of the cytosol, called the nucleoid. They also lack various internal structures bound with phospholipid membranes. Reproduce via asexual reproduction. Prokaryotes are typically <1.0 µm in diameter and structurally simple: They have an external part: the glycocalyx followed by the cell wall and the cytoplasmic membrane, and some have a motile part which is the flagellum. Then there is the cytoplasm, containing the ribosomes, the nucleic acids that are confined in the nucleoid, and eventually there are inclusions inside the cytoplasm. They live singly, in pairs, chained or in clusters, in almost every habitat. The external structure of bacterial cells: 1. The glycocalyx (sweet cup): It is a gelatinous, sticky structure made of carbohydrates polysaccharides, polypeptides or both, which surrounds the outside of the cell. There are 2 types of glycocalyx: Capsule: composed of organized repeating units of chemicals firmly attached to the cell’s surface. The presence of a capsule is extremely important in terms of pathogenesis of infections, because it may prevent the recognition by the host. Slime layer: it is a water-soluble glycocalyx that forms a sticky layer that is loosely attached to cell’s surface. It has a major role in promoting the attachment of the microbe to surfaces, including our mucosae. Function: 1. Attachment to surfaces, in particular forming colonies/communities which are called biofilms: they are communities of different microbes living altogether and they use their glycocalyx to attach. 11.10.21 Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri 2. The glycocalyx, and in particular the capsule, may protect the microbes from desiccation, so microbes that have a capsule are more resistant in the environment. 3. It prevents the phagocytosis of microbes by protecting the microbe from being recognized by the phagocytes. For example: streptococcus pneumoniae and klebsiella pneumoniae both have a very thick capsule and are responsible for severe infections in humans; the reason for their capacity to cause such an infection is their capsule, which prevents the phagocytosis by neutrophils and macrophages. 2. The Flagellum-Flagella: Responsible for microbes’ movement and motility in the environment. This structure senses the wetness in the environment, and allows the microorganism to move. The flagellum is composed of: • • • A filament: hollow structure, 20nm in diameter, made of globular protein units called flagellin (our immune system perceives the presence of an invading microbe by recognizing this protein), which are deposed in a clockwise helix at the lengthening tip. There are different types of flagellins in different species. A hook: it is a curved protein structure, which associates the filament to the cell wall of the microbe. A basal body: protein made, anchors the filament and the hook to the cell wall and the cytoplasmic membrane, through integral proteins. The 2 major groups of microbes, gram positive and gram negative, differ in the type of cell wall and in the structure that anchors the filament to the cell wall. These differences in flagellum proteins allow classification in SEROVARS (different strains of the same species, which means that the serum of the host may discriminate these variances) Functions: • Rotation propels bacterium through environment • The flagella can move in both directions: counterclockwise or clockwise • Bacteria move in response to stimuli (taxis). These stimuli can be light (phototaxis) or chemicals (chemotaxis), and can be positive or negative. Receptors for light and chemicals on the cell surface send signals to the flagella regulating speed and direction of rotation. 11.10.21 Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri Bacteria move through: runs: occur when all of the flagella rotate counterclockwise and become bundled. and tumbles: occur when the flagella rotate clockwise, and become unbundled. The flagellum rotates 360°with 100,000 rpm, changing direction, which allows a speed of 60 cell lengths per second. Flagellar arrangement: The flagella could be arranged in deferent ways depend on the microbes: single polar flagellum Tuft of polar flagellum peritrichous flagella Axial filament: Peculiar flagellar arrangement, characteristic of the spirochetes (spiral-shaped bacteria). Located between the outer membrane and the cytoplasmic membrane, it is distributed along the length of the microbe. Its movement allows the microbe to penetrate tissues through corkscrew-like movements. These bacteria are especially important because they are responsible for 2 major diseases in humans:   Syphilis (by treponema pallidum) Lyme disease (Borrelia burgdorferi) 11.10.21 Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri 3. Fimbiae (fimbria) and pili (pilus): Fimbriae (fimbria): They are filaments present on the surface of some bacteria, and responsible for cell motility across a surface. They are the main mechanism of virulence, which is the capacity of the bacteria to enter the host and cause disease (they allow the microbe to adhere to the host’s mucosae). Structure: - They are much shorter and smaller than flagella. Rodlike shaped and sticky, with short extensions which adhere to one another or to substances. They are present in hundreds per cell e.g. Neisseria gonorrhoeae Functions: i. They are important for the adhesion to mucous membranes (mechanism of virulence) ii. Allow the movement across a surface through attachment and retraction. iii. Adhesion to a substrate (mucosae) forming BIOFILMS (slimy masses of microbes adhering to a substrate, they represent 99% of bacteria in nature). iv. Conduction of electrical signals among cells in a biofilm. Pili (pilus) or conjugation pilus: Pili are longer than a fimbria and shorter than a flagellum. Can be considered as a special type of fimbria. There are just one or few pili per cell. They are a very important structure, because they are used to transfer DNA from one cell to another, in a process called conjugation. Bacteria replicate in their lives so to transmit their genome to their progeny, but they are also able to form conjugation pili and allow some part of their nucleic acids to move to the neighboring cell (which is not necessarily of the same species). 11.10.21 Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri Biofilms: Organized, layered systems of bacteria and other microbes attached to a surface (e.g., dental plaque, catheters). They are responsible for diseases, drain clogging in patients with catheter (in the bladder/vein), and lung infections in cystic fibrosis patients. Some are useful in controlling industrial pollution. Biofilm bacteria communicate via chemical and electrical signals: organization and formation of three-dimensional structures. They also condition each other in terms of which gene to express, whether they are resistant or not to antimicrobial drugs, etc. Thus, bacteria in biofilms behave differently from individual bacteria: e.g. expression of flagella vs pili, or resistance to antibiotics. 4. The Cell Wall: Functions: It confers the structure and the shape of microbes. It can be: • Spherical (called cocci). Can be present as: single chains: streptococci clusters: staphylococci cuboidal packets: sarcinae • Rod shaped. Can be present as: single chains The cell wall also has an important role in protecting the microbe from osmotic forces, and is responsible for the attachment of the cells together. it. It is also the reason behind the resistance feature to antimicrobial drugs. Structure: The cell wall is composed of peptidoglycan, which is a polysaccharide composed of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). These blocks are linked together covalently forming long chains (glycans). The long chains are bound through short chains of 4 amino acids (tetrapeptide) or by other short chains of amino acids (peptido). 11.10.21 Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri This structure is so solid and resistant that it allows the microorganism to survive in harsh conditions in the environment. There are few bacteria, like Mycoplasma that, lack a cell wall. Gram-Positive Bacterial cell wall: The peptidoglycan layer is very thick, and contains teichoic acids al lipoteichoic acids (polysaccharides) that anchor the peptidoglycan to the cytoplasmic membrane. Negative charge. These acids are peculiar in their chemical characteristics; for example, in Mycobacterium tuberculosis we have mycolic acid, a waxy lipid, that protects the microbes from desiccation. Also, the metabolism to generate these waxy lipids is so energy consuming that the mycobacterium tuberculosis replicates very slowly. Therefore, because of the slow replication rate and the waxy acids, it is difficult to grow and treat these bacteria, so the treatment of patients with tuberculosis takes months instead of normal bacterial infection. The thick cell wall also cause a difficulty in staining this type of bacteria with waterbased dyes; therefore, it is common to use acid-fast stain (crystal violet dye: purple). Gram-Negative bacterial cell wall: The peptidoglycan layer is much smaller/thinner. Above the peptidoglycan layer there is an external layer called the outer bilayer membrane which is made of: - Inner leaflet: phospholipids and proteins - Outer leaflet: lipopolysaccharides (lipid A or endotoxin + sugar). The presence of the lipopolysaccharide is one of the features that discriminates gram negative from gram positive bacteria. It also contains porins to allow the transfer of molecules. We also have the periplasmic space is made of peptidoglycans + periplasm (gel containing water, nutrients, digestive enzymes and proteins). The presence of lipopolysaccharides is what really discriminates Gram+ from Grambacteria. 11.10.21 Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri Lipopolysaccharide (LPS): It has an active domain which is lipid A, and it is made of three portions: i. O Side chain (O antigens): glycan polymer which may differ between species or strains of the same species (our antibodies recognize these differences). Core domain: made of non-carbohydrate components (exp. phosphate), and an oligosaccharide component Lipid A: it is a phosphorylated glucosamine disaccharide that is bound to different number of fatty acids (number depends on the species) that anchor the LPS to the membrane. When a patient has an infection from a gramnegative bacterium, the problem is Lipid A, which is released when bacteria die or when they replicate. This molecule is able to recognize a specific receptor in the host called TLR4 (Toll Like Receptor 4), and this receptor is responsible for the inflammation developed after the infection (e.g. fever, vasodilation, inflammation, shock, blood clotting) ii. iii.  There are some bacteria without cell walls that are often mistaken for viruses due to their small size. These bacteria have other features of prokaryotic cells, such as ribosomes. 5. Bacterial Cytoplasmic Membrane: Structure: • • • • Phospholipids: form a phospholipid bilayer. Hydrophilic heads and hydrocarbon hydrophobic tails: bipolar nature. Proteins: integral proteins or peripheral proteins (they form receptors, carriers, enzymes and channels). Sterol-like molecules. Fluid mosaic model: proteins and lipids are free to flow laterally within a membrane. Functions: • Energy storage • Harvest light energy in photosynthetic bacteria • Naturally impermeable to most substances • Proteins allow substances to cross membrane 11.10.21 • • Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri Maintain concentration and electrical gradient Selectively permeable, which causes a differential concentration of ions inside and outside the cell, thus creating an electrical potential. There are proteins which generate this selective permeability of compounds, so there may be a passive process of transport (movement down the concentration or electrochemical gradients): Diffusion for small or lipid soluble chemicals (e.g., oxygen, CO2, alcohol, FAs) through the phospholipid bilayer Facilitated diffusion through nonspecific channel proteins or permeases. Osmosis: diffusion of water across a semipermeable membrane.  Effects of isotonic, hypertonic and hypotonic solution on cells: If the cell is in an isotonic solution where the ions concentration is the same inside as outside of the cell there, is a normal balanced flux of H2O inside and outside. The same effect is in cells with and without a wall. Instead, when the cell is in a hypertonic solution, the water will flow out of the cell, causing it to shrink. Finally, in a hypotonic solution the water will flow inside the cell, eventually causing it to burst. The process of shrinking and bursting however is counteracted by the cell wall, therefore microorganisms with a cell wall are more resistant in these environments. This is significant because some drugs used to alter the cell wall (like penicillin) lead to the death of bacteria due to osmotic pressure. In addition to the passive process there is an active process of transport (movement against concentration or electrochemical gradient, requires use of energy): Active transport through transmembrane permease proteins + ATP (gated channels or ports). Group translocation: the substance is chemically changed during transport (e.g. glucose, glucose 6 phosphate). Uniport: allows the passage of 1 substance at a time Antiport: allows the passage of two substances in opposite directions Symport: allows the passage of two substances in the same direction 11.10.21 Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri 6. Cytoplasm of Bacteria • Cytosol: - • • Inclusions: - Liquid portion of cytoplasm mostly water ions, carbohydrates, proteins, lipids and wastes contains cell's DNA in region called the nucleoid may include reserved deposits of chemicals lipids, starch, glycogen, compounds containing sulfur, nitrogen, phosphate, lipids polymers, gas vesicles…). We can use these deposits to diagnose which are the microbes involved in specific process. Endospores: - A structure generated only by two microorganisms: Bacillus and Clostridium. - This structure is a defensive strategy that the microorganisms use against hostile or unfavorable conditions (lack of nutrients, chemicals...etc.). Essentially, the bacterium becomes a vegetative cell without any metabolic activity, and it may remain in the hostile environment and stay in this vegetative state for decades. - It is not a reproductive structure - Sporulation occurs in 8-10 hour's - These endospores can be central, subterminal or terminal - Extremely resistant to drying, heat, chemicals and radiations. - Double membrane, spore coat, dipicolinic acid, calcium, DNAbinding proteins confer resistance. - A very serious concern, since the endospore-forming bacteria produce deadly toxins (such as tetanus, anthrax, and gangrene). 11.10.21 Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri Formation of Endospores: Sporulation 1. 2. 3. 4. 5. 6. 7. 8. DNA replication DNA aligns along the cell's long axis cytoplasmic membrane invagination to form forespore. cytoplasmic membrane grows and engulfs forespore within a second membrane. Vegetative cell's DNA disintegrates The cortex of peptidoglycan is deposited between the membranes, meanwhile dipicolinic acid and calcium ions accumulate within the center of the endospore. spore coat forms around the endospore and becomes more complex endospore matures: completion of spore coat and increase in resistance to heat and chemicals by unknown process. endospore is released from original cell, while the original cell dies. 7. Non-membranous Organelles: • Ribosomes: o Site of protein synthesis. o Size is expressed in svedbergs, determined by sedimentation rate. prokaryote ribosomes are 70S, eukaryote ribosomes are 80S. o bacterial 70S ribosomes are target for antimicrobial drugs. They are composed of two subunits: → 30S, polypeptides and one ribosomal rRNA. → 50S, polypeptides and 2 ribosomal rRNA. we can use this specificity since they are different from our ribosomes for antimicrobial drugs. • Cytoskeleton: few types of protein fibers. Involved in cell division, orientation and deposition of strands of NAGs and NAMs sugars, segregation of DNA and cell movement. 11.10.21 Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri Archea: Other group of microorganisms included in prokaryotes. They are not pathogenic (did not show to cause diseases) although they live in biofilms. They have: • Glycocalyces: archaea can be found associated to biofilms through glycocalyces; however, no archeon has been shown to be pathogenic. • Flagella/fimbriae: structure, composition, protein sequence, mode of rotation are different in Archaea and bacteria…. Analogous structures, not phylogenetically linked. • Cell wall: made of proteins and polysaccharides, not of peptidoglycan. • Cytoplasmic membrane: NOT made by phospholipid bilayer. • Cytoplasm: 70S ribosomes, cytoskeleton, circular DNA. Typical Eukaryotic cell: • • • • • The eukaryotic cell is more complex. It has a nucleus and nuclear membrane that separate the nucleic acids from the rest of the cell. Contains membrane-bound organelles Glycocalyx: o is anchored to cell membrane and helps anchoring cells to each other o protects the cell o important for cell-cell communication and recognition o absent in cells with cell wall Cell wall: o present in fungi, algae and plants o provides protection, shape and support against osmotic pressure o it is made of polysaccharides: i. in fungi it is cellulose, chitin and glucomannan. When there is a suspected fungal infection a lab test for glucomannan is requested, because there is no other cell in human body that produces glucomannan. ii. in algae, the cell wall is used to produce agar (for bacterial culture) 11.10.21 Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri • • Cytoplasmic membrane: o consists of phospholipids and proteins o it also contains steroid lipids that are involved in maintaining fluidity o they are able to move and to perform processes like: i. endocytosis: through pseudopods ii. pinocytosis: when the molecules are liquids iii. phagocytosis: when the molecules are solids iv. exocytosis: vesicles containing various substances fuse with the cytoplasmic membrane and the content is eliminated outside of the cell. Flagella: o eukaryotic cells may have flagella o they differ from the bacterial flagella because of their composition. In eukaryotes, flagella are within the cytoplasmic membrane, and are composed of a protein called tubulin, which is arranged to form microtubules (9+2). o eukaryotic flagella have no hook: the filaments are anchored to the cell by the basal body (9 triplets) o may be single/multiple; they are generally found at one pole of the cell o flagella do not rotate, but it undulate rhythmically • Cilia: o shorter and more numerous than flagella o coordinated beating propels the cells through their environment o also used to move substances past the surface of the cell • Other non-membranous organelles: o ribosomes: 80S=40S+60S it is difficult to use these ribosomes as a therapeutic target, since they are similar to humans. o cytoskeleton: consists of microtubules of tubulin, microfilaments of actin and intermediate filaments o Centrioles & Centrosome: are present only in some eukaryotic microorganisms, and the function is the same as in humans. 11.10.21 • Microbiology1 Sbobinator: Adan Abu Rashid Reviewer: Lucia Baglieri Membranous organelles: o nucleus: has a semi liquid part, the nucleoplasm, ≥ 1 nucleoli, chromatin, a nuclear envelope composed of two phospholipid bilayers, and nuclear pores o Lysosomes: contain catabolic and digestive enzymes. o ER and Golgi apparatus o Peroxisomes: from the ER, oxidase and catalase are used to degrade free radicals and hydrogen peroxide o Vacuoles, and vesicles: store and transfer chemicals within the cell. Used to perform endocytosis and phagocytosis. o Phagocytosis: bacterium internalized by phagocytosis → formation of a vacuole called phagosome→ fusion with vesicle coming from Golgi, containing digestive enzymes→ the microbe is killed by the enzymes → the remaining content is eliminated outside of the cell through exocytosis. There are microbes that developed mechanisms to prevent the fusion of lysosome with the phagosome, therefore the microorganism may remain alive in the vacuole, causing disease. o Mitochondria o Chloroplasts: light harvesting structure in photosynthetic eukaryotes, which produce ATP. Possess a phospholipid bilayer. The endosymbiotic theory: Eukaryotes originated from the fusion of small aerobic prokaryotes with larger anaerobic prokaryotes. The smaller prokaryotes became parasites that lost the ability to exist independently, while the larger cell became dependent on parasites for aerobic ATP production. This theory is not universally accepted.