L1 Bacterial Structure & Growth Classification PDF

Bacterial Structure & Growth + Classification Dr. Ardita Dewi Roslani Microbiology IMS  TOPIC LEARNING OUTCOMES TLO1: Describe the characteristics & properties of the bacterial cell which make them unique pathogens. TLO2: Discuss t...

Bacterial Structure & Growth + Classification Dr. Ardita Dewi Roslani Microbiology IMS  TOPIC LEARNING OUTCOMES TLO1: Describe the characteristics & properties of the bacterial cell which make them unique pathogens. TLO2: Discuss the structure and composition of medically- important bacterial cells. TLO3: Explain the nomenclature and classification of bacterial cells. TLO4: Discuss in brief the physical and nutritional requirements of bacteria. TLO5: Explain the terms related to pathogenesis of infection.  Bacteria Content: STRUCTURE OF BACTERIA: 1. Cytoplasmic Structures (Nucleoid, Inclusion Bodies, Volutin Granule) 2. Cell envelope 3. Cell membrane 4. Cell wall (Differences between Gram-positive & Gram-negative) 5. Glycocalyx, capsule & slime layer 6. External Structures (Flagella, Pili, Fimbriae, Endospores) CLASSIFICATION OF BACTERIA: I. Taxonomy of Bacteria (Identification, Classification, Nomenclature) II. Criteria for Identification (Growth on Media, Bacterial Microscopy, Nutrition, Biochemical & Immunological Tests, Genetic Diversity). TERMS RELATED TO PATHOGENESIS OF INFECTIONS Figure 1 Diagrammatic structure of a typical bacterial cell.  Structure of Bacteria Bacteria are single-celled prokaryotes; their DNA forming a long circular molecule not contained within a defined nucleus. Many are motile, using a unique pattern of flagella. A bacterial cell is surrounded by a complex cell wall and often a capsule. They reproduce by binary fission. 1. Cytoplasmic Structures of Bacterial Cells: a) The Nucleoid: Prokaryotes have no true nuclei; instead they package their DNA in a structure known as the nucleoid. The DNA is neutralized by magnesium ions, but histone-like proteins (HLP) exist in bacteria – bind DNA; regulatory function etc. Rapidly-growing bacteria have more nucleoids per cell than slowly- growing ones. A FIGURE 2 The nucleoid. A: Colour-enhanced transmission electron micrograph of Escherichia coli with the DNA shown in red. B: Chromosome released from a gently lysed cell of E. coli. Note how tightly packaged the DNA must be inside the bacterium.  Structure of Bacteria b) Inclusion Bodies: They are insoluble granules which bacteria often use to store energy and as a reservoir of structural building blocks. Inclusion bodies are bounded by a thin lipid membrane to separate them from the cytoplasm. c) Volutin Granules: Many bacteria accumulate large reserves of inorganic phosphate in the form of granules of polyphosphate. These granules are sources of phosphate for nucleic acid synthesis to support growth. These granules are sometimes termed metachromatic granules. They are characteristic features of corynebacteria.  Structure of Bacteria 2. Cell Envelope: Prokaryotic cells are surrounded by complex envelope outermost layers - differ in composition among the major groups. These structures protect the organisms from hostile environments, such as extreme osmolarity, harsh chemicals, and even antibiotics, maintains its shape and enable cell processes. The envelope consists of: a. The cytoplasmic (cell) membrane b. The cell wall +/- outer membrane (if present e.g. Gram-negatives)  Structure of Bacteria 3. Cell Membrane: The bacterial cell / cytoplasmic membrane, is a typical “unit membrane” composed of phospholipids and upward of 200 different kinds of proteins. The major functions of the cytoplasmic membrane are: (1) Selective permeability and transport of solutes; (2) Electron transport and oxidative phosphorylation in aerobic species; (3) Excretion of hydrolytic exoenzymes; (4) Bearing the enzymes and carrier molecules for biosynthesis of DNA; (5) Bearing the receptors of chemotactic and other transduction systems. FIGURE 3 The structure of a prokaryotic cytoplasmic membrane: a phospholipid bilayer.  Structure of Bacteria 4. Cell Wall: i. Peptidoglycan layer (in both Gram-positives & negatives): Bacterial cell wall owes its strength to a layer composed of a substance referred to as PEPTIDOGLYCAN. Peptidoglycan is a complex polymer consisting of three parts: a backbone of N-acetylglucosamine & N-acetylmuramic acid; identical tetrapeptide side chains attached to N-acetylmuramic acid; identical peptide cross-bridges. Bacteria are classified according to their response to the Gram-staining process of this cell wall. Gram-positive bacteria look purple under the microscope, and Gram-negative bacteria look red (pink). The Gram staining process involves 2 stains and 2 other reagents. STEPS FOR GRAM- STAINING Fixes TABLE 1 Comparison of Features of Gram-Positive & Gram-Negative Bacteria. Figure 3.1 (a) The Gram-positive cell wall has a thick layer of peptidoglycan and lipoteichoic acids that anchor the wall to the cytoplasmic membrane. Figure 3.1 (b) The Gram-negative cell wall has a thin layer of peptidoglycan and an outer membrane composed of lipopolysaccharide (LPS), phospholipids, and proteins.  Structure of Bacteria ii. Teichoic and teichuronic acids (Special components of Gram-positive cell walls): Most Gram-positive cell walls contain considerable amounts of teichoic and teichuronic acids, which may contain a variety of sugars. There are two types of teichoic acids: wall teichoic acid (WTA) and membrane lipoteichoic acids (LTA). iii. Polysaccharides: Hydrolysis of Gram-positive walls has yielded sugars such as mannose & arabinose; these sugars exist as subunits of polysaccharides in the cell wall. Some polysaccharides may serve as a functional replacement for wall teichoic acids.  Structure of Bacteria 5. OUTER MEMBRANE (Special components of Gram-negative cells) It is a bilayered structure; its inner leaflet composition resembles that of the cell membrane, and its outer leaflet contains a distinctive component, a lipopolysaccharide (LPS). As a result, the leaflets of this membrane are asymmetrical.  Structure of Bacteria a. Lipopolysaccharide (LPS): consists of a complex glycolipid, called lipid A. Each Gram-negative species contains a unique repeat unit (referred to as O antigen). LPS is extremely toxic - called the endotoxin of Gram- negative bacteria - it is released only when the cells are lysed. b. Lipoprotein: The most abundant protein of Gram-negative cells. It stabilizes the outer membrane and anchors it to peptidoglycan layer. c. The periplasmic space: The space between the inner and outer membranes, which contains the thin peptidoglycan layer and a gel-like solution of proteins. Figure 4 Lipopolysaccharide structure. A: The lipopolysaccharide from Salmonella. B: Molecular model of an Escherichia coli lipopolysaccharide.  Structure of Bacteria 6. Glycocalyx: Glycocalyx - the polysaccharide-containing material produced inside the cell and extruded onto the cell’s surface when growing in their natural environments. The glycocalyx plays a role in the adherence of bacteria to surfaces in their environment, forming BIOFILMS. Glycocalyx includes both slime layers + capsule (polysaccharide) Slime layer & Capsule polysaccharide: Capsule: thick; tightly-packed organised layer; tightly-associated with envelope; contributes to the invasiveness of pathogenic bacteria (virulence factor); protects from phagocytosis. Slime layer: thin; loosely or unorganised layer; loosely-associated with cell.  Structure of Bacteria 7. External Structures of Bacterial Cells: a) Flagella: Flagella are thread-like appendages composed of several thousand protein subunits called flagellin. The flagellin of different bacterial species differ in structure. They are highly antigenic (H antigens), and some of the immune responses to infection are directed against them. The flagellum is attached to the bacterial cell by a complex structure consisting of a hook & basal body. There are 4 flagellar arrangements: Monotrichous (single polar flagellum on one side) Amphitrichous (single polar flagellum on both sides) Lophotrichous (a tuft of multiple polar flagella) Peritrichous (flagella distributed over the entire cell).  Gram + Gram - FIGURE 5 Proximal structure of bacterial flagella. (a) Detail of flagellar structure of a Gram-positive cell. (b) Detail of flagellum of a Gram-negative bacterium.  Structure of Bacteria Bacteria move with alternating series of: TUMBLEs - clockwise flagellar rotations that produce movements where each flagellum rotates independently. RUNS - counterclockwise flagellar rotations that produce “bundled” flagella, leading to movements of cell in one direction Movement toward a favorable stimulus is positive taxis, whereas movement away from an unfavorable stimulus is negative taxis. Spirochetes (subgroup of bacteria) have endoflagella at both ends that spiral tightly around the cell. Rotation of endoflagella causes the axial filament to rotate around the cell, causing a “corkscrew” movement. FIGURE 6 Motion of a peritrichous bacterium. In peritrichous bacteria, runs occur when all of the flagella rotate counterclockwise and become bundled. Tumbles occur when the flagella rotate clockwise, become unbundled, and the cell spins randomly.  Structure of Bacteria b) Pili: They are surface appendages shorter than flagella and composed of protein subunits termed PILINS. There are TWO types of pili:  Ordinary pili: responsible for the adherence of bacteria to host cells;  Sex pili: responsible for the attachment of donor and recipient cells during the process of bacterial conjugation. The virulence of certain pathogenic bacteria depends on the production of these “colonization antigens” i.e. ordinary pili that provide the cells with adherent properties. These pili elicit the formation of host antibodies. *Antibodies against pili of one bacterial species will NOT prevent the attachment of another species.  Structure of Bacteria c) Fimbriae: Fimbriae are rod-like proteinaceous extensions. These sticky, bristle-like projections adhere to one another and to substances in the environment. There may be hundreds of fimbriae per cell. *e.g. Neisseria gonorrhoeae is a bacterium with fimbriae able to colonise the mucous membrane of the reproductive tract. Fimbriae also have an important function in biofilms (slimy masses or communities of microbes, enclosed in polymeric matrix, adhering to a substrate or foreign surface, via fimbriae). They act as electrical wires, conducting electrical signals among cells in a biofilm. FIGURE 7 Proteus vulgaris FIGURE 8 Two Salmonella cells has flagella and fimbriae. are connected by a pilus.  Structure of Bacteria d) Endospores: Bacillus spp. and Clostridium spp. are capable of forming endospores. They undergo a cycle of differentiation in response to environmental conditions: The process of ‘sporulation’, is triggered by near depletion of several nutrients (carbon, nitrogen, or phosphorous). Each cell forms a single internal spore - liberated when the mother cell undergoes autolysis. The spore is highly resistant to heat and chemical agents; when returned to favorable nutritional & environmental conditions, the spore germinates to produce a single vegetative cell. Vegetative cell Spore FIGURE 9 The formation of an endospore. The steps depicted occur over a period of 8–10 hours.  Classification of Bacteria I. Taxonomy of Bacteria: The following are interrelated areas of bacterial taxonomy: Identification: is the use of a classification scheme to (I) isolate and distinguish specific organisms among the mix of complex microbial flora, (II) verify the special properties of a culture in a clinical setting, and (III) isolate the causative agent of a disease. Classification: is the categorisation of organisms into taxonomic groups by using morphological, biochemical and genetic properties. Nomenclature: is the naming of an organism by an established group of scientific and medical professionals. Figure 10 How the structural and biological characteristics of bacteria can be used in classification, taking Gram-positive bacteria as an example.  Classification of Bacteria II. Criteria for Identification a) Growth on Media (Culture): Bacterial classification includes growth on different types of bacteriological culture media which can be classified into several types: e.g. enriched media such as blood agar, selective media such as bile salt agar, and differential media such as MacConkey agar. FIGURE 11 Bacterial colonies on a blood agar Differences in colony size, shape, and color indicate the presence of different species.  Classification of Bacteria b) Bacterial Microscopy: The Gram stain, together with visualization by light microscopy, has been among the most informative methods for classifying bacteria. This staining divides bacteria on the basis of fundamental differences in the structure of their cell walls (Gram negative or positive). c) Biochemical Tests: Many types i. The oxidase test is used to distinguish organisms on the basis of the presence or absence of a respiratory enzyme, e.g. Enterobacteriaceae [-] from other Gram-negative rods. ii. The catalase test is used to distinguish between the Gram-positive cocci (i.e. staphylococci [+] and streptococci [-]). Figure 12 The three basic shapes of bacterial cells.  Classification of Bacteria d) Nutritional Requirements: All bacteria obtain energy by oxidizing proteins & carbohydrates from their environment. There are TWO types of metabolism: 1. Aerobic metabolism is the complete utilization of an energy source. 2. Anaerobic metabolism is the incomplete utilization of an energy source. e) Immunological Tests: The word “sero” indicates the use of antibodies that react with specific bacterial cell surface structures such as LPS, flagella, or capsule (antigens). The terms “serotype,” “serogroups,” and “serovars” use the specificity of these antibodies to sub-divide bacterial strains. During an epidemic, it is important to identify a particular strain by these methods. OTHER IMPORTANT JARGONS/ TERMS RELATED TO THE PATHOGENESIS OF INFECTION Nonpathogen: o A microorganism that does not cause disease; may be part of the normal flora. Pathogen: o A microorganism capable of causing disease. Opportunistic pathogen: o A microorganism capable of causing disease only when the host's resistance is impaired (when the patient is "immunocompromised"). Carrier: o A person or animal with asymptomatic infection, that can be transmitted to another susceptible person or animal. Adherence (adhesion, attachment): o The process by which bacteria stick to the surfaces of host cells. o Once bacteria have entered the body, adherence is a major initial step in the infection process. Invasion: o The process whereby bacteria, viruses and fungi enter the host cells or tissues and spread in the body. Toxigenicity: o The ability of a microorganism to produce a toxin that contributes to the development of disease. Virulence: o The quantitative ability of a microorganism to cause disease. – measured relative to a standard (another microbe/host) o Virulent organisms cause disease when introduced into the host in small numbers. Virulence involves adherence, invasion and toxigenicity. Pathogenicity: o The ability of an infectious microorganism to cause disease. Infection: o Multiplication of an infectious microorganism within the body. However:  Multiplication of the bacteria that are part of the normal flora of the GIT, skin, etc. is generally not considered an infection.  Multiplication of pathogenic bacteria even if the person is asymptomatic, is deemed an infection. How Do Bacteria Cause Disease? Bacterial pathogens often have special structures or physiological characteristics that improve the chances of successful host invasion and infection. Virulence factors are structural or physiological characteristics that help organisms to cause infection and disease. Examples of Virulence factors include: 1) Adherence factors – e.g. pili 2) Bacterial exotoxins & endotoxins 3) Enzymes # Tissue-degrading enzymes – e.g. coagulase # IgA1 proteases 4) Antiphagocytic factors – e.g. capsule, surface antigens 5) Intracellular pathogenicity – ability to survive within phagocytes 6) Antigenic heterogeneity / genetic variation 7) Requirement for iron (essential nutrient for mircroorganisms; affects virulence) 8) Bacterial biofilms  References Carroll K.C., Morse S.A., Mietzner T., Miller S. (2016). Jawetz, Melnick & Adelberg’s Medical Microbiology (27th Edition). SECTION I: Fundamentals of Microbiology. (pp 1- 126). USA: McGraw Hill. Goering R.V., Dockrell H.M., Zuckerman M., Roitt I.M., Chiodini P.L. (2013). Mims' Medical Microbiology (5th Edition). SECTION 1: The Adversaries – Microbes. (pp 3-63). USA: Elsevier Saunders. Bauman R.W. (2015). Microbiology with Diseases by Body System (4th Edition). (pp 56- 94). USA: Pearson Education.