Bacterial Genetics, Replication & Antibacterials

Preface

• This book intends to be a comprehensive and up-to-date guide to medical microbiology and immunology.
• It is designed for undergraduate medical students and postgraduates preparing for higher degrees.
• The book covers the aspects of medical microbiology and immunology: Bacteriology, Virology, Mycology, and Immunology
• This is in four separate volumes in addition to a book encompassing laboratory diagnostic methods of infectious diseases and navigates the reader to this ever-expanding science.
• Volume I covers General Microbiology (Bacteriology, Virology, and Mycology).
• Volume II covers Systematic Bacteriology.
• Volume III covers Systematic Virology, Systematic Mycology, and some topics of clinical relevance.
• Volume IV covers Laboratory diagnostic methods of infectious diseases.
• Volume V covers Immunology.
• The primary objective was to supply the reader with a concise, updated source reflecting the progress in the field of microbiology and immunology.

Introduction to Microorganisms

• Microorganisms are microscopic organisms, only seen through a microscope.
• Microorganisms include algae, protozoa, fungi, bacteria, archaea, viruses, and prions.
• Algae, protozoa, and fungi are eukaryotic microorganisms, made from larger, more complex cells with DNA enclosed in a nuclear membrane, forming the nucleus.
• Bacteria and archaea are prokaryotic microorganisms, single-celled organisms without a membrane-bound nucleus.
• DNA exists as a long, folded thread suspended in cytoplasm called nucleoid, and lacks mitochondria and membrane-bound organelles.
• Viruses are the smallest infective agents, obligate intracellular parasites lacking metabolic machinery, depending on host cells for vital functions.
• Viruses contain nucleic acid (DNA or RNA) surrounded by a protein coat.
• Prions are infectious proteins without nucleic acid, implicated in causing various diseases.

Bacterial Structure

• Bacteria are found in air, water, and soil and in or on human bodies, animals and plants.
• Bacteria are differentiated into major categories, based on shape, size, arrangement, staining characteristics, and motility.
• Bacteria size is measured in µm.

Bacterial Shape and Arrangement

• Cocci (singular: coccus) describes spherical organisms arranged in pairs, clusters, or chains.
• Bacilli (singular: bacillus=stick) describes rod-shaped organisms that may occur singly, in pairs, or in chains.
• Spiral bacteria describes coiled organisms, e.g., spirochaetes that are flexible.

Staining Characteristics

• Simple stains: single dye like methylene blue, staining cells and structures with the same color to reveal characteristics of size, shape, and arrangement.
• Differential stains: using more than one dye, distinguish between different types of bacteria by giving them different colors.
• Gram stain divides bacteria into Gram-positive (violet-staining) and Gram-negative (red staining), and is the most important differential stain in clinical microbiology.

Motility

• Ability of bacteria to move independently.
• Some bacteria are non-motile; others are motile.
• Different modes of motility include darting, corkscrew or swarming motility and may help in the identification of an organism.

Bacterial Ultra-Structures

• All bacteria have a nucleoid, ribosomes, and a cytoplasmic membrane.
• Most have a cell wall; some are further enveloped by a capsule or slime layer.
• Some have cytoplasmic inclusions and appendages like flagella and pili, and subcellular structures are best revealed by electron microscopy.

Cytoplasm

• Contains a few morphologically distinct components.

Nucleoid

• Genetic information is in a single circular molecule of double-stranded DNA that constitutes the bacterial chromosome.

Plasmids

• Additional genetic information is contained on small circular extrachromosomal DNA molecules that can replicate independently.

Ribosomes

• Protein synthesis site, consisting of protein and RNA.
• Prokaryotic ribosomes have a sedimentation constant of 70S, smaller than the 80S ribosomes of eukaryotes.
• Difference makes bacterial ribosomes a selective target for antibiotic action.

Inclusion Granules

• Granules of nutrient materials, e.g., carbohydrates and lipids.

Cytoplasmic Membrane

• Limited externally by a thin elastic cytoplasmic membrane, a phospholipid protein bilayer similar to eukaryotic cells but lacks sterols.
• Selective transport: Molecules move across via simple diffusion, facilitated diffusion, and active transport.
• Secretion of extracellular enzymes: Hydrolytic enzymes digest large food molecules into subunits, and enzymes destroy harmful chemicals like antibiotics.
• Respiration: Respiratory enzymes are located in the cytoplasmic membrane, a functional analogue of the mitochondria in eukaryotes.
• Cell wall biosynthesis: Site of cell wall biosynthesis enzymes.
• Reproduction: A specific protein attaches to the DNA and separates the duplicated chromosomes, with a septum forming to separate cytoplasm of daughter cells.

Cell Wall

• Bacterial cell wall surrounds the cytoplasmic membrane, and is strong, relatively rigid, and has some elasticity.
• Its strength is primarily due to peptidoglycan, a complex polymer of carbohydrates and amino acids
• Additional components divide bacteria into Gram-positive and Gram-negative.

Gram-positive Cell Wall

• Composed of:
  • Peptidoglycan: Many sheets, comprising up to 50% of the cell wall material, allow chemicals to pass through.
  • Teichoic acids: Fibers protrude outside the peptidoglycan and along with cell wall-associated proteins are the major surface antigens.

Gram-negative Cell Wall

• Composed of:
  • Peptidoglycan: Much thinner, composed of one or two sheets comprising 5-10% of the cell wall material.
  • Outer membrane: A phospholipid protein bilayer outside the peptidoglycan, carrying lipopolysaccharide (LPS) molecules, consisting of Lipid A (endotoxin) and polysaccharides (major surface antigens/somatic or O antigen).

Functions of the Cell Wall

• Maintains the characteristic shape of the bacterium.
• Supports the weak cytoplasmic membrane against the high internal osmotic pressure of the protoplasm.
• Plays an important role in cell division.
• It is responsible for the staining affinity of the organism.

Wall Deficient Variants

• Mycoplasma: The only group of bacteria that exists naturally without a cell wall.
• Mycoplasmas do not assume a defined recognizable shape, because they are naturally resistant to cell wall inhibitors, such as penicillins and cephalosporins.
• L-Forms: Wall-defective or wall-deficient bacteria.
  • "L" stands for Lister Institute in London, where they were first discovered.
  • They can develop from cells that normally possess a cell wall, when exposed to hydrolysis by lysozyme or by blocking peptidoglycan biosynthesis with antibiotics, such as penicillin, provided that they are present in an isotonic medium.
  • Some L-forms resynthesize their walls once the inducing stimulus is removed, resulting relapses; others permanently lose the capacity to produce a cell wall.
  • L-forms may survive therapy with cell wall inhibitors.

Glycocalyx

• Many bacteria secrete extracellular polymers outside their cell walls called glycocalyx, composed of polysaccharides and sometimes protein.
• Glycocalyx forms an additional layer that may come in one of two forms:
  • Slime Layer: Thin glycocalyx layer that is loosely bound to the cell wall, and involved in attachment of bacteria to other cells/inanimate surfaces to form biofilms, defined as aggregates of microorganisms adhering to each other on a surface embedded within a matrix of extracellular polysaccharide. Importance of biofilms: Protect bacteria from host defenses (e.g., antibodies), resist penetration of antibiotics and detergents, and facilitate the exchange of antibiotic resistance genes.

Capsule

• Thick glycocalyx layer that is firmly attached to the cell wall.
• Importance: Protect bacteria from phagocytic cells & antibacterial agents (e.g., bacteriophages) and provide bacterial adhesion to target surfaces for infection.
• Demonstrated using a negative staining technique (the bacterial cells and the background are stained, capsule has a clear halo).

Appendages

• Several structures project through the cell wall of bacteria to form surface appendages, the most important of which are flagella and pili.

Flagella

• Hair-like appendages, too small to be detected by light microscope, and demonstrated clearly with the electron microscope.
• Location/number of flagella on a cell varies according to bacterial species.
• Organisms may be monotrichous (single polar flagellum), lophotrichous (multiple polar flagella) or peritrichous (flagella distributed over the entire cell surface).
• Flagella consist of a protein called flagellin, which differs in different bacterial species.
• Flagellins constitute the H antigens and motile bacteria migrate towards regions with a higher concentration of nutrients and away from harmful substances.

Pili or Fimbriae

• Protein tubes that extend from the cells, shorter/thinner than flagella, and composed of structural protein subunits termed pilins.
• Adherence: function of the short pili (fimbriae) that occur in great numbers around the cell, enabling to attach to the surfaces, thus contributing to infection.
• Conjugation: A special long pilus called the sex pilus is involved in the transfer of DNA between bacteria, a process known as conjugation.

Bacterial Spores (Endospores)

• Some bacteria develop a highly resistant resting phase called endospore, that does not grow or reproduce, and exhibits absolute dormancy (sporulation).
• Triggered by unfavorable environmental conditions (depletion of nutrients, accumulation of metabolites, changes in growth requirements, etc.)
• Cytoplasmic membrane invaginates, enclosing a section of the cytoplasm that contains the bacterial chromosome, some ribosomes, and other cytoplasmic materials, acquiring a thick cortex and thin but tough outer spore coat.
• Spores are more resistant to disinfectants, drying, and heating; moist heat at 121°C for 10-20 minutes is needed to kill spores while 60°C suffices to kill vegetative forms.
• Respond quickly to favorable environmental conditions returning to the vegetative state within 15 minutes, absorb water and swell, the protective coat disintegrates, and a single vegetative cell emerges.
• Staining: the spore remains uncolored and seen as a clear area within the stained cell, or stained using special procedures.
• Position: Spores may be central, terminal, or subterminal.
• Shape: Spores may be oval or rounded.
• Size: Spores may be large (bulging) or small (non-bulging).
• Position and shape are characteristic and help in microscopic identification.

Bacterial Growth

• Bacteria reproduce asexually by binary fission, where a single cell divides to form two genetically identical daughter cells.

Steps of Binary Fission

• The cell grows in size, usually elongating.
• The two strands of the bacterial chromosome separate, and each strand acts as a template for the formation of a new complementary strand
• Results in the formation of two copies of double-stranded DNA molecules (one "old" and one "new").
• The two copies become attached to the two opposite ends of the cytoplasmic membrane.
• Protoplasm divides into two equal parts by the growth of a transverse septum from the cytoplasmic membrane and cell wall, giving rise to two identical daughter cells.
• Generation time (doubling time) is the time required for a population of bacteria to double in number (as short as 13 minutes, up to 24 hours).

Environmental Factors Affecting Bacterial Growth

• Nutrients: Classified as autotrophs (synthesize complex organic substances from simple inorganic materials, e.g. CO2) or heterotrophs (require organic sources for carbon, cannot synthesize complex organic substances from simple inorganic sources).
• Oxygen: Strict or obligate aerobes require oxygen for growth, strict or obligate anaerobes require complete absence of oxygen forming toxic molecules.
• Facultative anaerobes grow better in presence of oxygen; micro-aerophilic organisms require reduced oxygen level; aerotolerant anaerobes have an anaerobic pattern of metabolism but tolerate oxygen.
• Carbon dioxide (CO2): Minute amount of CO2 in air is sufficient for most bacteria, certain species require higher concentrations (5-10%) of CO2 for growth.
• Temperature: Most organisms grow within 20-40°C, with an optimum of 37°C.
• Some can grow at refrigeration temperature, while others grow best at high temperatures.
• Hydrogen ion concentration (pH): Optimal pH close to human body (pH 7.2), but some grow better alkaline (8-9) or acidic (4 or less).

Bacterial Viruses (Bacteriophages)

• Bacteriophages/phages are viruses that infect bacteria, where the bacterial cell acts as a host.
• Morphology includes a head containing a nucleic acid core (DNA, or rarely RNA) surrounded by a protein coat (capsid), and a tail consisting of a hollow core surrounded by a contractile sheath ending in a base plate and tail fibres.

Replication of Bacteriophages

• Two cycles for phage replication.

Lytic (vegetative) cycle

• So-called because it ends in lysis of the bacterial host cell and the release of the newly formed phages. Adsorption: The phage attaches by its tail to specific receptors on the bacterial cell, thus specificity determines the susceptibility to different phages. Penetration: The tail sheath contracts; nucleic acid is injected into the cell, the empty head and the tail are left outside. Eclipse phase: No phage components are detected inside the cell through the time of viral nucleic acid directs the host cell metabolism to synthesize the enzymes and proteins required for phage synthesis. Replication: Hundreds of phage components including nucleic acids, capsids and tails are synthesized. Assembly: The phage components combine to form complete phage particles which mature into infectious phages. Release: The bacterial cell bursts, liberating many phage particles to infect new cells.

Temperate (lysogenic) cycle

• The phage (temperate phage) does not replicate and lyse the bacteria and phage DNA integrates within the bacterial chromosome and divides with it to pass daughter cells.
• The integrated phage genome (prophage) and the bacteria carrying it (lysogenic bacteria).
• Lysogenic bacteria are immune to infection by another phage and acquire new properties (e.g., toxin production/resistance to antibiotics) called lysogenic conversion/phage conversion and lost when phage is lost.
• The prophage is carried inside the bacterial cell indefinitely passing to daughter cells or induced to detach from the bacterial chromosome and start lytic cycle spontaneously, or achieved by an inducer (e.g. U.V. light).

Transduction

• The transfer of DNA from one bacterial cell to another by means of bacteriophage.

Generalized Transduction

• During lytic phage cycle, the bacterial DNA is fragmented; any fragment (whether chromosomal or plasmid) is accidentally incorporated into phage head in place of phage DNA.
• The phage transfer then transfers incorporated bacterial DNA into another bacterial host.

Specialized Transduction

• Takes place with a prophage in a lysogenized cell is induced to detach from the chromosome to start the lytic cycle, with it carrying an adjacent piece of chromosomal DNA (in -addition to phage DNA) and transferring to another bacterial cell.

Bacterial Genetics

• Bacterial genome, the total set of genes present inside the bacterial cell, comprises the bacterial chromosome and genes for bacterial growth.
• Additional genes may be carried on plasmids, bacteriophage DNA (prophage), and transposable genetic elements.

Bacterial Chromosome

• Single, circular, supercoiled, double-stranded DNA molecule.
• Lacking a nuclear membrane, and DNA is concentrated in a region in the cytoplasm called nucleoid.
• Follows the same rules of gene expression and protein synthesis as higher organisms and replicates as previously described.

Plasmids

• Extrachromosomal, circular, double-stranded DNA molecules dispersed in the cytoplasm.
• Smaller than bacterial chromosome, these genes encode properties beneficial but not essential therefore are considered dispensable.
• They replicate independently and multiple copies of the same plasmid may exist.
• Classified according to functions: Fertility (F) plasmids carries fertility (F) factors coding for a sex pilus mediate gene transfer during conjugation and conjugative plasmids.
• Resistance (R) plasmids carry genes for R-factors to antimicrobial drugs and are transferable. Virulence plasmids encode exotoxins, adhesins or invasion factors.
• A single cell can contain several plasmids; F plasmids are major carriers of antibiotic resistance genes in Gram-negative bacteria.

Bacteriophage DNA

• integrated in the chromosome of a lysogenic bacterial cell (i.e. the prophage) is considered a genome part.
• Bacterial variations are changes in bacterial characters, which can be phenotypic or genotypic.

Phenotypic

• Responds to environment changes with no genetic change as in the formation of L-forms when bacteria are exposed to lysozyme, and reversible (transient)/non-heritable to daughter cells.

Genotypic

• Changes in the genetic constitution through mutation/gene transfer as in insertion sequences and transposons and irreversible (permanent)/heritable to daughter cells.

Mutation

• Permanent, heritable change in the nucleotide base sequence of a DNA molecule that occurs spontaneously or induced by radiation/chemical agents. Induced mutations can be used to manipulate viral genomes for vaccine production/gene therapy.

Gene Transfer

• There are 3 methods for gene transfer among bacteria.
  • Transformation is the uptake of naked DNA from the environment by a bacterial cell occurs when dying bacteria release DNA and causes "transformation" of the recipient cell
  • Transduction is the transfer of DNA by a bacteriophage.
  • Conjugation is one bacterium transfer of genetic material to another through direct cell-to-cell contact by donors (F+) with possession of fertility (F) and recipients (F-) lacking the F factor.
  • The F factor carries genes for the synthesis of the sex pilus which then acts as a conjugation tube, the donor and recipient separate and copies a strand by DNA replication becoming F+.
  • Conjugation more frequently observes mechanism of DNA transfer among bacteria.

Antibacterial Agents (Antibiotics)

• Chemical substances that kill or inhibit bacterial cells, including antiseptics, antibacterial soaps, chemical disinfectants, and antibiotics used in medicine.
• Probiotics are live, non-pathogenic bacteria effective in the treatment/prevention of disease by excluding the pathogen or enhancing the immune response.
• Antibiotics are antibacterial substances produced by microorganisms (e.g. Streptomyces, Penicillium) or synthetically.
• Bacteriostatic: Antibiotic inhibits multiplication, which resumes upon removal.
• Bactericidal: Antibiotic kills bacteria, no resumption of multiplication.
• Selective toxicity is the antimicrobial agent's ability to harm a pathogen without harming the host.
• Spectrum is range of microorganisms affected by a certain antibiotic: broad if they kill or Gram-positive/negative, narrow if mainly effective against one.

Mechanisms of Action of Antibiotics

• Inhibition of bacterial cell wall synthesis: Bactericidal with minimal toxicity (drugs inhibit last steps of peptidoglycan biosynthesis, e.g., ß-lactam antibiotics binding to penicillin-binding proteins).
• Drugs inhibit early steps in peptidoglycan biosynthesis inside the cytoplasmic membrane, e.g, glycopeptides, and can be used against ß-lactam-resistant staphylococci.
• Interference with cell membrane function: Disrupt cytoplasmic membrane function (e.g., polymyxins) and are microbicidal and highly toxic.
• Inhibition of bacterial protein synthesis: Target is selective for bacterial ribosomes 70S which differ from mammalian ribosomes 80S, with agents acting on the 30S ribosomal subunit, e.g., tetracycline, and 50S ribosomal subunit, e.g., macrolides
• Inhibition of bacterial nucleic acid synthesis occurs by preventing RNA synthesis inhibiting RNA polymerase e.g. in some bacteria or preventing DNA synthesis blocking e.g., DNA gyrase.
• Inhibition of bacterial metabolic pathways: act as antimetabolites blocking bacterial biosynthesis of folic acid (examples are sulfonamides and trimethoprim).
• Microorganisms vary in susceptibility to chemotherapeutic agents, determined by testing in vitro .

Empiric Antibiotic Therapy

• "best guess" directed against likely causing infection
• In seriously ill patients, but after collecting specimens for culture, or in closed lesions that have no available sample.

Combined Antibiotic Therapy

• One drug per infections usually necessary due to conditions.
• Reasons include to treat seriously suspected infections, delay emergence, or increase synergistic action to kill and treat mixed infections.

Antimicrobial Chemoprophylaxis

• An effective antimicrobial agent that prevents vs that treats in an infection to prevent development including, long acting penicillin (or erythromycin) and rifampicin to close contacts.
• Complications of antibiotic therapy can include that are dose dependent or non-dose dependent, allergies (may cause uticaria, or serum sickness, superinfection, or resistance
• antibiotic resistance is a global problem with the increasing frequency in the lack of need for resistance genes can caused by gram negative large molecule.

Acquired resistance

• Mutations due to selective antibiotic and horizontal or lateral transfer of plasmid.
• Decreasing reduce influx by blocking entrance, reduce to allow high intracellular.
• Also with modify beta that can't be destroyed or alter the and high concentrations may be needed

Bacterial Pathogenesis

• Is a process by which the organism relationship with the host, most microbial end without manifestations as clinical disease terms silent or infections in host.
• classified among in soil or water, and commensals pathogens that do not immunocompromised Patients, or when they location.

Stages of the Infectious Process

• of Infection which may be man, animal or Transplacental e.g., skin multiplication within which damage factors e.g., blood, urine factors such gene carries a species the outcome relationship to the pathogenecy
• factors are often

Virulence factors of Bacteria

• Structures e.g., that capsule a Toxin disease strains lacking they adhesion prevent, and are often and the tissue causing spread cell walls for the organism as capsule and for or as enzymes which it toxin or
• the endotoxin

Clinical classification

• The outer of the skin deeper so implantation in organ

Antimicrobial Drug

• the selective

Explore bacterial genetics and their replication process. Discuss the distinctions between eukaryotes and prokaryotes and the impact of bacterial genetics and pathogenesis on antibacterial agent development.