Lecture 1 - Bacterial Classification, Growth and Pathogenesis PDF

Microbial World and You; Bacterial Clasṁmsification, Structure, Growth and Pathogenesis Natia Tamarashvili, PhD What is Microbiology Microbiology – study of microorganisms (simple forms of life visible only with a microscope) Microorganisms may be Normal flora Pathogenic Microbes make the Universe 3...

Microbial World and You; Bacterial Clasṁmsification, Structure, Growth and Pathogenesis Natia Tamarashvili, PhD What is Microbiology Microbiology – study of microorganisms (simple forms of life visible only with a microscope) Microorganisms may be Normal flora Pathogenic Microbes make the Universe 30 There are > 5 x 10 types Microbes in the world Humans have intimate relation with Microbes > 90% of the cells in our Body are Microbes 5 Impact of pathogens Nearly 2,000 different microbes cause diseases 10 B infections/year worldwide 13 M deaths from infections/year worldwide Microorganisms Nomenclature Non-cellular organism Virus Prokaryotes Bacterium Eukaryotes Others Fungi Prions Viroid Structure The eukaryotic cell has: ▪ a true nucleus with multiple chromosomes surrounded by a nuclear membrane ▪ and uses a mitotic apparatus to ensure equal allocation of the chromosomes to progeny cells. The nucleoid of a prokaryotic cell consists of ▪ a single circular molecule of loosely organized DNA, ▪ lacking a nuclear membrane and mitotic apparatus Viruses have an inner core of genetic material (either DNA or RNA) but no cytoplasm, ▪ they depend on host cells to provide the machinery for protein synthesis and energy generation. Eukaryotes Vs. Prokaryotes Eukaryotic cells contain organelles, such as mitochondria and lysosomes, and larger (80S) ribosomes, whereas prokaryotes contain no organelles and smaller (70S) ribosomes. Most prokaryotes have a rigid external cell wall that contains peptidoglycan, a polymer of amino acids and sugars, as its unique structural component. Eukaryotes on the other hand, do not contain peptidoglycan. Either they are bound by a flexible cell membrane, or, in the case of fungi, they have a rigid cell wall with chitin, a homopolymer of N-acetylglucosamine, typically forming the framework. The eukaryotic cell membrane contains sterols, whereas no prokaryote, except the wall-less Mycoplasma, has sterols in its membranes. Nature of the nucleic acid The eukaryotic cells contain both DNA and RNA, viruses contain either DNA or RNA but not both. Method of replication Prokaryotic cells (e.g., bacteria) replicate by binary fission whereas eukaryotic cells replicate by mitosis. In contrast, viruses disassemble, produce many copies of their nucleic acid and protein, and then reassemble into multiple progeny viruses. Reproduction Prokaryotic cell division is binary fission. Single DNA molecule that first replicates. Attaches each copy to a different part of the cell membrane. Cell begins to pull apart. There are then two cells of identical genetic composition. Characteristics of microbes Structure of Bacterial Cells Shape & Size of Bacteria Structure of Bacteria Cell Wall Cytoplasmic Membrane Cytoplasm Structures Outside the Cell Wall Bacterial Spores Shape & Size of Bacteria Bacteria are classified by shape into three basic groups: cocci, bacilli, and spirochetes The cocci are round, the bacilli are rods, and the spirochetes are spiral-shaped. Some bacteria are variable in shape and are said to be pleomorphic (many-shaped). The shape of a bacterium is determined by its rigid cell wall. The microscopic appearance of a bacterium is one of the most important criteria used in its identification. The arrangement of bacteria certain cocci occur in pairs (diplococci) some in chains (streptococci), and others in grapelike clusters (staphylococci). These arrangements are determined by the orientation and degree of attachment of the bacteria at the time of cell division. SHAPE & SIZE OF BACTERIA 22 Sizes of representative bacteria, viruses, yeasts, protozoa, and human red cells STRUCTURE OF BACTERIA Cell wall Peptidoglycan N acetyl glucosamine & N acetyl Muramic acid Protect the cell from osmotic changes Rigidity Multilayered in Gram positive Mono to bi layered in Gram negative Peptidoglycan N acetyl glucosamine (NAG) & N acetyl muramic acid (NAM). Peptidoglycan Because peptidoglycan is present in bacteria but not in human cells, it is a good target for antibacterial drugs. Several of these drugs, such as penicillins, cephalosporins, and vancomycin, inhibit the synthesis of peptidoglycan by inhibiting the transpeptidase that makes the cross-links between the two adjacent tetrapeptides. Cell Walls of Gram-Positive and Gram-Negative Bacteria Cell Walls of Gram-Positive and Gram-Negative Bacteria GRAM STAIN Gram stain is the most important staining procedure. Gram positive bacteria stain purple, whereas gram-negative bacteria stain pink. This difference is due to the ability of gram-positive bacteria to retain the crystal violet–iodine complex in the presence of a lipid solvent, usually acetone–alcohol. Gram-negative bacteria, because they have an outer lipid-containing membrane and thin peptidoglycan, lose the purple dye when treated with acetone–alcohol. They become colorless and then stain pink when exposed to a red dye such as safranin. The Gram stain is useful in two ways: (1) In the identification of many bacteria. (2)In influencing the choice of antibiotic because, in general, grampositive bacteria are more susceptible to penicillin G than are gram- negative bacteria. The Gram stain Note that it takes approximately 100,000 bacteria/mL to see 1 bacterium per microscopic field using the oil immersion (100×) lens. So the sensitivity of the Gram stain procedure is low. This explains why a patient’s blood is rarely stained immediately but rather is incubated in blood cultures overnight to allow the bacteria to multiply. One important exception to this is meningococcemia in which very high concentrations of Neisseria meningitidis can occur in the blood. Medically Important Bacteria That Cannot Be Seen in the Gram Stain Lipopolysaccharide The LPS is composed of three distinct units (1) A phospholipid called lipid A, which is responsible for the toxic effects. (2) A core polysaccharide of five sugars linked to lipid A. (3) An outer polysaccharide consisting of up to 25 repeating units of three to five sugars - O antigen of several gram negative bacteria. Lipopolysaccharide The lipopolysaccharide (LPS) of the outer membrane of the cell wall of gram negative bacteria is endotoxin. It is responsible for many of the features of disease, such as fever and shock (especially hypotension), caused by these organisms. It is called endotoxin because it is an integral part of the cell wall, in contrast to exotoxins, which are actively secreted from the bacteria. The constellation of symptoms caused by the endotoxin of one gramnegative bacteria is similar to another, but the severity of the symptoms can differ greatly. In contrast, the symptoms caused by exotoxins of different bacteria are usually quite different Special components of Gram positive cell wall Teichoic Acid Polymers containing ribitolor glycerol residues there are 2 types of teichoic acids a) wall teichoic acid b) membrane teichoic acid (lipoteichoic acid) linked to membrane glycolipid The medical importance of teichoic acids lies in their ability to induce septic shock when caused by certain gram-positive bacteria; that is, they activate the same pathways as does endotoxin (LPS) in gram- negative bacteria. Teichoic acids also mediate the attachment of staphylococci to mucosal cells. Gram-negative bacteria do not have teichoic acids The periplasmic space The space between the inner and outer membranes, called the periplasmic space Contains: the peptidoglycan layer and a gel-like solution of proteins. The periplasmic space Cytoplasmic Membrane Lipid bi layer Selective permeability Site of ATP production Cytoplasm The cytoplasm has two distinct areas when seen in the electron microscope: (1) An amorphous matrix that contains ribosomes, nutrient granules, metabolites, and plasmids. (2) An inner, nucleoid region composed of DNA. Structures inside Cytoplasm Ribosomes Granules Nucleoid Plasmids Transposons The cytoplasm of a prokaryote contains numerous 70s ribosomes; ribosomes consist of rRNA and protein. Protein synthesis occurs at ribosomes; it can be inhibited by certain antibiotics. The difference between prokaryotic (70s) and eukaryotic (80s) ribosomes allows antibiotics to selectively target the prokaryotic ribosomes while sparing eukaryotic ribosomes. Dr. Dalia Mohsen Associate prof. of microbiology Nucleoid The nucleoid is the area of the cytoplasm in which DNA is located. The DNA of prokaryotes is a single, circular molecule that contains about 2000 genes. One major difference between bacterial DNA and eukaryotic DNA is that bacterial DNA has no introns, whereas eukaryotic DNA does. Plasmids Plasmids are extrachromosomal, double-stranded, circular DNA molecules that are capable of replicating independently of the bacterial chromosome (1) Transmissible plasmids can be transferred from cell to cell by conjugation. (2) Nontransmissible plasmids do not contain the transfer genes; Plasmids carry the genes for the following functions and structures of medical importance: (1) Antibiotic resistance, which is mediated by a variety of enzymes. (2) Resistance to heavy metals, such as mercury, the active component of some antiseptics and silver, which is mediated by a reductase enzyme. (3) Resistance to ultraviolet light, which is mediated by DNA repair enzymes. (4) Pili (fimbriae), which mediate the adherence of bacteria to epithelial cells. (5) Exotoxins, including several enterotoxins Structures Outside the Cell Wall Capsule - a gelatinous layer covering the entire bacterium. It is composed of polysaccharide 1) It is a determinant of virulence of many bacteria since it limits the ability of phagocytes to engulf the bacteria 2) Specific identification of an organism can be made by using antiserum against the capsular polysaccharide. 3) Capsular polysaccharides are used as the antigens in certain vaccines. 4) The capsule may play a role in the adherence of bacteria to human tissues. Flagella Long (3 to 12 µm), filamentous surface appendages Organs of locomotion Chemically, composed of proteins called flagellins The number and distribution of flagella on the bacterial surface are characteristic for a given species - hence are useful in identifying and classifying bacteria Flagella may serve as antigenic determinants (e.g. the H antigens of Gram-negative enteric bacteria) Pili (Fimbriae) Pili are hair like filaments that extend from the cell surface. They are found mainly on gram-negative organisms. Pili have two important roles: (1) They mediate the attachment of bacteria to specific receptors on the human cell surface, which is a necessary step in the initiation of infection for some organisms. (2) A specialized kind of pilus, the sex pilus, forms the attachment between the male (donor) and the female (recipient) bacteria during conjugation. Glycocalyx (Slime Layer) The glycocalyx is a polysaccharide “slime layer” coating that is secreted by many bacteria. It covers surfaces like a film and allows the bacteria to adhere firmly to various structures (e.g., skin, heart valves, prosthetic joints, and catheters). The glycocalyx is an important component of biofilms. Spores Additional Organelles Highly resistant resting stages formed during adverse environment (depletion of nutrients). Formed inside the parent cell, hence called Endospores. Very resistant to heat, radiation and drying and can remain dormant for hundreds of years. Formed by bacteria like Clostridia, bacillus. Keratin like coat is responsible for the remarkable resistance of the spore to heat, dehydration, radiation, and chemicals. Identification of Bacteria Morphology Biochemical properties Bacterial Growth Physical Requirements – Temperature Psychrophiles Mesophiles Thermophiles pH Osmotic Pressure Acidophiles- Grow between pH 0 and 5.5. Examples: Ferroplasma, Thiobacillus Sulfolobus acidocaldarius, etc. thioxidans, Alkalophiles- Grow between pH range of 7.5 to 14. Examples: Thermococcus alcaliphilus, etc. Neutrophiles- Grow between pH 5.5 to 8.0 Examples:Lactobacillus acidophillus, Pseudomonas aerunginosa, etc. Bacteria prefer media of pH near neutrality, cannot tolerate pH values much below 4-5. E. coli, and usually The Requirements for Growth PHYSICAL REQUIREMENTS Temperature pH Oxygen Hydrostatic Pressure Osmotic pressure CHEMICAL REQUIREMENTS (NUTRITIONAL FACTORS) Carbon Nitrogen, sulfur, and phosphorous Trace elements (iron, copper, zinc) Oxygen Organic growth factor Bacterial Growth Chemical Requirements Carbon Oxygen Nitrogen Sulfur Phosphorus Obligate (strict) vs. facultative “Obligate” (or “strict”) means that a given condition is required for growth. “Facultative” means that the organism can grow under the condition, but doesn’t require it. The term “facultative” is often applied to suboptimal conditions. For example, an obligate thermophile requires elevated temperatures for growth, while a facultative thermophile may grow in either elevated temperatures or lower temperatures. Binary Fission Binary Fission is a type of Asexual reproduction in single celled organisms Bacterial Doubling Time Escherichia coli Mycobacterium tuberculosis Mycobacterium leprae 20 minutes 18 hours 14 days The doubling time varies not only with the species, but also with the amount of nutrients, the temperature, the pH, and other environmental factors. Stages of Bacterial Growth Lag Log (logarithmic) Stationary Death Lag phase The first is the lag phase, during which vigorous metabolic activity occurs but cells do not divide. This can last for a few minutes up to many hours. An initial stationary phase represents the time from the moment of seeding the bacteria on the nutrient medium. bacteria absorb nutrients, synthesize enzymes and prepare for cell division. Logarithmic growth phase Phase of exponential (logarithmic) growth which is characterized by a maximal division rate and decrease in cell size. The log phase is always brief, unless the rapidly dividing culture is maintained by constant addition of nutrients and frequent removal of waste products β-Lactam drugs, such as penicillin, act during this phase because the drugs are effective when cells are making peptidoglycan (i.e., when they are dividing). Logarithmic or Exponential Growth The population of bacterial cells divide at a constant rate so that the total number of cells doubles with each division. Because one cell gives rise to two progeny cells, bacteria are said to undergo exponential growth (logarithmic growth). Bacteria Undergo Exponenti al Growth Bacterial Population growth curve A population growth curve for any particular species of bacteria may be determined by: ▪ growing a pure culture of the organism ▪ in a liquid medium ▪ at a constant temperature. Stationary phase The stationary phase occurs when the nutrients in the liquid medium are used up and the concentration of toxic waste products from the metabolizing bacteria build up, the rate of division slows, such that the number of bacteria that are dividing equals the number that are dying. Death phase The final phase is the death phase, which is marked by a decline in the number of viable bacteria. The mas overcrowding occurs the concentration of toxic waste products continues to increase and the nutrient supply decreases. Microorganisms die at a rapid rate. Types of Pathogens A microorganism is pathogenic if it is capable of producing a disease in an immunocompetent person. True Pathogens a) b) Highly Pathogens Less Pathogens Definitions Pathogenicity and Virulence Pathogenicity The ability of a microbe to cause disease This term is often used to describe or compare species. Virulence The degree of pathogenicity in a microorganism This term is often used to describe or compare strains within a species. Virulence Virulence: defined as quantitative measure of pathogenicity, and is measured by minimum number of bacteria to cause the disease. LD 50 (The 50% lethal dose): Number of organisms required to kill 50% hosts. ID 50 (50% infectious dose): Number of organisms required to cause infection in 50% of host population. Virulenc e Organisms with lower LD 50 are more virulent than higher ID 50, as fewer bacteria are required to cause death or disease. Infectious dose of an organism required to cause disease varies greatly among the pathogenic bacteria. Shigella (ID 50 less than 100) and Salmonella (100,000) cause diarrhea by producing infection in GIT. Infectious dose infective dose (ID) amount of a pathogenic agent that will cause infection in susceptible Subjects. The infectious dose of bacteria depends primarily on their virulence factors. Virulence factor: Infectious dose of bacteria depends upon virulence factor i.e. whether a bacteria has: Pilli Toxin Capsule Invasive properties Pathogenic bacteria can be grouped into three categories on the basis of their invasive properties for eukaryotic cells: 1. 2. 3. Extracellular bacteria Facultative intracellular bacteria Obligate intracellular bacteria Extracellular bacterial pathogens do not invade cells and proliferate instead in the extracellular environment. Some of extracellular bacteria even don’t penetrate body tissues (e.g. V. cholerae) but adhere to epithelial surfaces and cause disease by secreting potent toxins. TYPES OF BACTERIAL INFECTIONS The word infection does not have to be equated with disease. Bacteria cause disease by two major mechanisms: (1) toxin production and (2) invasion and inflammation. Types of Bacterial Infections Non communicable (e.g. botulism) Communicable, spreads from host to host via airborne, droplets and coughing ( e.g. Tuberculosis) Contagious, Highly communicable (e.g. Influenza) Asymptomatic / inapparent. Chroniccarriers, the organisms continue to grow with or without producing the symptoms in the host. Epidemiological Classification of Bacterial Infections Endemic: Infection at persistently low level in specific geographical area. Epidemic: Higher than the usual. Pandemic: Spreads rapidly over large population area and is considered emergency which requires immediate remedial measures to control it. STAGES OF BACTERIAL PATHOGENESIS 1) 2) 3) 4) 5) Transmission from an external source into the portal of entry. Evasion of primary host defenses such as skin or stomach acid. Adherence to mucous membranes, usually by bacterial pili. Colonization by growth of the bacteria at the site of adherence. Disease symptoms caused by toxin production or invasion accompanied by inflammation. 6) Host responses, both nonspecific and specific (immunity), during steps 3, 4, and 5. 7) Progression or resolution of the disease. MECHANISMS OF PATHOGENICITY Portal of Entry Adherence Penetration/invasion of host defense Damage to host cell Important portals of entry 1. Mucus Membranes ▪ Respiratory tract ▪ Gastrointestinal tract ▪ Genital 2. Skin 3. Parentarel Common Diseases contracted via the Respiratory Tract Common cold Flu Tuberculosis Pneumonia Measles Diphtheria Common diseases contracted via the G.I. Salmonellosis Tract Salmonella sp. Shigellosis Shigella sp. Cholera Vibrio cholorea Ulcers Helicobacter pylori Botulism Clostridium botulinum Clostridium botulinum 3rd Portal of Entry: Parentarel Microorganisms are deposited into the tissues below the skin or mucus membranes Punctures and scratches injections bites surgery Adherence to Cell Surfaces Adherence (attachment) is often an essential step in bacterial pathogenesis or infection, required for colonizing a new host - Process by which microorganisms attach themselves to cells. Requires the participation of two factors: a receptor and an ligand Adherence to Cell Surfaces Certain bacteria have specialized structures: ▪ Pili ( fimbriae) ▪ Capsules ▪ Glycocalyces Capsules Streptococcus pneumoniae Klebsiella pneumoniae Haemophilus influenzae Bacillus anthracis Streptococcus mutans K. pneumoniae Biofilm s Production of biofilm is initiated after the attachment of bacteria to the cell surface. Biofilm is polysacchride and protein in nature and protect bacteria from both antibiotics and host immune defenses such as antibodies and neutrophils. Biofilms have been found to be involved in a wide variety of microbial infections in the body, by one estimate 80% of all infections The development of a biofilm may allow for an aggregate cell colony (or colonies) to be increasingly antibiotic resistant.. Transmissio n Means how the organism transmits the disease to the body cells: Human to human. Non human to human (fomites, animals, soil and water). Normal flora can become pathogenic at times but the infection is mostly acquired from the external sources. Vertical transmission Bacteria, virus and other microbes can be transmitted via vertical transmission (mother to child). Across the placenta Birth canal Breast milk Transmitted via horizontal transmission (person to person). Transmissio n Bacterial diseases can be transmitted via food and insects. Animals can also act as a source (reservoir) or the mode of transmission (vector) of certain organisms. Diseases for which animals are the reservoirs are called zoonoses. Zoonotic Diseases Caused by Bacteria Toxigenesi s The ability to produce toxins. Bacteria may produce two types of toxins called exotoxins and endotoxins. Exotoxins are released from bacterial cells and may act at tissue sites removed from the site of bacterial growth. Endotoxins are cell-associated substance. (In a classic sense, the term endotoxin refers to the lipopolysaccharide component of the outer membrane of Gram-negative bacteria). References Gerard J. Tortora, Berdell R. Funke, Christine L. Case - Microbiology_ an introduction-Pearson (2018)