Theoretical Medical Microbiology (L-1 & L-2) PDF

Theoretical Medical microbiology L-1 & L-2 ______________________________________________ INTRODUCTION TO MICROBIOLOGY Medical microbiology is a branch of microbiology that deals with the study of microorganisms including bacteria, viruses, fungi, and p...

Theoretical Medical microbiology L-1 & L-2 ______________________________________________ INTRODUCTION TO MICROBIOLOGY Medical microbiology is a branch of microbiology that deals with the study of microorganisms including bacteria, viruses, fungi, and parasites of medical importance that are capable of causing diseases in humans. It also includes the study of microbial pathogenesis, disease pathology, immunology, and epidemiology of diseases. BRANCHES OF MICROBIOLOGY - Bacteriology: Study of bacteria—the smallest, simplest single celled organisms. - Mycology: The study of fungi which includes both microscopic forms (molds and yeasts) and larger forms (mushrooms). - Parasitology: Study of parasites which traditionally includes pathogenic protozoa and helminths. - Virology: Study of viruses-minute particles that parasitize living things. - Immunology: Study of systems of body defenses that protect against infection. - Microbial taxonomy: Study of classification, naming and identification of microorganisms. - Biotechnology: This discipline includes any process in which humans use systems or process of organisms to arrive at a desired product. - Genetic engineering and recombinant DNA technology: This involves techniques that deliberately alter the genetic make-up of organisms to introduce new compounds, different genetic combinations and even unique organisms. 1 Theoretical Medical microbiology L-1 & L-2 ______________________________________________ The Universal Tree of Life On the basis of small subunit ribosomal RNA (ssrRNA) analysis, the tree of life gives rise to three cellular domains of life: Archaea, Bacteria, and Eukarya. Bacteria and Archaea share the procaryotic type of cellular configuration, but otherwise are not related to one another any more closely than they are to the eukaryotic domain, Eukarya. Eukarya consists of all eukaryotic cell-types, including protista, fungi, plants and animals. Members of the domain Bacteria are unicellular and prokaryotic. Their cells have thick, rigid walls that surround a cell membrane and contain a substance known as peptidoglycan. These bacteria are ecologically diverse, ranging from free-living soil organisms to deadly parasites. Some photosynthesize, while others do not. Some need oxygen to survive, while others are killed by oxygen. 2 Theoretical Medical microbiology L-1 & L-2 ______________________________________________ Ranks or Levels of Bacterial Taxonomy The most commonly used levels or ranks (in descending order) are: Domain: collection of similar kingdoms. Kingdom: collection of similar phyla or divisions. Phylum or Division: collection of similar classes Classes: a collection of similar orders. Orders: a collection of similar families. Families: a collection of similar genera. Genera: a collection of related species. Species: a group of related isolates or strains. An example is the classification of E. coli: Domain Bacteria Kingdom Eubacteria Phylum Proteobacteria Class Gammaproteobacteria Order Enterobacteriales Family Enterobacteriaceae Genus Escherichia Species E. coli Binomial nomenclature Organisms are named using binomial nomenclature. Binomial nomenclature employs the names of the two-level taxa, genus and species, to name a species. Binomial nomenclature includes: i. Genus comes before species (e.g., Escherichia coli) ii. Genus name is always capitalized (e.g., Escherichia) iii. Species name is never capitalized (e.g., coli) iv. Both names are always either italicized or underlined (e.g Escherichia coli) v. The genus name may be used alone, but not the species name (i.e saying or writing “Escherichia “ alone is legitimate while saying or writing “ coli” is not). 3 Theoretical Medical microbiology L-1 & L-2 ______________________________________________ THE STRUCTURE OF BACTERIAL COMPONENTS AND THEIR FUNCTION 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. Bacterial morphology: A: Cocci: in clusters (A-1); chains (A-2); in pairs with pointed ends (A-3); in pairs with kidney bean shape (A-4). B: Rods (bacilli): with square ends (B-1); with rounded ends (B-2); club-shaped (B-3); fusiform (B-4); comma-shaped (B-5). C: Spirochetes: relaxed coil (C-1); tightly coiled (C-2). In addition to their characteristic shapes, the arrangement of bacteria is important. For example, 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. 4 Theoretical Medical microbiology L-1 & L-2 ______________________________________________ Bacteria range in size from about 0.2 to 5 μm. The smallest bacteria (Mycoplasma) are about the same size as the largest viruses (poxviruses) and are the smallest organisms capable of existing outside a host. The longest bacteria rods are the size of some yeasts and human red blood cells (7 μm). STRUCTURE OF BACTERIA Prokaryotic cells have three architectural regions: a cell envelope consisting of a capsule, cell wall and plasma membrane; a cytoplasmic region that contains the cell genome (DNA) and ribosomes and various sorts of inclusions; and appendages (proteins attached to the cell surface) in the form of flagella and pili. Cell Wall The cell wall is the outermost component common to all bacteria (except Mycoplasma species, which are bounded by a cell membrane, not a cell wall). Some bacteria have surface features external to the cell wall, such as a capsule, flagella, and pili, which are less common components. The cell wall is located external to the cytoplasmic membrane and is composed of peptidoglycan. The peptidoglycan provides structural support and maintains the characteristic shape of the cell. 5 Theoretical Medical microbiology L-1 & L-2 ______________________________________________ Cell Walls of Gram-Positive and Gram-Negative Bacteria The structure, chemical composition, and thickness of the cell wall differ in gram positive and gram-negative bacteria. (1) The peptidoglycan layer is much thicker in gram-positive than in gram negative bacteria. Many gram-positive bacteria also have fibers of teichoic acid, which protrude outside the peptidoglycan, whereas gram-negative bacteria do not have teichoic acids. (2) In contrast, the gram-negative bacteria have a complex outer layer consisting of lipopolysaccharide, lipoprotein, and phospholipid. Lying between the outer membrane layer and the cytoplasmic membrane in gram-negative bacteria is the periplasmic space, which is the site, in some species, of enzymes called β- lactamases that degrade penicillins and other β-lactam drugs. The cell wall has several other important properties: (1) In gram-negative bacteria, it contains endotoxin, a lipopolysaccharide. (2) Its polysaccharides and proteins are antigens that are useful in laboratory identification. (3) Its porin proteins play a role in facilitating the passage of small, hydrophilic molecules into the cell. 6 Theoretical Medical microbiology L-1 & L-2 ______________________________________________ Cell Walls of Acid-Fast Bacteria Mycobacteria (e.g., Mycobacterium tuberculosis) have an unusual cell wall, resulting in their inability to be Gram-stained. These bacteria are said to be acid fast because they resist decolorization with acid–alcohol after being stained with carbolfuchsin. This property is related to the high concentration of lipids, called mycolic acids, in the cell wall of mycobacteria. In view of their importance, three components of the cell wall (i.e., peptidoglycan, lipopolysaccharide, and teichoic acid) are discussed in detail here. Peptidoglycan Peptidoglycan is a complex, interwoven network that surrounds the entire cell. It is found only in bacterial cell walls. It provides rigid support for the cell, is important in maintaining the characteristic shape of the cell, and allows the cell to withstand media of low osmotic pressure, such as water. The term peptidoglycan is derived from the peptides and the sugars (glycan) that make up the molecule. Synonyms for peptidoglycan are murein and mucopeptide. 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. Protoplasts and Spheroplasts The form of bacteria which are devoid of peptidoglycan component of cell envelope and which are thus sensitive to the osmolarity of the medium belong to the groups of 7 Theoretical Medical microbiology L-1 & L-2 ______________________________________________ spheroplasts or protoplasts. When the surface of bacterium is completely free of cell wall components, it is called as protoplast (in gram-positive bacteria) whereas in spheroplasts outer membrane is present (in gram-negative bacteria). Protoplasts cannot revert to normal morphology by re-forming their cell walls but spheroplasts can do so under suitable conditions. 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 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. This outer polymer is the important somatic, or O, antigen of several gram-negative bacteria that is used to identify certain organisms in the clinical laboratory. 8 Theoretical Medical microbiology L-1 & L-2 ______________________________________________ Teichoic Acid Teichoic acids are fibers located in the outer layer of the gram-positive cell wall and extend from it. They are composed of polymers of either glycerol phosphate or ribitol phosphate. Some polymers of glycerol teichoic acid penetrate the peptidoglycan layer and are covalently linked to the lipid in the cytoplasmic membrane, in which case they are called lipoteichoic acid; others anchor to the muramic acid of the peptidoglycan. 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. Cytoplasmic Membrane Just inside the peptidoglycan layer of the cell wall lies the cytoplasmic membrane (also called a cell membrane or plasma membrane) which is composed of a phospholipid bilayer similar in microscopic appearance to that in eukaryotic cells. They are chemically similar, but eukaryotic membranes contain sterols, whereas prokaryotes generally do not. The only prokaryotes that have sterols in their membranes are members of the genus Mycoplasma. The membrane has four important functions: (1) Active transport of molecules into the cell. (2) Energy generation by oxidative phosphorylation. (3) Synthesis of precursors of the cell wall. (4) Secretion of enzymes and toxins. Capsule An organized layer of glycocalyx, firmly attached to the outer surface of a bacterial cell wall. It may be composed of complex polysaccharide (pneumococci and 9 Theoretical Medical microbiology L-1 & L-2 ______________________________________________ Klebsiella) or polypeptide (Bacillus anthracis) or hyaluronic acid (Streptococcus pyogenes). The sugar components of the polysaccharide vary from one species of bacteria to another and frequently determine the serologic type (serotype) within a species. For example, there are 84 different serotypes of Streptococcus pneumoniae, which are distinguished by the antigenic differences of the sugars in the polysaccharide capsule. The capsule has several functions: (1) It limits the ability of phagocytes to engulf the bacteria. Negative charges on the capsular polysaccharide repel the negatively charged cell membrane of the neutrophil and prevent it from ingesting the bacteria. (2) The capsule may play a role in the adherence of bacteria to human tissues, which is an important initial step in causing infection. Slime Layer An unorganized, loosely attached layer of glycocalyx surrounding a bacterial cell. The glycocalyx is a polysaccharide 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. It also mediates adherence of certain bacteria, such as Streptococcus mutans, to the surface of teeth. This plays an important role in the formation of plaque, the precursor of dental caries. 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. 10 Theoretical Medical microbiology L-1 & L-2 ______________________________________________ Ribosomes Bacterial ribosomes are the site of protein synthesis as in eukaryotic cells, but they differ from eukaryotic ribosomes in size and chemical composition. Bacterial ribosomes are 70S in size, with 50S and 30S subunits, whereas eukaryotic ribosomes are 80S in size, with 60S and 40S subunits. The differences in both the ribosomal RNAs and proteins constitute the basis of the selective action of several antibiotics that inhibit bacterial, but not human, protein synthesis. Granules The cytoplasm contains several different types of granules that serve as storage areas for nutrients and stain characteristically with certain dyes. For example, volutin is a reserve of high energy stored in the form of polymerized metaphosphate. It appears as a “metachromatic” granule since it stains red with methylene blue dye instead of blue as one would expect. Metachromatic granules are a characteristic feature of Corynebacterium diphtheriae, the cause of diphtheria. Nucleoid The nucleoid is the area of the cytoplasm in which DNA is located. The DNA of prokaryotes is a single, circular molecule that has a molecular weight (MW) of approximately 2 × 109 and contains about 2000 genes. Because the nucleoid contains no nuclear membrane, no nucleolus, no mitotic spindle, and no histones, there is little resemblance to the eukaryotic nucleus. One major difference between bacterial DNA and eukaryotic DNA is that bacterial DNA has no introns, whereas eukaryotic DNA does. 11 Theoretical Medical microbiology L-1 & L-2 ______________________________________________ Plasmids Plasmids are extrachromosomal, double-stranded, circular DNA molecules that are capable of replicating independently of the bacterial chromosome. Although plasmids are usually extrachromosomal, they can be integrated into the bacterial chromosome. Several different types of plasmids can exist in one cell: (1) Transmissible plasmids can be transferred from cell to cell by conjugation. They are large, since they contain about a dozen genes responsible for synthesis of the sex pilus and for the enzymes required for transfer. They are usually present in a few (1–3) copies per cell. (2) Nontransmissible plasmids are small, since they do not contain the transfer genes; they are frequently present in many (10–60) copies per cell. 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 and silver. (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. Other plasmid-encoded products of interest are as follows: (1) Bacteriocins are toxic proteins produced by certain bacteria that are lethal for other bacteria. (2) Several antibiotics produced by Streptomyces. (3) A variety of degradative enzymes. Transposons Transposons are pieces of DNA that move readily from one site to another either 12 Theoretical Medical microbiology L-1 & L-2 ______________________________________________ within or between the DNAs of bacteria, plasmids, and bacteriophages. Because of their unusual ability to move, they are nicknamed “jumping genes.” Some transposons move by replicating their DNA and inserting the new copy into another site (replicative transposition), whereas others are excised from the site without replicating and then inserted into the new site (direct transposition). Transposons can code for drug-resistant enzymes, toxins, or a variety of metabolic enzymes and can either cause mutations in the gene into which they insert or alter the expression of nearby genes. In contrast to plasmids, transposons are not capable of independent replication; they replicate as part of the DNA in which they are integrated. Flagella Flagella are long, whiplike appendages that move the bacteria toward nutrients and other attractants, a process called chemotaxis. The long filament, which acts as a propeller, is composed of many subunits of a single protein, flagellin, arranged in several intertwined chains. Flagellated bacteria have a characteristic number and location of flagella: - Monotrichous: Bacteria which have one polar flagellum. - Lophotrichous: Bacteria with a tuft of several polar flagella. - Amphitrichous: Bacteria with flagella at both the ends. - Peritrichous: Bacteria with flagella distributed all over the surface of the bacterium. - Atrichous: Bacteria which do not possess any flagellum. 13 Theoretical Medical microbiology L-1 & L-2 ______________________________________________ Only certain bacteria have flagella. Many rods do, but most cocci do not and are therefore nonmotile. Spirochetes move by using a flagellum like structure called the axial filament, which wraps around the spiral-shaped cell to produce an undulating motion. Pili (Fimbriae) Pili are hairlike filaments that extend from the cell surface. They are shorter and straighter than flagella and are composed of subunits of pilin, a protein arranged in helical strands. 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. Bacterial Spores These highly resistant structures are formed in response to adverse conditions by two genera of medically important gram-positive rods: the genus Bacillus and the genus Clostridium. Spore formation (sporulation) occurs when nutrients, such as sources of carbon and nitrogen, are depleted. The spore forms inside the cell and contains bacterial DNA, a small amount of cytoplasm, cell membrane, peptidoglycan, very little water, and most importantly, a thick, keratinlike coat that is responsible for the remarkable resistance of the spore to heat, dehydration, radiation, and chemicals. Once formed, the spore has no metabolic activity and can remain dormant for many years. Upon exposure to water and the appropriate nutrients, specific enzymes degrade the coat, water and nutrients enter, and germination into a potentially pathogenic bacterial cell occurs. 14 Theoretical Medical microbiology L-1 & L-2 ______________________________________________ Stages of sporulation Shape of spore may be: 1. Spherical central 2. Oval central 3. Oval sub-terminal not bulging 4. Oval sub-terminal with bulging 5. Oval terminal with bulging 6. Terminal spherical with bulging 15 Theoretical Medical microbiology L-1 & L-2 ______________________________________________ 16