Microbiology Lecture Notes PDF

MICROBIOLOGY LECTURE 1 NOTES Classification of Protista a. Higher Protist (Eukaryotic) 1. Algae except (blue green algae) 2. Protozoa 3. Fungi b. Lower Protist (Prokaryotic) 1. Bacteria 2. Blue green algae 1cm= 10mm 1mm=...

MICROBIOLOGY LECTURE 1 NOTES Classification of Protista a. Higher Protist (Eukaryotic) 1. Algae except (blue green algae) 2. Protozoa 3. Fungi b. Lower Protist (Prokaryotic) 1. Bacteria 2. Blue green algae 1cm= 10mm 1mm= 1000 micro.m = 1 million nano m Microorganisms can be classified into major groups: - algae, - protozoa, - fungi, - bacteria, - viruses and a number of organisms - intermediate between bacteria and viruses (e.g. rickettsiae, chlamydiae and more recently described mimiviruses- so called as they mimic viruses). Bacteria, fungi and protozoa belong to the kingdom of protists and are differentiated from animals and plants by being unicellular or relatively simple multicellular organisms leading a parasitic existence. - Viruses are unique, acellular, metabolically inert organisms and therefore can replicate only within living cells. (Obligate intracellular parasite) Other differences between viruses and cellular organisms include: Structure. Cells possess a nucleus or, in the case of bacteria, a nucleoid with DNA. This is surrounded by the cytoplasm where: - energy is generated and - proteins are synthesized. In viruses the inner core of genetic material is either DNA or RNA, but they have no cytoplasm and hence depend on the host for their - energy and - proteins (i.e. they are metabolically inert) Reproduction. Bacteria reproduce by binary fission (a parent cell divides into two similar cells), but viruses disassemble, produce copies of their nucleic acid and proteins, and then reassemble to produce another generation of viruses. As viruses are metabolically inert they must replicate within host cells. Bacteria, however, can replicate extracellularly (except rickettsiae and chlamydiae, which are bacteria that also require living cells for growth). (Obligate intracellular parasite) Bacteria divide by binary fission The major characteristics of bacteria, mycoplasmas, rickettsiae, chlamydiae and viruses are given in Table 2.1. EUKARYOTES AND PROKARYOTES Another way of classifying cellular organisms is to divide them into prokaryotes and eukaryotes (Greek karyon: nucleus). Fungi, protozoa and humans, for instance, are eukaryotic, whereas bacteria are prokaryotic. In prokaryotes the bacterial genome, or chromosome, is a single, circular molecule of double-stranded DNA, lacking a nuclear membrane (smaller, single or multiple circular DNA molecules called plasmids may also be present in bacteria), whereas the eukaryotic cell has a true nucleus with multiple chromosomes surrounded by a nuclear membrane. The main differences between prokaryotes and eukaryotes are listed in Table 2.2. Theodor Svedberg was awarded the 1926 Nobel Prize for chemistry for his work in colloid chemistry, but his name is best known for the “Svedberg unit,” the unit of measurement of the velocity of sedimentation and, thus, the molecular weight of proteins. Prokaryotes can again be divided into eubacteria and archaebacteria (synonym: archaea). - Eubacteria comprise the familiar groups of bacteria, including the vast majority of human pathogens, whilst - archaebacteria appear - rarely to cause human disease and - live in extreme environments (e.g. high temperature or salt concentrations). Archaebacteria received little attention traditionally as they cannot be easily cultured in the laboratory. Interestingly, recent studies have uncovered the presence of archaebacteria in the oral cavity. Their role, if any, in oral health or disease is yet to be determined. MORPHOLOGY Shape and size The shape of a bacterium is determined by its rigid cell wall. Bacteria are classified by shape into three basic groups (Fig. 2.1): I. cocci (spherical) 2. bacilli (rod-shaped) 3. spirochaetes (helical). Some bacteria with variable shapes, appearing both as coccal and bacillary forms, are called pleomorphic (pleo: many, morphic: shaped) in appearance. The size of bacteria ranges from about 0.2 to 5 /μ (micro.meter) The smallest bacteria approximate the size of the largest viruses (poxviruses), whereas the longest bacilli attain the same length as some yeasts and human RBCs (red blood cells) 7μ (micro.m) Common bacterial forms. A coccus; B capsulated diplococci; C, D cocci in chains (e.g. streptococcus) and clusters (e.g. staphylococcus); E bacillus; F, G capsulated and flagellated bacillus (e.g. Escherichia coli); H curved bacilli (e.g. Vibrio spp.);  I spore-bearing bacilli (e.g. Clostridium tetani); J spirochaete. Arrangement Bacteria, whichever shape they may be, arrange themselves (usually according to the plane of successive cell division) as: Gram-staining characteristics Bacteria can be classified into two major subgroups according to the staining characteristics of their cell walls. The stain used, called the Gram stain (first developed by a Danish physician, Christian Gram), divides the bacteria into The Gram-staining property of bacteria is useful both for their - identification and in the - therapy of bacterial infections because, in general, Gram-positive bacteria are more susceptible: to penicillins than Gram-negative bacteria. Structure Bacteria have a rigid cell wall protecting a fluid protoplast. Comprising a cytoplasmic membrane and a variety of other components (described below). Structures external to the cell wall - Flagella. Flagella are whip-like filaments which act as propellers and guide the bacteria toward nutritional and other sources. (Organ of locomotion: motility) The filaments are composed of many subunits of a single protein, flagellin. - monotrichous, a single polar flagellum;  - amphitrichous, single flagellum attached to each end, - lophotrichous, tufts of flagella at one or both end or - peritrichous all over the outer surface. Many bacilli (rods) have flagella, but most cocci do not and are therefore non-motile. Spirochaetes move by using a flagellum-like structure called the axial filament, which wraps around the cell to produce an undulating motion. - Fimbriae and pili. Fimbriae and pili are fine, hair-like filaments, shorter than flagella, that extend from the cell surface. Pili, found mainly on Gram-negative organisms, are composed of subunits of a protein, pilin, and mediate the adhesion of bacteria to receptors on the human cell surface a necessary first step in the initiation of infection. A specialized type of pilus, the sex pilus, forms the attachment between the male (donor) and the female (recipient) bacteria during conjugation, when genes are transferred from one bacterium to another. - Glycocalyx (slime layer). The glycocalyx is a polysaccharide coating that covers the outer surfaces of many bacteria and allows the bacteria to adhere firmly to various structures, e.g.. - oral mucosa, - teeth, - heart valves and - catheters This is especially true in the case of Streptococcus mutans, a major cariogenic (Cause tooth caries) organism, which has the ability to produce vast quantities of extracellular polysaccharide in the presence of dietary sugars such as sucrose. Capsule. An amorphous (without defined shape), gelatinous layer (Jelly like) (usually more substantial than the glycocalyx) surrounds the entire bacterium; it is composed of - polysaccharide, and sometimes - protein (e.g. Bacillus anthracis: anthrax disease). The sugar components of the polysaccharide vary in different bacterial species and frequently determine the serological type within a species (e.g. 84 different serological types of Streptococcus pneumoniae can be distinguished by the antigenic differences of the sugars in the polysaccharide capsule). The capsule is important because: It mediates the adhesion of bacteria to human tissues or prosthesis such as dentures or implants - a prerequisite for colonization and infection Anti-phagocytosis: virulence It hinders or inhibits phagocytosis; hence, the presence of a capsule correlates with virulence It helps in laboratory identification of organisms (in the presence of antiserum against the capsular polysaccharide the capsule will swell greatly – a phenomenon called the Quellung reaction) 84 different serological types of S. pneumoniae Its polysaccharides are used as antigens in certain vaccines because they elicit protective antibodies (e.g. polysaccharide vaccine of S. pneumoniae). Cell wall The cell wall confers rigidity upon the bacterial cell. It is a multilayered structure outside the cytoplasmic membrane. It is porous and permeable to substances of low molecular weight. The cell wall is made of peptidoglycan (protein + sugar) and is covered by an outer membrane (in G-ve). The cell wall varies in thickness and chemical composition. (Fig. 2.4). The term 'peptidoglycan' is derived from the peptides and the sugars (glycan) that make up the molecule. (Synonyms for peptidoglycan are murein and mucopeptide.) The cell walls of Gram-positive and Gram-negative bacteria have important structural and chemical differences: The peptidoglycan layer is common to both Gram-positive and Gram- negative bacteria but is much thicker in the Gram-positive bacteria Gram-negative organisms have a complex outer membrane composed of lipopolysaccharide (LPS), lipoprotein and phospholipid. These form porins, through which hydrophilic molecules are transported in and out of the organism. The 0 antigen of the LPS and the lipid A component are also embedded in the outer membrane. Lying between the outer membrane and the cytoplasmic membrane of Gram-negative bacteria is the periplasmic space. It is in this space that some bacterial species produce enzymes that destroy drugs such as penicillins (e.g. -lactamases)  The LPS of Gram-negative bacteria, which is extremely toxic, has been called the endotoxin. (Hence, by definition, endotoxins cannot be produced by Gram-positive bacteria as they do not have LPS in their cell walls.) LPS is bound to the cell surface and is only released when it is lysed. It is responsible for many of the features of disease, such as fever and shock (see Ch. 5) The cell walls of some bacteria (e.g. Mycobacterium tuberculosis) contain lipids called mycolic acids which cannot be Gram-stained, and hence are called acid-fast (i.e. they resist decolorization with acid alcohol after being stained with carbolfuchsin). Bacteria with defective cell walls. Some bacteria can survive with defective cell walls. These include mycoplasmas, L-forms, (spheroplasts and protoplasts). Mycoplasmas do not possess a cell wall and do not need hypertonic media for their survival. They occur in nature and may cause human disease (e.g. pneumonia). L-forms are usually produced in the laboratory and may totally or partially lack cell walls. They may be produced in patients treated with penicillin. Both - spheroplasts (derived from Gram-negative bacteria) and - protoplasts (derived from Gram-positive bacteria) - lack cell walls, - cannot replicate on laboratory media and are - unstable and osmotically fragile. They require hypertonic conditions for maintenance and are produced in the laboratory by the action of enzymes or antibiotics. Cytoplasmic membrane (phospholipid bilayer) sterols The cytoplasmic membrane lies just inside the peptidoglycan layer of the cell wall and is composed of a phospholipid bilayer similar in appearance to that of eukaryotic cells. However, eukaryotic membranes contain sterols (cholesterol), whereas prokaryotes generally do not. The membrane has the following major functions: active transport and selective diffusion of molecules and solutes into and out of the cell electron transport and oxidative phosphorylation, in aerobic species synthesis of cell wall precursors secretion of enzymes and toxins (periplasmic space: -lactamases (penicillinase) supporting the receptors and other proteins of the chemotactic and sensory transduction systems. Mesosome. This is a convoluted invagination of the cytoplasmic membrane which functions as the origin of the transverse septum that divides the cell in half during cell division. It is also the binding site of the DNA which will become the genetic material of each daughter cell. Cytoplasm The cytoplasm comprises an inner, nucleoid region (composed of DNA) which is surrounded by an amorphous matrix that contains ribosomes, nutrient granules, metabolites and various ions. Nuclear material or nucleoid Bacterial DNA comprises a single, supercoiled, circular chromosome that contains about 2000 genes, approximately I mm long in the unfolded state. (It is analogous to a single, haploid chromosome.) During cell division it undergoes semiconservative replication bi-directionally from a fixed point. Ribosomes. Ribosomes are the sites of protein synthesis. Bacterial ribosomes differ from those of eukaryotic cells in both - size and - chemical composition. They are organized in units of - 70S, compared with eukaryotic ribosomes of - 80S. These differences are the basis of the selective action of some antibiotics that inhibit bacterial, but not human, protein-synthesis. (Tetracycline 30S) Cytoplasmic inclusions. The cytoplasm contains different types of inclusions, which serve as sources of stored energy; examples include - polymetaphosphate, - polysaccharide and - B-hydroxybutyrate (made by the body, provides energy when not enough sugars have been eaten) Bacterial spores Spores are formed in response to adverse conditions by the medically important bacteria that belong to the; - genus Bacillus (which includes the agent of anthrax) and the - genus Clostridium (which includes the agents of tetanus and botulism). These bacteria sporulate (form spores) when - nutrients, such as sources of carbon and nitrogen, are scarce (Fig. 2.6). The spore develops at the expense of the vegetative cell and contains - bacterial DNA, - a small amount of cytoplasm, - cell membrane, peptidoglycan, - very little water and, most importantly, - a thick, keratin-like coat. (contains a high concentration of calcium dipicolinate) Spore formation Spore forming bacteria grow as vegetative cells and divide by binary fission when nutrients are available and environmental conditions are not adverse. When nutrients are depleted or conditions become adverse spore formation is initiated. The DNA condenses and aligns it self in the center of the cell. The vegetative cell is now referred to as mother cell. Next, the DNA divides into two complete copies and the mother cell membrane invaginates to form the developing fore spore. The mother cell membrane continues to grow and engulfs the developing spore and the developing spore is now surrounded by two membrane layers. Next , peptidoglycan is laid down between the two membranes of the developing spore to form the cortex. The dipiclonic acid is formed inside the spore and calcium enter from the outside and water is removed to the outside. A protein coat is formed exterior to the cortex and spore becomes mature. Some spores forms additional layer called the exosporium. A mature spore is resistant to environmental conditions. Finally, lytic enzymes destroy the mother cell and the mature spore is released. This coat, which contains a high concentration of calcium dipicolinate, is remarkably resistant to  - heat, - dehydration, - radiation and - chemicals. Once formed, the spore is metabolically inert and can remain dormant for many years. Spores are called either Central, terminal or subterminal, depending on their position in relation to the cell wall of the bacillus from which they developed. When appropriate conditions supervene (i.e. water, nutrients) there is enzymic degradation of the coat and the spore transforms itself into a metabolizing, reproducing bacterial cell once again (Fig. 2.6). Central, terminal or subterminal Endospores (spores) Clinical relevance of bacterial spores The clinical importance of spores lies in their extraordinary resistance to - heat and - chemicals. Because of this, - sterilization cannot be easily achieved by boiling; - other, more efficacious methods of sterilization, such as steam under pressure (autoclaving), are required to ensure the sterility of products used for surgical purposes. Bacterial spores are used for evaluating the sterilization efficacy of autoclaves; spores of Bacillus stearothermophilus and other species are used.. TAXONOMY The systematic classification and categorization of organisms into ordered groups are called taxonomy. A working knowledge of taxonomy is useful for diagnostic microbiology and for studies in epidemiology and pathogenicity. As mentioned at the beginning of this chapter, bacteria and fungi are protists. Bacterial classification is somewhat artificial in that they are categorized according to phenotypic (as opposed to genotypic) features, which facilitate their laboratory identification. Phenotypic taxonomy These phenotypic features comprise: morphology (cocci, bacilli, spirochaetes) staining properties (Gram-positive, Gram-negative) cultural requirements (aerobic, facultative anaerobic, anaerobic) biochemical reactions (saccharolytic and asaccharolytic, according to sugar fermentation reactions) antigenic structure (serotypes). Most of the medically and dentally important bacteria are classified according to their - morphology, - Gram-staining characteristics and - atmospheric requirements. A simple classification of medically important bacteria is given in Figures 2.7 and 2.8. Genotypic taxonomy Genotypic classification and speciation of organisms are becoming increasingly important and useful. Genotypic taxonomy exploits the genetic characteristics, which are more stable than phenotypic features of organisms. These methods essentially analyze bacterial DNA to speciate them, for example by: - Bacterial DNA sequence AGTCCCTTAATACGATGC - assessing molecular guanine and cytosine (GC) content, Additionally, recent research indicates that endogenous bacterial habitats in humans, including the oral cavity, harbour a flora that cannot be cultured using routine laboratory techniques. These so-called unculturable bacteria comprise mostly archaebacteria, mentioned before and can only be detected by molecular techniques (e.g. direct amplification of 16S RNA). The role of these totally new phylotypes of bacteria in either disease or health awaits clarification. How do organisms get their names? Organisms are named according to a hierarchical system, beginning with the taxonomic rank kingdom, followed by division, subdivision, order, family, genus and species (Table 2.3). The scientific name of an organism is classically a binomial of the last two ranks, i.e. a combination of the generic name followed by the species name, e.g. Streptococcus salivarius (note that the species name does not begin with a capital letter). The name is usually written in italics with the generic name abbreviated (e.g. S. salivarius). When bacterial names are used adjectivally or collectively the names are not italicized and do not begin with a capital letter (e.g. staphylococcal enzymes, staphylococci, lactobacilli).