Introduction to Bacteriology and Bacterial Structure/Function

INTRODUCTION TO BACTERIOLOGY AND BACTERIAL STRUCTURE/FUNCTION LEARNING OBJECTIVES To describe historical landmarks of medical microbiology To describe Koch’s Postulates To describe the characteristic structures and chemical nature of cellular constituents that distinguish eukaryotic and prokaryotic cells To describe chemical, structural, and functional components of the bacterial cytoplasmic and outer membranes, cell wall and surface appendages To name the general structures, and polymers that make up bacterial cell walls To explain the differences between gram negative and gram positive cells To describe the chemical composition, function and serological classification as H antigen of bacterial flagella and how they differ from flagella of eucaryotic cells To describe the chemical composition and function of pili To explain the unique chemical composition of bacterial spores To list medically relevant bacteria that form spores To explain the function of spores in terms of chemical and heat resistance To describe characteristics of different types of membrane transport To describe the exact cellular location and serological classification as O antigen of Lipopolysaccharide (LPS) To explain how the structure of LPS confers antigenic specificity and toxicity To describe the exact cellular location of Lipid A To explain the term endotoxin in terms of its chemical composition and location in bacterial cells INTRODUCTION TO BACTERIOLOGY 1. Two main threads in the history of bacteriology: 1) the natural history of bacteria and 2) the contagious nature of infectious diseases, were united in the latter half of the 19th century. During that period many of the bacteria that cause human disease were identified and characterized. 2. Individual bacteria were first observed microscopically by Antony van Leeuwenhoek at the end of the 17th century. 3. Bacteria are readily visible when present in large numbers because they make a turbid suspension. The controversy over spontaneous generation of bacterial life in liquid cultures led to the development of two important bacteriological procedures. a. Sterilization: the preparation of medium or instruments such that no living bacteria are present. b. Aseptic technique: laboratory technique that allows the manipulation of sterilized material without bacteriological contamination. 4. Bacteria are most easily studied in pure cultures in which only a single species is present. Pure cultures were originally produced by limiting dilution in liquid medium. Today pure cultures are usually prepared on medium solidified with agar, a gelling agent derived from seaweed. A mixed bacterial suspension is mechanically spread on the agar surface to yield isolated individual bacterial cells. These grow to yield macroscopic colonies (clones) that can be used to prepare pure cultures. 5. The ability to prepare pure cultures led to the study of bacterial classification and taxonomy. (A-1) a. The first basis for classification was shape. Round bacteria are called cocci (singular coccus). Rod shaped bacteria are called bacilli (singular bacillus). Other shapes will be considered later in the course. b. Bacteria are very difficult to study microscopically unless stained. The staining characteristics of bacteria in the Gram stain are very useful in classification. Gram positives are violet, while gram negatives are red. c. Bacterial taxonomy today depends upon the extent of DNA sequence homology. An important laboratory technique for the amplification and detection of specific DNA sequences (as, for example, in a bacterium or a virus) is the polymerase chain reaction (PCR). Examples of when PCR is used for clinical diagnostics will be considered later in this course. However, for routine laboratory diagnosis the most important bacterial characteristics are: 1. The morphology of colonies on appropriate agar medium. 2. Microscopic morphology and staining of individual bacteria. 3. Simple biochemical characteristics such as the ability to ferment a given carbohydrate. 4. Specific antigens detected by known antisera. 6. Koch’s Postulates. The use of pure cultures has made possible the identification of the bacterial etiology of many infectious diseases. The original rules for the proof of microbial etiology (Koch's Postulates): a. Find the bacteria in all cases of the disease. b. Grow the bacteria in pure cultures. c. Reproduce the disease (in animals) using the pure culture. d. Reisolate the bacteria in pure culture from the experimental infection. These rules cannot be applied to all infectious diseases. Some infectious diseases, such as obligate intracellular pathogens (i.e., those organisms that cannot grow on laboratory medium but require a host cell to grow) will not answer all of Koch’s postulates. 7. Koch’s Molecular Postulates. Koch’s Molecular Postulates were put forth by Stanley Falkow in 1988 to deal with defining the molecular basis by which a specific infectious disease is caused. a. The phenotype under investigation should be associated significantly more often with a pathogenic organism than with a nonpathogenic member or strain. b. Specific inactivation of a gene (or genes) associated with the suspected virulence trait should lead to a measurable decrease in virulence. c. Restoration of full pathogenicity should accompany replacement of the mutated gene with the wild type original. (A-2) BACTERIAL STRUCTURE AND FUNCTION: THE MICROBIAL WORLD (Introduction to the Procaryotic Cell) Reading assignment: Levinson, Chapter 1, 2 (omit plasmids and transposons until genetics lectures), and 5 Classes of Microorganisms (which classes contain human pathogens?) Distinguishing Characteristics Algae : no pathogens, all photosynthetic Fungi : some pathogens, nonphotosynthetic; rigid cell wall Protozoa : some pathogens, no rigid cell wall; unicellular, nonphotosynthetic (cysts have rigid walls) Bacteria : many pathogens; mostly require organic compounds as energy source but some of the non-pathogens are photosynthetic; all (but one) have a rigid cell wall Microorganisms have been traditionally differentiated from animals and true plants on the basis of their relatively simple biological-organization. The higher plants and animals are multicellular and develop distinct tissue regions that differ from one another with respect to the kinds of cells of which they are composed. A further level of internal complexity may be achieved by the combination of different tissues into a specialized local structure known as an organ (e.g. liver or leaf). Microorganisms are unicellular, but there is an increasing realization that they can act as multicellular groups and show differentiation into functionally distinct regions. Examples include stalk and spore formation in the soil microbe Myxococcus xanthus, and the formation of surface microbial communities on implants by pathogenic microbes. Microorganisms are divided into two subgroups on basis of structure of the individual cell. (This has clinical importance, since different classes of antibiotics are used to treat pathogens in each group.) Higher Microorganisms: Fungi, Protozoa, Algae (Eucaryotic cells) (Eucaryotic (Eukarya domain) = "true" nucleus) Lower Microorganisms: Bacteria (Procaryotic cells) (Bacterial domain) (A-3) Characteristics of structure and function exhibited by Eucaryotic as compared to Procaryotic cells. (These differences are often important for understanding the mechanism of action of chemotherapeutic agents. Antibiotics useful for combating bacterial infections are often useless against fungal infections.) 1. Chromosome(s) Eucaryotic: Each cell contains a number of different linear chromosomes contained Within the Nuclear Membrane. Mitosis occurs. Procaryotic: Each cell generally contains one circular chromosome. Not bound by a nuclear membrane. The mechanism of chromosome segregation during division does not involve mitosis. 2. Mitochondria and other membrane bound structures within the cytoplasm housing specific parts of the functional machinery of the cell. Eucaryotic: Eucaryotic mitochondrion contains the oxidative enzymes and carries out oxidative phosphorylation. Eucaryotic cells also contain other membrane-bound structures, such as vacuoles, peroxisomes, etc. Procaryotic: Procaryotic cells contain no mitochondria, although mitochondria likely evolved from prokaryotes (“endosymbiotic hypothesis”). Oxidative enzymes are associated with cytoplasmic membrane of cell. Oxidative phosphorylation is associated with the cytoplasmic membrane. A general characteristic of procaryotic cell: No membrane bound structures smaller than the cell itself. There are a few exceptions to this generalization, but there are no elaborate membrane bound structures inside of the cytoplasmic membrane in the procaryotic cell. Procaryotic cells do demonstrate organization at the level of protein localization (e.g., some proteins are localized to the pole of the cell and some to the center of the cell) 3. Mechanism of cellular movement—a third way to distinguish eucaryotic from procaryotic cells. Eucaryotic: Movement may be accomplished by cytoplasmic streaming (amoeboid movement) or by contraction of flagella or cilia. (The flagellum or cilium of eucaryotic cells, when present, is comprised of microtubules in a specific 9 doublet:2 singlet arrangement that is surrounded by a membrane continuous with the cell membrane.) Procaryotic: There is no cytoplasmic streaming or amoeboid movement. (In fact, the cytoplasm of the bacterial cell is very dense, due to a high content of ribosomes

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