Hart C. Anthony, Shears Paul. Color Atlas of Medical Microbiology.pdf

The Morphology and Fine Structure of Bacteria 155

The Cell Wall of Gram-Negative Bacteria

Lipopolysaccharide (LPS)

Outer membrane

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space

Murein

Cytoplasmic membrane

Fig. 3.11 Note the characteristic thin murein layer and the outer membrane connected to it by proteins (OmpA, murein lipoprotein). Many different proteins are localized in the outer membrane. Its outer layer is made up of closely packed lipopolysaccharide complexes (see Fig. 3.12).

& Outer membrane proteins.

—OmpA (outer membrane protein A) and the murein lipoprotein form a bond between outer membrane and murein.

—Porins, proteins that form pores in the outer membrane, allow passage of hydrophilic, low-molecular-weight substances into the periplasmic space.

—Outer membrane-associated proteins constitute specific structures that enable bacteria to attach to host cell receptors.

—A number of Omps are transport proteins. Examples include the LamB proteins for maltose transport and FepA for transport of the siderophore ferric (Fe3+) enterochelin in E. coli (see also p. 13).

& Lipopolysaccharide (LPS). This molecular complex, also known as endotoxin, is comprised of the lipoid A, the core polysaccharide, and the O-specific polysaccharide chain (Fig. 3.12).

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The Lipopolysaccharide Complex

Fig. 3.12 The three-part lipopolysaccharide complex (LPS) of Gram-negative bacteria is anchored in the outer membrane by means of its lipid moiety. LPS is also known as endotoxin.

—Lipoid A is responsible for the toxic effect. As a free substance, or bound up in the LPS complex, it stimulates—by binding together with the LPS binding protein (LBP) to the CD14 receptor of macrophages—the formation and secretion of cytokines that determine clinical endotoxin symptomatology. Interleukin 1 (IL-1) and tumor necrosis factor (TNF) induce an increased synthesis of prostaglandin E2 in the hypothalamus, thus setting the “thermostat” in the temperature control center higher, resulting in fever. Other direct and indirect endotoxin effects include granulopoiesis stimulation, aggregation and degeneration of thrombocytes, intravasal coagulation due to factor VII activation, a drop in blood pressure, and cachexia. LPS can also activate the alternative complement pathway. Release of large amounts of endotoxincanlead toseptic(endotoxic) shock. Endotoxinisnotinactivated byvapor sterilization. Therefore,the parentmaterials used inproduction of parenteral pharmaceuticals must be free of endotoxins (pyrogens).

—The O-specific polysaccharide chain is the so-called O antigen, the fine chemical structure of which results in a large number of antigenic variants useful in bacterial typing (e.g., detailed differentiation of salmonella types) (see p. 284f.).

L-forms (L = Lister Institute). The L-forms are bacteria with murein defects, e.g., resulting from the effects of betalactam antibiotics. L-forms are highly

The Morphology and Fine Structure of Bacteria 157

unstable when subjected to osmotic influences. They are totally resistant to betalactams, which block the biosynthesis of murein. The clinical significance of the L-forms is not clear. They may revert to the normal bacterial form when betalactam therapy is discontinued, resulting in a relapse.

Capsule

Flagella

Flagella give bacteria the ability to move about actively. The flagella (singular flagellum) are made up of a class of linear proteins called flagellins. Flagel-

Bacterial flagella

Fig. 3.13 a Flagellated bacterial cell (SEM, 13 000!). b Helical structure of bacterial flagella (SEM, 77 000!).

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Biofilm

A bacterial biofilm is a structured community of bacterial cells embedded in a self-produced polymer matrix and attached to either an inert surface or living tissue. Such films can develop considerable thickness (mm). The bacteria located deep within such a biofilm structure are effectively isolated from immune system cells, antibodies, and antibiotics. The polymers they secrete are frequently glycosides, from which the term glycocalyx (glycoside cup) for the matrix is derived.

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&Certain oral streptococci (S. mutans) bind to the proteins covering tooth enamel, then proceed to build a glucan matrix out of sucrose. Other bacteria then adhere to the matrix to form plaque (Fig. 3.14), the precondition for destruction of the enamel and formation of caries (see p. 243f.).

&Oral streptococci and other bacteria attach to the surface of the cardiac valves to form a biofilm. Professional phagocytes are attracted to the site and attempt, unsuccessfully, to phagocytize the bacteria. The frustrated phagocytes then release the tissue-damaging content of their lysosomes (see p. 23), resulting in an inflammatory reaction and the clinical picture of endocarditis.

Bacterial Spores

Bacterial spores (endospores) are purely dormant life forms. Their development from bacterial cells in a “vegetative” state does not involve assimilation of additional external nutrients. They are spherical to oval in shape and are characterized by a thick spore wall and a high level of resistance to chemical and physical noxae. Among human pathogen bacteria, only the genera Clostridium and Bacillus produce spores. The heat resistance of these spores is their most important quality from a medical point of view, since heat ster-

Dental Plaque

Fig. 3.14 Dental plaque can be rendered visible with an erythrosin stain.

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ilization procedures require very high temperatures to kill them effectively. Potential contributing factors to spore heat resistance include their thick wall structures, the dehydration of the spore, and crosslinking of the proteins by the calcium salt of pyridine-2,6-dicarboxylic acid, both of which render protein denaturing difficult. When a spore’s milieu once again provides favorable conditions (nutrient medium, temperature, osmotic pressure, etc.) it returns to the vegetative state in which spore-forming bacteria can reproduce.

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The Physiology of Metabolism and Growth in Bacteria

& Human pathogenic bacteria are chemosynthetic and organotrophic (chemo-organotrophic). They derive energy from the breakdown of organic nutrients and use this chemical energy both for resynthesis and secondary activities. Bacteria oxidize nutrient substrates by means of either respiration or fermentation. In respiration, O2 is the electron and proton acceptor, in fermentation an organic molecule performs this function. Human pathogenic bacteria are classified in terms of their O2 requirements and tolerance as facultative anaerobes, obligate aerobes, obligate anaerobes, or aerotolerant anaerobes. Nutrient broth or agar is used to cultivate bacteria. Nutrient agar contains the inert substrate agarose, which liquefies at 100 8C and gels at 45 8C. Selective and indicator mediums are used frequently in diagnostic bacteriology.

Bacteria reproduce by means of simple transverse binary fission. The time required for complete cell division is called generation time. The in-vitro generation time of rapidly proliferating species is 15–30 minutes. This time is much longer in vivo. The growth curve for proliferation in nutrient broth is normally characterized by the phases lag, log (or exponential) growth, stationary growth, and death. &

Bacterial Metabolism

Types of Metabolism

Metabolism is the totality of chemical reactions occurring in bacterial cells. They can be subdivided into anabolic (synthetic) reactions that consume energy and catabolic reactions that supply energy. In the anabolic, endergonic

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reactions, the energy requirement is consumed in the form of light or chemical energy—by photosynthetic or chemosynthetic bacteria, respectively. Catabolic reactions supply both energy and the basic structural elements for synthesis of specific bacterial molecules. Bacteria that feed on inorganic nutrients are said to be lithotrophic, those that feed on organic nutrients are organotrophic.

Human pathogenic bacteria are always chemosynthetic, organotrophic bacteria (or chemo-organotrophs).

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Catabolic Reactions

Organic nutrient substrates are catabolized in a wide variety of enzymatic processes that can be schematically divided into four phases:

Digestion. Bacterial exoenzymes split up the nutrient substrates into smaller molecules outside the cell. The exoenzymes represent important pathogenicity factors in some cases.

Uptake. Nutrients can be taken up by means of passive diffusion or, more frequently, specifically by active transport through the membrane(s). Cytoplasmic membrane permeases play an important role in these processes.

Preparation for oxidation. Splitting off of carboxyl and amino groups, phosphorylation, etc.

Oxidation. This process is defined as the removal of electrons and H+ ions. The substance to which the H2 atoms are transferred is called the hydrogen acceptor. The two basic forms of oxidation are defined by the final hydrogen acceptor (Fig. 3.15).

&Respiration. Here oxygen is the hydrogen acceptor. In anaerobic respira-

tion, the O2 that serves as the hydrogen acceptor is a component of an inorganic salt.

&Fermentation. Here an organic compound serves as the hydrogen acceptor.

The main difference between fermentation and respiration is the energy yield, which can be greater from respiration than from fermentation for a given nutrient substrate by as much as a factor of 10. Fermentation processes involving microorganisms are designated by the final product, e.g., alcoholic fermentation, butyric acid fermentation, etc.

The energy released by oxidation is stored as chemical energy in the form of a thioester (e.g., acetyl-CoA) or organic phosphates (e.g., ATP).

Fig. 3.15 In oxidation of organic nutrient substrates, protons (H+) and electrons (e–) are transferred in more or less long chains. The respiration is aerobic when the final electron acceptor is free oxygen. Anaerobic respiration is when the electrons are transferred to inorganically bound oxygen. Fermentation is the transfer of H+ and e– to an organic acceptor.

The role of oxygen. Oxygen is activated in one of three ways:

&Transfer of 4e– to O2, resulting in two oxygen ions (2 O2–).

&Transfer of 2e– to O2, resulting in one peroxide anion (1 O22–).

&Transfer of 1e– to O2, resulting in one superoxide anion (1 O2–).

Hydrogen peroxide and the highly reactive superoxide anion are toxic and therefore must undergo further conversion immediately (see Fig. 3.15).

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Bacteria are categorized as the following according to their O2-related behavior:

&Facultative anaerobes. These bacteria can oxidize nutrient substrates by means of both respiration and fermentation.

&Obligate aerobes. These bacteria can only reproduce in the presence of O2.

&Obligate anaerobes. These bacteria die in the presence of O2. Their

Anabolic Reactions

It is not possible to go into all of the biosynthetic feats of bacteria here. Suffice it to say that they are, on the whole, quite astounding. Some bacteria (E. coli) are capable of synthesizing all of the complex organic molecules that they are comprised of, from the simplest nutrients in a very short time. These capacities are utilized in the field of microbiological engineering. Antibiotics, amino acids, and vitamins are produced with the help of bacteria. Some bacteria are even capable of using aliphatic hydrocarbon compounds as an energy source. Such bacteria can “feed” on paraffin or even raw petroleum. It is hoped that the metabolic capabilities of these bacteria will help control the effects of oil spills in surface water. Bacteria have also been enlisted in the fight against hunger: certain bacteria and fungi are cultivated on aliphatic hydrocarbon substrates, which supply carbon and energy, then harvested and processed into a protein powder (single cell protein). Culturing of bacteria in nutrient mediums based on methanol is another approach being used to produce biomass.

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Metabolic Regulation

Bacteria are highly efficient metabolic regulators, coordinating each individual reaction with other cell activities and with the available nutrients as economically and rationally as possible. One form such control activity takes is regulation of the activities of existing enzymes. Many enzymes are allosteric proteins that can be inhibited or activated by the final products of metabolic pathways. One highly economical type of regulation controls the synthesis of

3enzymes at the genetic transcription or translation level (see the section on the molecular basis of bacterial genetics (p. 169ff.).

Growth and Culturing of Bacteria

Nutrients

The term bacterial culture refers to proliferation of bacteria with a suitable nutrient substrate. A nutrient medium (Table 3.2) in which chemoorganotrophs are to be cultivated must have organic energy sources (H2 donors) and H2 acceptors. Other necessities include sources of carbon and nitrogen for synthesis of specific bacterial compounds as well as minerals such as sulfur, phosphorus, calcium, magnesium, and trace elements as enzyme activators. Some bacteria also require “growth factors,” i.e., organic compounds they are unable to synthesize themselves. Depending on the bacterial species involved, the nutrient medium must contain certain amounts of O2 and CO2 and have certain pH and osmotic pressure levels.

Table 3.2 Nutrient Mediums for Culturing Bacteria