Medical Microbiology Prepared by Ass. Prof. Dr . Wasan A. Bakir

1. Medical Microbiology Prepared by Ass. Prof. Dr. Wasan A. Bakir

2. Bacteriology means the study of bacteria . Bacteria are prokaryotic cells (unicellular organisms with no a nucleus) Typically a few micrometres in length, bacteria have a number of shapes, ranging from spheres to rods and spirals. Mycoplasma, the smallest bacteria (and therefore the smallest cells).

3. The two classes of cells are distinguished by several other characteristics: • (1)Eukaryotic cells have nuclear envelope and nucleus, in prokaryotesthey lacks the nuclear envelope so there is no nucleus. (2) Eukaryotic cellsreproduce by simple and complex processes. But inprokaryotes reproduce by binary fission (simple dividing process). • (3) Eukaryotic cells contain organelles, such as mitochondria and lysosomes, and larger (80S) ribosomes, whereas prokaryotes contain no organelles and smaller (70S) ribosomes. • (4) Most prokaryotes have a rigid external cell wall that contains peptidoglycan, a polymer of amino acids and sugars, as its unique structural component. Eukaryotes, 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.

4. Differences between Eukaryotic and prokaryotes cells

5. Bacterial Structures A variety of structures are found in prokaryotic cells. • Flagella • Pili • Capsule • Plasma Membrane • Cytoplasm • Cell Wall • Lipopolysaccharides • Teichoic Acids • Inclusions • Spores Chapter 4

6. SHAPE OF BACTERIA Most bacterial species are either spherical, called cocci, or rod-shaped, called bacilli. Some bacteria, called vibrio, are shaped like slightly curved rods or comma-shaped; others can be spiral-shaped, called spirilla, or tightly coiled, called spirochaetes. A small number of other unusual shapes have been described, such as star-shaped bacteria. 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).

7. Shapes of Bacteria • Coccus • Chain = Streptoccus • Cluster = Staphylococcus • Bacillus • Chain = Streptobacillus • Coccobacillus • Vibrio = curved • Spirillum • Spirochete • Square • Star Chapter 4

8. Bacterial Cell Structure

9. Important Chemical Components of Surface Structures Cell Wall Peptidoglycans: Unique features of almost all prokaryotic cells. The cell wall is the outermost component common to all bacteria (except Mycoplasma species and Halobacterium halobium, which are bounded by a cell membrane, not a cell wall. Both Gram-positive and Gram-negative bacteria possess cell wall peptidoglycans, which confer the characteristic cell shape and provide the cell with mechanical protection. Peptidoglycan is found onlyin bacterial cells. It is a network that covers the entire bacterium and gives the organism its shape. It is composed of a sugar backbone (glycan) and peptide side chains (peptido).

10. Peptidoglycan structure. A: Peptidoglycan is composed of aglycanchain (NAM and NAG), a tetrapeptide chain, and a cross-link (peptide interbridge).

11. The ß-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine is specifically cleaved by the bacteriolytic enzyme lysozyme. Lysozyme, an enzyme present in human tears, mucus, and saliva, can cleave the peptidoglycan backbone by breaking its glycosyl bonds. Lysozyme-treated bacteria may swell and rupture as a result of the entry of water into the cells, which have a high internal osmotic pressure.protoplasts are formed. These protoplasts are able to survive and continue to grow on suitable media.Gram-negative bacteria treated similarly produce spheroplasts.

12. Cell walls of gram-positive and gram-negative bacteria. Note that the peptidoglycan in gram-positive bacteria is much thicker than in gram-negative • bacteria. Note also that only gram-negative bacteria have an outer membrane • containing endotoxin (lipopolysaccharide [LPS]) and have a periplasmic space • where β-lactamases are found. Several important gram-positive bacteria have teichoic acids.

13. Comparison of Cell Walls of Gram-Positive and Gram-Negative Bacteria

14. Lipopolysaccharides: • 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, incontrastto exotoxins, which are actively secreted from the bacteria.

15. Endotoxins possess an array of powerful biologic activities and play an important role in the pathogenesis of many Gram-negative bacterial infections. In addition to causing 1- endotoxicshock 2- LPS is pyrogenic 3- can activate macrophages and complement 4- it is mitogenic for B lymphocytes 5- induces interferon production 6- causes tissue necrosis. The LPS is composed of three distinct units 1- lipid A anchor2- polysaccharide core3- chains of carbohydrates (mainly penta sugars).

16. 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 through ketodeoxyoctulonate • (KDO) 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. Chapter 4

17. Teichoic Acids: • They are composed of polymers of either glycerol phosphate or ribitolphosphate. bearing a strong negative charge. located in the outer layer of the gram-positive cell wall and extend from it. They are covalently linked to the peptidoglycan in some Gram-positive bacteria. They are strongly antigenic, but are generally absent in Gram-negative bacteria. • Give - charge to outer surface of Gram+. • Some polymers of glycerol teichoicacid penetrate the peptidoglycan layer and are covalently linked to the lipid in the cytoplasmic membrane, in which case they are called lipoteichoicacid that are considered as bridges connecting cell membrane to cell wall in Gram positive bacteria. They are antigenic and cytotoxic.

18. Periplasmicspace: This is a layer found between the cell membrane and the cell wall, other authors say it lays between the peptydoglycan layer and the outer membrane layer of the gram negative bacterial cell wall, which plays a very important role in (1) maintaining cell’s osmotic pressure, and also (2) as reservoir for digestive enzymes before their secretion to the outside of the cell. • Cytoplasmic Structures; • Plasma Membrane: The bacterial plasma membrane is composed primarily of protein and phospholipid (about 3:1). It performs many functions, including (1) transport of molecules into the cell (2) synthesis of precursors of the cell wall(3) energygeneration (4) secretion of enzymes and toxins.

19. Cellular Organelles: 1- 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, whereas eukaryotic ribosomes are 80S in size.

20. 2- The Nucleoid Prokaryotic and eukaryotic cells were initially distinguished on the basis of structure: the prokaryotic nucleoid, the equivalent of the eukaryotic nucleus, is structurally simpler than the true eukaryotic nucleus. Because the nucleoid contains no nuclear membrane, no nucleolus, no mitotic spindle, there is little resemblance to the eukaryotic nucleus.. Under the light microscope, the nucleoid of the bacterial cell can be seen with the aid of Feulgen staining, which stains DNA. The DNA is then seen to be a single, continuous, circular molecule.

21. 3- Plasmids These are extra chromosomal circular DNA that are capable of replicating independent of the bacterial chromosome. The plasmids mainly carry genes responsible for characteristics like fertility, antibiotic resistance and production of bacteriocin (a protein that kills closely related bacteria). • The plasmids can be easily isolated from or introduced into the bacterial cells. They can be integrated with desired genes. Hence, plasmids are use in genetic engineering.

22. 4- Mesosomes Thin sections of Gram-positive bacteria reveal the presence of vesicular or tubular-vesicular membrane structures called mesosomes, which are apparently formed by an invagination of the plasma membrane. These structures are much more prominent in Gram-positive than in Gram-negative organisms.

23. Indeed, electron-microscopic studies have suggested that the mesosomes, as usually seen in thin sections, may arise from membrane perturbation and fixation artifacts. some evidence indicates that mesosomes may be related to events in the cell division cycle, as well as in sporulation.

24. Surface Appendages Two types of surface appendage can be recognized on certain bacterial species: the flagella, which are organs of locomotion, and pili, which are also known as fimbriae. . Flagella • Someprokaryoteshave distinct appendages that allow them to move about or adhere to solid surfaces. • Consist of delicate strands of proteins. • Flagella: Long, thin extensions that allow some bacteria to move about freely in aqueous environments.

25. Response to chemical stimuli involves a sensory system of receptors that are located in the cell surface and/or periplasm and that transmit information to methyl-accepting chemotaxis proteins that control the flagellar motor. The protein subunits of a flagellum (flagellins) are assembled in a helical shape to form a cylindrical structure with a hollow core. The ability of bacteria to swim by means of the propeller-like action of the flagella that move the bacteria toward nutrients and other attractants, a process called chemotaxis(movement in response to attractant and repellent substances in the environment)

26. Flagella are medically important for two reasons: 1-Some species of motile bacteria (e.g., E. coli and Proteus species) are common causes of urinary tract infections. Flagella may play a role in pathogenesis by propelling the bacteria up the urethra into the bladder. 2- Some species of bacteria (e.g., Salmonella species) are identified in the clinical laboratory by the use of specific antibodies against flagellar proteins. Flagellated bacteria have a characteristic number and location of flagella: some bacteria have one, and others have many; in some, the flagella are located at one end, and in others, they are all over the outer surface. Only certain bacteria have flagella. Spirochetes move by using a flagellum like structure called the axial filament, which wraps around the spiral-shaped cell to produce an undulating motion.

27. Typical arrangements of bacterial flagella

28. Pili (Fimbriae) Piliare 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. Pilihave two important roles: (1) Short, 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) Long, A specialized kind of pilus, the sex pilus, forms the attachment between the male (donor) and the female (recipient) bacteria during conjugation.

29. Some bacteria have both flagella and pili. The electron micrograph below, shows the characteristic wavy appearance of flagella and two types of pili on the surface of Escherichia coli. Pili. on an Escherichia coli cell. The short pili (fimbriae) mediate adherence; the sex pilus is involved in DNA transfer.

30. Electron micrograph of negatively stained E coli showing wavy flagella and numerous short, thinner, and more rigid hairlike structures, the pili

31. Capsules and Loose Slime • The capsule is a gelatinous layer covering the entire bacterium. It is composed of polysaccharide, except in the bacillus anthrax, which has a capsule of polymerized D-glutamic acid.Some organisms lack a well-defined capsule but have loose slime, slime layers external to the cell wall or cell envelope.

32. Not all bacterial species can produce capsules, however, the capsules of encapsulated pathogens are often important determinants of virulence. Encapsulated species are found among both Gram-positive and Gram-negative bacteria. In both groups, most capsules are composed of high molecular-weight viscous polysaccharides that are retained as a thick gel outside the cell wall or envelope. The capsule is important: 1- It is a determinant of virulence of many bacteria since it limits the ability of phagocytes to engulf 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.

33. 3- Specific identification of an organism can be made by using antiserum against • the capsular polysaccharide. In the presence of the antibody, the capsule will swell greatly. This swelling phenomenon, is called the quellungreaction, which is used in the clinical laboratory to identify certain organisms. • 4- Capsular polysaccharides are used as the antigens in certain vaccines

34. The glycocalyx (slime layer) 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 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.

35. Other Intracellular Components • Other granular inclusions randomly distributed in the cytoplasm of various species, that serve as storage areas for nutrients. include; metabolic reserve particles such as; poly-b-hydroxybutyrate(PHB), polysaccharide,glycogen-likegranules, and polymetaphosphateor metachromaticgranules. • Metachromatic granules are a characteristic feature of Corynebacteriumdiphtheriae, that cause of diphtheria.

36. Endospores • Endospores are highly heat-resistant, dehydrated resting cells formed intracellularly. These highly resistant structures are formed by two genera of medically important gram-positive rods: the genus Bacillus, which includes the agent of anthrax, and the genus Clostridium, which includes the agents of tetanus and botulism. 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 resistance of the spore to heat, dehydration, radiation, and chemicals.

37. Location The position of the endospore differs among bacterial species and is useful in identification. Terminalendospores are seen at the poles of cells, whereas central endospores are in the middle. Subterminalendospores are those between these two extremes. Examples of bacteria having terminal endospores include Clostridium tetani, the pathogen that causes the disease tetanus. Bacteria having a centrally placed endospore include Bacillus cereus.

38. Where it finally consisting of the following layers; 1- The central core: contains the minimized contents of the spore plus a single strand of DNA and high content of Caliciumdipicolinate. 2- The cortex layer: which contains a double layer of peptidoglycan, which is more resistant. 3- The spore coat:contains special hard resistant protein. 4- The keratinized exosporium: contains keratinized resistible

39. Bacterial Structures