BASIC MICROBIOLOGY

1. BASIC MICROBIOLOGY SAMUEL AGUAZIM ( MD)

3. Bacteria: Prokaryotes • Bacteria are prokaryotic cells. • in contrast to eukaryotic cells, they have no nucleus • the genetic material is restricted to an area of the cytoplasm called the nucleoid (area of the cytoplasm in which DNA is located. • DNA of prokaryotes is a single, circular molecule and contains about 2000 genes. • Human DNA has approximately 100,000 genes. • Do not have cytoplasmic compartment such as mitochondria and lysosomes that are found in eukaryotes. • A structure found in prokaryotes but not in eukaryotic animal cells is the cell wall which allows bacteria to resist osmotic stress.

4. Comparison of the nucleus of Eukaryotic and Prokaryotic cells • The Eukaryotic cell has a true nucleuswith multiple chromosomes surrounded by a nuclear membrane:uses a mitotic apparatus to ensure equal allocation of the chromosomes to progeny cells. • The nucleoidof a prokaryotic cell consists of a single circular molecule of loosely organized DNA lacking a nuclear membrane and mitotic apparatus • Eukaryotic cells contain organelles, such as mitochondria and lysosomes, and larger (80S) ribosomes, whereas prokaryotes contain no organelles and smaller (70S) ribosomes. • Prokaryotes have a rigid external cell wall that contains peptidoglycan, a polymer of amino acids and sugars, as its unique structural component. Eukaryotes, on the other

5. Eukayotic Cell

7. SHAPE & SIZE • Bacteria are classified by shape into three basic groups:   1. The cocci are round 2. The bacilli are rods 3. The spirochetes are spiral-shaped. • The arrangement of bacteria is also important. For example, certain cocci occur in: - pairs (diplococci) - chains (streptococci) - grapelike clusters (staphylococci).

8. STRUCTURE Cell Wall: • outermost component common to all bacteria (except Mycoplasmaspecies) • Eukaryotes are bounded by a cell membrane, not a cell wall).

9. The Cell Wall • multilayered structure located external to the cytoplasmic membrane. • inner layer of peptidoglycan • outer membrane (Gram negative) that varies in thickness and chemical composition depending upon the bacterial type. • Peptidoglycan provides structural support and maintains the characteristic shape of the cell.

11. Gram Positive Cell wall NAM NAG Thicker layer of peptidoglycan than Gram negative bacteria

12. Gram Negative Cell Wall LPS is an Endotoxin!! Thinner layer of peptidoglycan than Gram positive bacteria

13. Peptidoglycan Structure Important!!!

14. Gram-negative organisms have: • complex outer layer consisting of lipopolysaccharide, lipoprotein, and phospholipid. • periplasmic space, which is the site, in some species, of enzymes called -lactamases that degrade penicillins and other -lactam drugs. • endotoxin, a lipopolysaccharide (LPS). • polysaccharides and proteins are antigens that are useful in laboratory identification. • porin proteins in the outer membrane form a trimer that acts, as a channel to allow the entry of essential substances such as sugars, amino acids, vitamins, and metals, as well as many antimicrobial drugs such as penicillins.

15. The LPS is composed of three distinct units : see next slide for picture (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 25-40 repeating units of three to five sugars (antigen-O) involved with antigenic variety.

16. Structure of Lipopolysaccharide (LPS) - endotoxin Toxic Effects!!

17. Lipopolysaccharide (LPS): • The LPS of the outer membrane of the cell wall of gram-negative bacteria is endotoxin. • responsible for many of the features of disease, such as fever and shock (especially hypotension), caused by these organisms. • called endotoxin because it is an integral part of the cell wall, in contrast to exotoxins, which are freely released from the bacteria.

18. Amino acids of peptidoglycan Two of these amino acids are worthy of special mention: • diaminopimelic acid, which is unique to bacterial cell walls • D-alanine, which is involved in the cross-links between the tetrapeptides and in the action of penicillin. The other important component in this network is the peptide cross-link between the two tetrapeptides.

19. Peptidoglycan as a target for antibacterial drugs • 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 the penicillins and cephalosporins, inhibit its synthesis by inhibiting the transpeptidasethat makes the cross-links between the two adjacent tetrapeptides.

20. Cell Walls of Acid-Fast Bacteria • Mycobacteria, eg, 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 in the cell wall of lipids called mycolic acids.

21. lysozyme The enzyme lysozyme: • is in human tears, mucus, and saliva • can cleave the peptidoglycan backbone by breaking its glycosyl bonds • contributes to the natural resistance of the host to microbial infection.

22. Cytoplasmic Membrane: • Just inside the peptidoglycan layer of the cell wall • composed of a phospholipidbilayer similar in microscopic appearance to that in eukaryotic cells. • chemically similar, but eukaryotic membranes contain sterols, whereas prokaryotes generally do not. • The only prokaryotes that have sterols in their membrane are the members of the genus Mycoplasma.

23. Ribosomes: differ from eukaryotic ribosomes • Bacterial ribosomes are the site of protein synthesis as in eukaryotic cells but they differ in size and chemical composition. • Bacterial ribosomes are 70Sin size, with whereas eukaryotic ribosomes are 80S in size.

24. Bacterial Ribosome Why don’t they add up? 60 and 40 S in the eukaryotic cell

25. Plasmids: can code for drug resistance • extrachromosomal • double-stranded • circular DNA molecules • capable of replicating independently of the bacterial chromosome.

26. Transposons: • pieces of DNA that move readily from one site to another, either within or between the DNAs of bacteria, plasmids, and bacteriophages. • nicknamed jumping genes. • can code for drug resistance enzymes, toxins, or a variety of metabolic enzymes. • can either cause mutations in the gene into which they insert or alter the expression of nearby genes.

27. Transposons: A summary • Inverted Repeats = integration of the transposon into the recipient DNA. • Transposase gene = enzyme that mediates the excision and integration processes. • Repressor gene= Regulate synthesis of Transposase and Drug resistance gene. • Drug resistance gene product is usually an Enzyme.

28. Specialized Structures Outside the Cell Wall: Capsule: • gelatinous layer covering the entire bacterium. • composed of polysaccharide, except in the anthrax bacillus, which has a capsule of polymerized D-glutamic acid. • Capsules serve as antigen in vaccines (Pneumovax, H.influenzae B, meningococcal vaccines). Conjugation with protein increases immunogenicity and T-cell-dependent response. • sugar components of the polysaccharide vary from one species of bacteria to another and determine the serologic type within a species.

29. Encapsulated Bacteria • Positive quellungreaction - if encapsulated bug is present, capsule swells when specific anticapsularantisera are added. • Examples: Streptococcus pneumonia, Neisseria meningitis, Haemophilusinfluenzae (especially B serotype), and Klebsiella pneumonia • Some NastiesHave Kapsules

32. Flagella:Flagella are long, whip like appendages that move the bacteria toward nutrients and other attractants. Pili (Fimbriae): • hairlike filaments that extend from the cell surface. • shorter and straighter than flagella • composed of subunits of a protein, pilin, arranged in helical strands. They are found mainly on gram-negative organisms.

33. Glycocalyx (slime layer): • polysaccharide coating that is secreted by many bacteria. • covers surfaces like a film and allows the bacteria to adhere firmlyto various structures. e.g.. skin, heart valves, and catheters.

34. Spores: • highly resistant structures are formed in response to adverse conditions • Bacillus - includes the agent of anthrax • Clostridium - includes the agents of tetanus and botulism. • Spore formation - when nutrients, such as sources of carbon and nitrogen, are depleted The spore forms inside the cell and contains: • bacterial DNA • small amount of cytoplasm • cell membrane • Peptidoglycan • very little water • most importantly a thick,keratin like coat that is responsible for the remarkable resistance of the spore to heat, dehydration, radiation, and chemicals

35. Spores: • spores have no metabolic activity and can remain dormant for many years. • exposure to water and the appropriate nutrients, specific enzymes degrade the coat; water and nutrients enter; and germination into a metabolizing, reproducing bacterial cell occurs. • extraordinary resistance to heat and chemicals. • As a result of their resistance to heat, sterilization cannot be achieved by boiling. • Steam-heating under pressure (autoclaving) at 121 0C, usually for 30 minutes, is required to ensure the sterility of products for medical use.

36. Spore Development

37. Growth of Bacterial Cells The first is thelag phase, during which vigorous metabolic activity occurs but cells do not divide. (2) Thelog phase is when rapid cell division occurs. • Beta lactam drugs, such as penicillin, act during the log phase because the drugs are effective when cells are making peptidoglycan (dividing) (3)The stationary phase occurs when nutrient depletion or toxic products cause growth to slow until the number of new cells produced balances the number of the cells that die. (4) The death phase, which is marked by a decline in the number of viable bacteria

38. The bacterial growth curve: # New cells = # of Cell Deaths Death phase a. Lag phase c. Stationary phase b. Log phase d. Death phase : Metabolic activity : Rapid cell division

39. Aerobic and anaerobic growth • For most organisms, an adequate supply of • oxygen enhances metabolism and growth. • The oxygen acts as the hydrogen acceptor in the final steps of energy production catalyzed by the flavoproteins and cytochromes. • Because the use of oxygen generates two toxic molecules, hydrogen peroxide (H2O2) and the free radical superoxide (o.2), bacteria require two enzymes to utilize oxygen.

40. The first enzyme is superoxide • dismutase,which catalyzes the reaction: • free radical superoxidesuperoxide dismutasehydrogen peroxide • 2o.2 + 2H ------------------------------> H2O2 + 02 • 2. second is catalase or peroxidase, which catalyzes the reaction: • hydrogen peroxidecatalase or peroxidase • 2H202---------------------------------------------------------->.2H20 + 02

41. The response to oxygen is important for classifying: • (1)M. tuberculosis: obligate aerobes • require oxygen to grow because their ATP-generating system is dependent on oxygen as the hydrogen acceptor.  • (2) E coli:facultative anaerobes (facultatives) • utilize oxygen to generate energy by respiration • can use the fermentation pathway to synthesize ATP in the absence of sufficient oxygen. • (3) Clostridium tetani:obligate anaerobes • cannot grow in the presence of oxygen because they lack either superoxide dismutase or catalase, or both. • Obligate anaerobes vary in their response to oxygen exposure; some can survive but are not able to grow, whereas others are killed rapidly.

42. Fermentation: • breakdown of a sugar, such as glucose or maltose, to pyruvic acid and then, usually, to lactic acid. • breakdown of a monosaccharide such as glucose, mannose, or galactose. • glycolyticcycle(glyco sugar, lytic breakdown) • the process by which facultative bacteria generate ATP in the absence of oxygen.

43. When oxygen is present • pyruvate produced by fermentation enters the Krebs cycle (oxidation cycle, tricarboxylic acid cycle) • metabolized to two final products, CO2 and H2O. • Krebs cycle generates much moreATP than the glycolytic cycle • facultative bacteria grow faster in the presence of oxygen.