Lecture 1 Notes (chapter 1)

MICROBIOLOGY 20: UNIT 1M.M.Youssef, MD, MSc

Introduction To Microbiology and Bacteria

-This unit aim to introduce Microbiology as a science and bacteria as a cell.

Note Outline for this session

-What is Microbiology?The study of microbes, which are microscopic organisms.

-What is the importance of studying Microbiology?-Microorganisms are part of the human environment and related to human health and sickness.-Microorganisms are responsible for wide range of diseases, known as infectious diseases; also they play an important role in some malignant diseases (tumors)

-Microorganisms not always harmful, less than 1% of known microorganisms are pathogenic (disease causing).-In fact Microorganisms are important to the human environment.

-What are the kinds of Microorganisms? -Algae ,Fungi , and protozoa also Helminths(worms) and Arthropods (insects) have microscopic stages in their life cycles (Eukaryotes) -Bacteria (prokaryotes) -Viruses ,Viroids , and Prions ( Acellular microorganisms) Germ Theory of DiseaseMicroorganisms (germs) can invade other organisms and cause disease (19th century).It wasnt very accepted when it came to life.The problem was how do you know something is there that you cannot see? The key answer was the Microscope, which opened the door of the microbial world.Anton Van Leeuwenhook invented a microscope that magnified 100-300X.Still the question where do the microbes come from?

Spontaneous Generation TheoryMicroorganisms come from nonliving material.Do you think that life come from non-life?What types of observation would lead to this theory?

Louis Pasteur is the one who succeeded to disapprove the spontaneous generation theory (1860).He used three flasks with broth;(1) Left open to air(2) Boiled and left open to air(3) Boiled and left open with curved outlet to air (swan-necked flask)

(1) and (2) get turbid (3) stay clean.

So,because of Pasteurs experiments spontaneous generation theory is refused.Kochs Postulates :Provided method of establishing the specific infectious cause of a disease;

1-The specific causative agent must be found in EVERY case of disease.

2-The disease causing organism must be isolated in a pure culture.

3-Inoculation of a sample of the culture into a healthy susceptible animal must produce the same disease.4-The disease causing organisms must be recovered from the inoculated animal.According to these postulates he assumed that an infectious disease is caused by a single organism (one organism-one disease concept)

Do you think it is perfect? Why?

LECTURE 2 NOTES (chapter 4)

What Is a Cell? Cells are the fundamental units of life and carry out all the basic functions of living things. (Cell Theory)

What do the cells need?1-Barrier from the environment (cell membrane)2-Genetic infomations (DNA, Nucleolus)3-Proteins to do stuff.

Two types of cells:PROKARYOTIC - have no membrane enclosed organelles, only one membrane surrounding entire cell, they all are single celled organisms (bacteria)EUKARYOTIC have membrane enclosed organelles, a nucleolus, multicellular or single celled organisms

Prokaryotic cells (bacteria) varies in size, shape, and arrangements, but they all multiply by Binary Fission, rather than mitosis or meiosis

Bacterial cell wall: semi rigid cell wall lies outside cell membrane in most bacteria, and it performs two main functions: 1-Maintain the characteristic shape of the cell.2-prevents the cell from bursting by fluid diffusion into the cell.

-Peptidoglycan: The major component of a cell wall.

Large polymer ,covalently linked molecule, like a large, multiple layers of chain-link fence surrounding the bacterial cell (This is a very specific bacteria thing)

It is composed of sugar and peptides.

Sugars (backbone): N-acetylglucosamine (glu NAC) N-acetylmuramic acid (mur NAC)

Cross-linking: Tetra peptide, (chains of four amino acids joined together),[L-alanine, D-glutamic acid, Diamino Pimellic acid (replaced by Lysine in most gm +ve bacteria and D-alanine]

Gram-positive bacteria have an additional molecule in the cell wall, called Teichoic acid; its a huge polymer that sticks out of cells (extends beyond the rest of the cell wall, providing attachment sites for bacteriophages)

N.B. Peptidolycan, forms a large supporting network around the bacterium, but doesnt hold things in and out it is porous.It is found in both gram-positive and gram-negative bacteria, but it is much thicker in gram-positive cells.

-Outer Membrane:It is a bilayer membrane, found primarily in gram-negative bacteria.It forms the outermost layer of the cell wall and is attached to peptidoglycan by small lipoproteins molecules.Outer membrane protects the cell, exerts little control over the movement of substances in and out the cell, it also inhibits the entrance of penicillin into gram-negative bacteria. Lipo polysaccharide (LPS)It is also called Endotoxin. It is important part of cell membrane and it is an integral part of the cell wall and it is not released until the death of bacteria (cell wall lyses).It is composed of polysaccharide (sugars that identify gm-ve cells) and lipid A (toxic part), its release causes fever and blood vessels dilatation, which causes drop of blood pressure (hypotenstion).

N.B. Antibiotics given late in gm-negative infection may worsen the condition and cause death.

-Periplasmic space:Highly evident in gram-negative bacteria, it is a gap between the cell membrane and the cell wall.It represents a very active area of cell metabolism, as it contains peptidoglycan. Many digestive enzymes and transport proteins,that destroy harmful substances and transport metabolites into bacterial cytoplasm.

In gram-positive, the periplasm (not periplasmic space) is where metabolic digestion occurs and where new cell wall peptidoglycan is attached (it is more like a part of the cell wall).

Distinguishing bacteria by cell wallsCertain properties of cell walls produce different staining reactions, which can distinguish between gm-positive, gm-negative and acid fast bacteria.

1- Gram-positive bacteria: Peptidoglycan layer periplasm . Cell membrane

Cell wall is relatively a thick layer of peptidoglycan (60-90% of cell wall), which is closely attached to the outer surface of cell membrane (no periplasmic space) They retain gram stains because of their thick layer walls and their structures. At time, cell walls are weaker breakdown of peptidoglycan), which could become Gram-variable or even Gram-negative.

2-Gram-Negative Bacteria: Cell wall is thinner but more complex than gram-positive bacteria.Only 10-20% of cell wall is peptidoglycan, the remainder consists of polysaccharides, proteins and lipids.

They fail to retain Crystal Violet-iodine dye because of their thin walls and its structure (lipoproteins and lipopolysaccharides).

3-Acid-Fast Bacteria (Mycobacteria):

-Lipids around cell protects from most stains (60% of cell wall).-Lipid wall likely inhibits nutrients, so it grows slowly.-They stain gram-positive.-Large layer of lipids in the cell wall protects from acids, alkalis and detergent. But requires a lot of nutrients.

Members of Mycobacteria family include Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacteria avium.

Wall-deficient Organisms

Mycoplasma have no cell walls. The cell membrane contains sterols (more like Eukaryotic cells). They dont have cell shape (pleomorphism) and are still subject to osmolarity changes.

L-Forms are wall-deficient bacteria (naturally or by chemical treatment). They can cause chronic or recurrent infection, if they rebuild their cell walls (they dont killed by antibiotics that affect cell wall.

Lecture 3 notes(chapter 4)

Cell Membrane (Plasma membrane, Cytoplasm membrane)

It is a living membrane that forms a barrier between the cell and its environment. Bacterial cell membrane has a similar structure to other cell membranes ( Eukaryotic cell). It consists mainly of phospholipid and proteins, where, phospholipids in the membrane are in fluid state and proteins are dispersed among them. (Fluid-mosaic model).Phospholipid membrane forms a bilayer with:-Phosphate ends that extends toward the membrane surface. They are charged and hydrophilic (water-loving). So, they can interact with watery environment.-Fatty acid ends extend inward, they are non-charged and hydrophobic (water-fearing). So, they form a barrier between the cell and its environment. Protein molecules are interspersed among the lipid molecules.Cell membranes are dynamic, materials move through it in a sensitive way. What are the functions of cell membranes?

Internal Structure: Cytoplasm:It is a semi fluid substance inside the cell membrane. It is made up of 80% water and 20% of dissolved stuff; it is like a suspension that contains enzymes, proteins, carbohydrates, lipids, and inorganic ions.

Nuclear regions:Unlike Eukaryote, there is no nuclear membrane. It contains one large circular piece of DNA (chromosome).There are sometimes smaller circular pieces called plasmids(plasmid is supplementary and not critical to cell survival).

Ribosome:Ribosome Consists of RNA and proteins. -Can be grouped in long chains called polyribosomes. -They are the sites of protein synthesis. -Ribosomes are spherical in shape, they contain two subunits, large (50 S) and small (30S). Both together are (70S), which is smaller than Eukaryotic ribosomes (80S).

N.B. Certain antibiotics, e.g. Erthromycin and Streptomycin, bind to 70S ribosomes and disrupt bacterial protein synthesis without affecting the host cells.

Endospores:Vegetative cellsactively metabolizing nutrients Resting cellsNo nutrients (Resting stages)..Endospores

Endospores are very common in Bacilli and ClostridiumBactreial endospores (single per cell),they help organism to survive, unlike fugal spores, that are for reproductionEndospores ,are formed within cells,they contain very little water,they are highly resistant To heat,drying,acids,bases,radiations,and disinfectants.They help bacteria to survive for long times in harsh conditions.

External Structures.Flagella (movement structures in motile bacteria), Axial Filaments (in Spirochetes), Pilli (attachment and conjugation), and Glyocalyx.

Let us move on to a Eukaryotic cell:Plasma membrane, comparable to cell membraneSterols, used in plasma membrane to keep rigidity and still retain fluid mosaic model

Cell NucleusSurrounded by nuclear envelopeNuclear pores let things in and out (like what)Nucleoplasm is the inside plasma of the nucleusNucleoli are sites of ribosome assembly (made up of RNA and proteins)Paired chromosomes of DNA, which are compacted together with proteins called histones. Chromatin is the protein/DNA mix that gives the nucleus a granular appearance.

Division of eukaryotes is by Mitosis and Meiosis.

MitochondriaPowerhouse of the cellsMany in each cellHave Outer membrane, inner membrane and a matrix insideThese organelles make ATP (the energy source for cells)

RibosomesBigger than bacterial, made up of RNA and protein (80S)

Endoplasmic ReticulumNetwork of membranesSmooth is where lipids are madeRough has ribosomes attached for making proteins

Golgi Apparatus (named after Golgi)A stacks of membranous sacs, gets stuff from ER and alters their chemical structures, and packing them in secretory vesicles. It also makes the Lysosomes, which are vesicles that contain digestive enzymes.

Lecture 4 Notes(chapter 5)Bacterial metabolism:

Metabolism is the sum of all chemical processes carried out by living organism.It includes anabolism and catabolism.Anabolism is the reactions that require energy to synthesize complex molecules from simpler ones.Catabolism is the reactions that release energy by breaking complex molecules into simpler ones.All catabolic reactions involve electron transfer, which allow energy to be captured in high-energy bonds in ATP.

_Oxidation is loss of electrons._Reduction is gain of electrons.

How microorganisms obtain energy?Microorganisms are classified as:Autotrophy (self-feeding) or Hetrotrophy (other-feeding)Nearly all microorganism infections are chemoheterotrophs.

Metabolic pathways can be catabolic (produces energy that cell can use), or anabolic (uses energy to make structural complexes, enzymes and other functional molecules.ATP molecules are the links that couple anabolic and catabolic pathways.

Enzymes are special category of proteins found in all living organisms.Chemical reactions that release energy needs energy to start the reaction, which is called Activation energy.Enzymes lower the amount of activation energy needed to initiate the reaction, thus they make it possible to occur at relatively low temperatures that living cells tolerate.Enzymes also provide a surface on which reactions take place. Each enzyme has a certain area on its surface called the Active site (binding site); it is the region at which the enzyme binds (forms a loose association) with its substrate.When the substrate binds to the active site of the enzyme, they form enzyme-substrate complex. Which leads to a chemical change in substrate that results in product formation and enzyme detachment.Enzymes have a high degree of specificity, they catalyze only one type of reaction, and most act on only one particular substrate.The active site accounts for the enzyme specificity.Enzymes are usually named by adding (suffix-ase) to the name of the substrate on which they act.Enzymes can be divided into endoenzymes and exoenzymes according to their site of action. Many enzymes need to associate with non-protein substances called coenzymes or cofactors, so they can catalyze their reactions.A Coenzyme is a non-protein organic molecule that bounds to an enzyme. Many of them are synthesized from vitamins, e.g. coenzyme A, NAD, and FAD.A cofactor is usually an inorganic ion, e.g. iron, zinc, or magnesium. Cofactors often improves the fit of an enzyme with its substrate, also they are essential for the reaction to proceed.

N.B. Carrier molecules such as coenzymes carry hydrogen atoms and electrons in many oxidative reactions.

The cells need to inhibit (control) its enzyme activities by different methods;A- Competitive inhibitionB- Non-Competitive inhibitionC- Feedback inhibition

Factors that affect the rate of enzyme reactions include: temperature, PH, and concentrations of substrate, product and enzyme.

Most human enzymes and those of human- infecting microbes have an optimum temperature near normal body temperature and an optimum PH near neutral, at which they catalyze reactions most rapidly.

LECTURE 5 NOTES(chapter 5)

Glycolysis, Fermentation, and Aerobic respiration are the metabolic processes used by most microorganisms to capture energy.

-Glycolysis ( Embden-Meyerhof pathway) is the metabolic pathway used by most organisms (both aerobes and anaerobes) to breakdown glucose.It consists of 10 steps, but the most important steps are:1- Phosphorylation of glucose (transfer of phosphate group from ATP to glucose).2- Breaking of glucose (six-carbon molecule) into two (three carbon molecule).3- Transfer of two electrons to the coenzyme NAD.4- The capture of energy in ATP.

Glycolysis (glucose oxidation or breakdown) result in a net energy capture of only two ATPs per glucose molecule, as one glucose molecule is metabolized by glycolysis into two molecules of pyruvic acid. Also, two molecules of reduced NAD (NADH) are also produced.Once the sugar has entered glycolysis , it is metabolized to pyruvic acid and then fermented or metabolized aerobically.

-Fermentation, is a process by which pyruvic acid undergoes subsequent metabolism in absence of oxygen.Fermentation aim to pass the electrons from reduced NAD (NADH) off to other molecules (NAD recycling). It doesnt result in capture of energy in ATP, but it helps energy capture indirectly by keeping glycolysis going.The most important and commonly occurring pathways are Homolactic acid fermentation and Alcholic fermentation.Other kinds of fermentations are performed by some microorganisms can be important in diagnosis of microbial infection and identifying the causative agent. Aerobic organisms obtain some energy from glycolysis and they use it to produce more energy from glucose by aerobic respiration via the krebs cycle and oxidative phosphorylation .

-Krebs cycle ( tricarboxylic acid cycle or citric acid cycle)It is a sequence of reactions in which, acetyl groups are oxidized to carbon dioxide. Hydrogen atoms are also removed, and their electrons are transferred to coenzymes that serve as electron carriers. (The hydrogens are eventually combined with oxygen to form water).Certain events in the krebs cycle are of special significance:-Oxidation of carbon. (Give carbon dioxide)-Transfer of electrons to coenzymes.-Substrate level energy captures.

Electron transport and oxidative phosporylation:

They can be modeled as a waterfall series. As the electrons are passed from carrier to carrier in the chain, they decrease in energy, and some of the energy they lose is directed to make ATP molecules (energy stores).

From the metabolism of a single glucose molecule, 10 paris of electrons are transported by NAD and only 2 pairs by FAD.Krebs cycle give atotal yield of 38 ATPs per glucose molecule(10 pairs of electrons from NAD produce 30 ATPs, 2 pairs from FAD produce 4 ATPs, in addition to 2 ATPs from glycolysis and 2 GTPs from krebs cycle that transferred eventually into 2 ATPs)

Oxidative phosphorylation generates much more energy than fermentation, which produces only 2 ATPs (about 5%).

The theory of chemiosmosis explains how energy is used to synthesize ATP.

Anaerobic respiration done in anaerobes that dont use free oxygen as their final electron acceptor.They only use parts of kerbs cycle and the electron transport chain; thus, they produce less ATP molecules than aerobic organisms.

Fat metabolism . fats (lipids) are hydrolyzed into glycerol and fatty acids. The glycerol is metabolized by glycolysis and the fatty acids are oxidized by beta-oxidation after they combine with coenzyme A ,forming Acetyl-CoA that enters the krebs cycle.

Proteins can also be metabolized for energy. They are first hydrolyzed into amino acids that are deaminated (amino group is removed), and then the deaminated molecules can enter glycolysis, fermentation or the krebs cycle.

What are the uses of energy in microorganism?

Lecture 6&7 (chapter 7) Microbial genetics

Gene (a specific segment of DNA) is the basic unit of heredity; it is a linear sequence of nucleotides that form a functional unit of chromosome.

DNA structure is a:Double helixAnti-parallel (5-> 3 and 337.8>100.5Rectal>38.4>101.5Infection Max40104.5Mortal Max43109.4

The hypothalamus controls much of the physiology of the body including temperatureFever is the resetting of the normal body temperature caused by infection, vaccinations, tissue injury. It is Not increasing the temp, but resetting to a higher temp.

Caused by pyrogen release into the blood.Exogenous pyrogens are from infectious agents (like a toxin from a microbe)Endogenous pyrogens are from the body, often released by the detection of a microbe or exogenous pyrogen. Chief amongst these is Interleukin 1 (IL1)This protein goes to the hypothalamus and causes the neuronal secretion of prostoglandins (sound familiar right)Within about 20 minutes, the prostoglandins reset the normal body temperature to the new temperature.Also leads to an increase in involuntary muscle contraction and increased in vasoconstriction (blood vessels shrink)Involuntary muscle contraction increases heat productionIncreased vasoconstriction insulates the body from heat lossSo our bodies now have a New TemperatureThus anything at normal 98.6 fells cold to us! This is the chills

When there is a loss of infection , there is a decrease in prostoglandins which quickly resets the temperature of the body so we begin to sweat and have vasodilation. This rapidly brings up back to normal.

What is the function of Fever?(1) Raise Body Temp Normal body temp is optimum for microbe growth, now they can not grow right(2) Inactivates some bacterial toxins and enzymes(3) Increases our bodys chemical reactions and increases the immune system functions (some of our enzymes function well at higher temps)(4) Forces a rest (febrile), we feel weak, we are forced to rest and get better.

Aspirin again!Aspiring stop protoglandin production, right (look at bradykinin). And is often given to reduce pain AND fever. Now, it is recommended to let the fever run its course, unless the fever is very high. Why?

INTERFERONSMainly antiviral proteins made by the body in response to viral infectionTwo types Type I and Type IIType I alpha and beta, block virus replication (produce AVPs into nearby cells).Type II gamma, activate macrophages, lymphocytes and NK cells.Both Type I and II are directly antiviralThey tell cells around the infected cell to enter an Anti-viral state

First administered as a drug in 1986 against Hairy Cell Leukemia. Which is caused by HTLV. Interferon alpha was used here. It was a treatement, NOT a cure

HCV infection is treated with Interferon alpha. Interferon alpha is not stable in the blood. Interferon alpha causes many side effects such as fatigues nausea headaches vomit, weight loss.

COMPLEMENTComplement system is a set of more than 20 large regulatory that plays a key role in host defense. They are produced by liver and circulate to blood.Functions of the complement system:1-Inhance the process of phagocytosis (by Opsonization)2-kill bacteria and enveloped viruses by causing lysis of their membranes (immune cytolysis)3-Regulate the inflammation and immuneresponse.

Host-Microbe Relationships and the Disease ProcessSigns are observable by someone. These include (1) Swelling, (2) Redness, (3) Rash, (4) Cough, (5) Runny nose, (6) Puss, (7) Fever, (8) Vomit, or (9) Diarrhea Just think of anything that you can visible see or notice.

Symptoms are what a patient feels. These include (1) pain, (2) shortness of breath, (3) nausea, (4) sore throat, (5) headache, or (6) malaise (feeling discomfort). Just think of things that you have to ask a patient about.

Syndrome is a combination of the S&S (Signs and Symptoms) that are unique for a specific disease

Response is seen as a fever, malaise, swollen lymph nodes and leukocytosis. We didnt mention leukocytosis in the class a lot, but you should be able to think of it. Leukocytosis is the state of having a high level of WBC in the blood

If after recovery from an infection, there is another infection of the same kind, it is called a sequelae.

TYPES OF INFECTIONS

Acute Rapid, often severe. Examples are measles, a cold, the flu etcChronic Slow, often less severe at first (can turn worse as time progresses). Examples are tuberculosis and leprosy.Subacute Between the Acute and Chronic Gingivitis is an exampleLatent Disease Quiet form of a disease. Often cycles between acute and latent disease. Herpes Simplex (HSV) is an example. Also Chickenpox is an acute disease that then goes latent for years and then becomes the acute disease shingles after a person gets older (and the immune system goes down)Local Infection Restricted to a particular areaFocal Infection One main site, but microbes or toxins are released all over the system. An example is an abseced tooth which can lead to bacteria in the blood

IMPORTANT TERMSBacteremia Bacteria in the bloodViremia Virus in the blood Septicemia Pathogen in the blood or blood poisoningToxemia Released toxins in the bloodSapremia fungal release of toxinsWhy are these terms so important? Remember: When an infection occurs, it must be walled off from the rest of the body to provide an effective response against it. Bacteremia/Viremia.. Any of the -emias show that this defense has failed and now bacteria or other microbes are now systemic. This is bad. Patient may go into septic shock (gram negative bacteria because of their LPS).

Primary infection this is the first infection of a previously healthy individualSecondary infection is another infection in the individual, possible because the patient was weak from the first infection. Remember, the immune system often focuses on one infection at a time and will leave the body open to attack from different types of microbes

Subclinical infection This is an infection by a particular microbe that does not show the full spectrum of S&S of the normal infection.

THE END

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