Bacteriology General PDF

Bacteriology General  Bacteriology Bacteriology course Introduction: Microbiology is the study of microorganisms. Difference between microorganisms: Bacteria ➡ prokaryotes Viruses ➡ not a microorganism outside the host cell Fungi and parasites ➡...

Bacteriology General  Bacteriology Bacteriology course Introduction: Microbiology is the study of microorganisms. Difference between microorganisms: Bacteria ➡ prokaryotes Viruses ➡ not a microorganism outside the host cell Fungi and parasites ➡ eukaryotes Parasites and fungi are in general larger in size than others It is a study we encounter in our everyday life: There are 100 million times as many bacteria in the ocean as there are stars in the known universe. Humans also have an intimate relationship with MO: more than 90% of our cells are microbes! The number of genes contained within the gut flora outnumbers that contained in our genome. Furthermore 8% of our DNA is derived from the remnants of viral genome. *read only Microorganisms included are bacteria, fungi, parasites and viruses (which are the only non-cellular organisms). All of these need to be viewed on special microscopy except for some parasites that are macroscopic such as: taeniea solium |Page1 Infection vs. disease: Infection occur when MO (not normal flora MO) enter our body and multiply. An infection may cause no symptoms and is called asymptomatic or subclinical or may cause symptoms (manifestations) and damage our cells and thus is named clinical or symptomatic disease. The danger of asymptomatic carrier is that he can transmit the disease through fomites or any other root. Example: 2 people acquired the common cold virus: 1. The first person has a cold and thus has a Symptomatic disease. 2. The second person is infected but with no manifestations thus he has a Subclinical infection. Characteristics of MO: 1. Continuity: MO can adapt to a variety of environments that include external sources such as soil, water and organic matter etc... In doing so the bacteria ensure their survival and enhance the possibility of transmission. By producing asymptomatic infection MO enhance the possibility of transmission from one person to another susceptible person. Note: an asymptotic infection such as Herpes virus, kissing disease stay in the body this is called chronic or latent infection where we become carriers and the disease can be reactivated due to stress, immunosuppression etc... And can be transmitted as symptomatic or asymptotic infection with this latter being the best source for continuity and transmission. 2. Infectious dose: Amount of microorganisms that have ability to produce disease. It is primary and varies between types of bacteria. Ex: Dysentery caused by Shigella dysentery bacteria transmitted via fecal contamination followed by simple hand shaking, is transmitted via a small amount of bacteria unlike E. coli which heeds a much higher dose. |Page2  Breast feeding provides the baby Pregnancy itself is a state with immunoglobulin (IgA/IgG) of immunosuppression or along with the virus sometimes, else the baby would be so diseases become milder and considered a foreign his immune system becomes body. fortified. The same MO can cause different pathogenesis according to: Geographic area Age group Season A MO relationship with specific pathogenesis can be described by any of these 3 terms: Endemic: a disease that exist permanently in a region or population. Example: Malaria in Africa Epidemic: An outbreak of disease that attacks many peoples at about the same time and may spread through one or several communities (coming from outside), also an endemic disease may become epidemic when it covers a large population. Pandemic: When an epidemic spreads throughout the world. |Page3 Glossary: Adherence (adhesion, attachment): The process by which bacteria stick to the surfaces of host cells. After bacteria have entered the body, adherence is a major initial step of the infection process. The terms adherence, adhesion, and attachment are often used interchangeably. Carrier: A person or animal with asymptomatic infection that can be transmitted to another susceptible person or animal. Infection: Multiplication of an infectious agent within the body. Multiplication of the bacteria that are part of the normal flora of the gastrointestinal tract, skin, and so on is generally not considered an infection; on the other hand, multiplication of pathogenic bacteria (e.g., Salmonella species)—even if the person is asymptomatic—is deemed an infection. Invasion: The process whereby bacteria, animal parasites ,fungi, and viruses enter host cells or tissues and spread in the body. Micro biota: Microbial flora harbored by normal, healthy individuals. Nonpathogenic: A microorganism that does not cause disease ;may be part of the normal micro biota. Opportunistic pathogen: An agent capable of causing disease only when the host’s resistance is impaired (i.e. ,when the patient is “immunocompromised” ) Pathogen: A microorganism capable of causing disease. Pathogenicity: The ability of an infectious agent to cause disease. Super antigens: Protein toxins that activate the immune system by binding to major histocompatibility complex( MHC) molecules and T-cell receptors (TCR) and stimulate large numbers of T cells to produce massive quantities of cytokines. Toxigenicity: The ability of a microorganism to produce a toxin that contributes to the development of disease. Virulence: The quantitative ability of an agent to cause disease. Virulent agents cause disease when introduced into the host in small numbers. Virulence involves adherence, persistence, invasion, and toxigenicity. Nosocomial infection: hospital acquired infection or toxin (interchangeably used with Hospital- acquired infection). The most famous example is pseudomonas aeruginosa: when someone needs a respirator for example, and this respirator is not properly disinfected those aerobic bacteria are introduced into lungs directly causing pathogenesis. Note that they can be found anywhere in environment not only in hospitals due to their high resistance. Bacteria found in hospital are stronger and have multi-drug resistance e.g.: staph aureus in hospital is stronger than that in environment. Prophylaxis: treatment given or action taken to prevent disease -‫الوقاية‬ |Page4  Section 1: Bacterial structure 1.1- Shapes and colonies A) Introduction: What is a bacterium? Bacteria are microscopic single-celled organisms that thrive in diverse environments. They can live within soil, in the ocean and inside the human gut. Humans' relationship with bacteria is complex. Sometimes they lend a helping hand, by curdling milk into yogurt, or helping with our digestion. At other times they are destructive causing diseases like pneumonia. Name: bacterium Division: prokaryotes Size: range between 0.2-5 micrometers Reproduction: binary fission (different rate between strains) Cellularity: unicellular-microscopic Habitat: they can live outside cells (by themselves), or intracellular (facultative intracellular) and some are energy parasites: deplete the cell from ATP for their own sake (produce their ATP for survival). What is the importance of knowing the bacterial size? Bacterial size can either enhance bacterial spread and invasion or repress it. Larger bacteria = 5 micrometers are usually prohibited from crossing the anatomical barrier of our respiratory tract and thus cannot cause lower respiratory tract infections. Also small bacteria = 0.2 micrometers need special microscopes to be seen (will be mentioned later). |Page5 Criterion to specify living organisms? They have to multiply They have to produce their own energy (either by themselves or picked up by the host cells) Outside a living cell a virus is considered an infectious agent and not a microorganism, it is inert. But when it enters a living cell it can multiply and it will produce energy. What is the difference between eukaryotes and prokaryotes? Prokaryotes eukaryotes DNA Circular DNA Linear DNA No introns Introns exist DNA Naked DNA bound to proteins Organelles No membrane bounded Membrane bounded organelles organelles , ex: mitochondria No nucleus Nucleus exist 70S ribosome 80S ribosomes Reproduction By binary fission Mitosis and Meiosis haploidy diploidy Average size Smaller (0.2-5 micrometers) larger |Page6 Knowing this difference can help in? Targeting the Antibiotic: the difference of the sedimentation coefficient between those two can help targeting the antibiotic against bacterial ribosome only and thus will only affect bacterial protein synthesis without damaging the host's cells. Note: Antibiotics work on Multiplying bacteria only. Peptidoglycan layer targeting: this structure is unique to bacteria, other prokaryotes ex: plants have their own cell wall but is made of cellulose rather than peptidoglycan; this help target this peptidoglycan wall to destruct the bacteria without harming the host. |Page7 B) Bacterial Shapes: Bacteria can have four different shapes and are named according to their shape: Rod-shaped bacteria are called bacilli (singular: bacillus), e.g., Bacillus anthracis, which causes the disease anthrax. Spherical bacteria are called cocci (singular: coccus), e.g., pneumonia is caused by Streptococcus pneumonia. Then there are spiral bacteria, called spirilla (singular: spirillum), e.g. treponema pallidum that causes syphilis comma-shaped ones, called vibrios (singular: vibrio) We also have polymorphic bacteria named coccobacilli, i.e. it changes its form during its life cycle, e.g. Proteus species, and pseudomonas… this is dangerous because we might think that we have bacterial combination under the microscope but in fact we only have one (false diagnosis). These shapes are microscopic and cannot be obtained by naked eye. o Coccobacilli sometimes appear as cocci, others as bacilli or coccobacilli. o Bacilli under the microscope can be seen as individual rods or palisades arrangement ( chinease arrangement). Shapes of other bacteria: Bacillus anathracis: rectangular shape Fusobacteria: rocket like Pseudomonas: longer bacilli |Page8 C) Arrangement and Colonies: Bacteria need a proper medium for its growth; this growth means colony formation and can be seen by naked eyes. A colony is defined as a visible mass of microorganisms all originating from a single mother cell. The importance of the colonies is that it can help distinguish between different types of bacteria. By performing gram staining later we will see these 2 important gram + cocci: Staphylococci: they can divide three dimensionally; so we can see: - Individual cells - Diplococcus (two cells still attached and dividing) - Tetrad (typical for staphylococci, 4 attached dividing cells) - Sarcinae (a grape-like cluster, shows the three dimensional division) Streptococci: they only divide longitudinally in one direction, not three dimensionally. So we can see: single cells, diplococci and long chains. Colonial morphology of other bacteria: Bacillus anathracis: chain morphology (not cocci) Note: Colonies don't differ by morphology only but also with color (opaque, translucent, transparent…), size… so to identify a bacterium we look at the color, shape and arrangement. |Page9 D) Methods of bacterial identification: a) Staining: very general (technique discussed later) b) DNA or RNA analysis: highly specific and very sensitive, the presence of a certain nucleic acid sequence or specific gene is interpreted as definitive identification of an organism. Note that bacteria have both DNA and RNA unlike viruses. c) Antibiogram: Tables showing how susceptible a series of organisms are to different antimicrobials. Also named susceptibility testing. Note that a contaminated culture may yield a wrong antibiogram. d) Colonial appearance: some bacteria appear as mucoid (not clear) e.g.: Klebsiella and pseudomonas. Keep in mind: the trending DNA testing help identify bacteria but not the antibiotic we use against it. | P a g e 10 1.2- Bacterial cell components A) Nucleoid: Bacteria have neither a nucleus nor a nuclear membrane but rather a nucleoid where DNA and RNA along with Ribosomes and plasmids are dispersed, also called protoplasm. The osmotic pressure inside the bacterial cell is higher by 5-20 atmospheric pressure than the external medium; this is important because it prevent leakage from inside the cell and directs the flow from outside to inside (from low to high osmotic pressure). [if we place a bacterium in a medium with osmotic pressure equal to that inside the bacteria the bacteria will lyse] It is negatively charged which affects staining. DNA: haploid, usually circular, not enclosed inside a membrane bounded nucleus but inside the cytoplasm. This means that translation; transcription and DNA replication occur in the same compartment and interact with other cytoplasmic compartments like ribosomes. Neither histones nor introns exist in the DNA. Their supercoiled nature is still unclear. Plasmids: small independent DNA that replicate independently (considered Replicon). Carry genes that may benefit the survival of the organism such as antibiotic resistance can be transmitted between bacteria (mechanisms discussed later). Ribosomes: 70S (50S big subunit, 30S large subunit), it is the most numerous intracellular structure in bacterial cell. | P a g e 11 B) Plasma membrane: Also named cytoplasmic membrane, is composed of a phospholipid bilayer and thus has all the general functions of a cell membrane: 1. Acting as a selective barrier for permeability and transport. 2. The receptors acting as chemo attractants or chemo repulses are present on this membrane. 3. Site of lipid carriers 4. Contains DNA replication machinery 5. Site of enzymes required for biosynthesis of bacterial structure. 6. Site of lipopolysaccharide synthesis 7. Does not contain sterols 8. There are foldings of the plasma membrane named mesosome it is thought to be artifacts that exist due to chemical preparation preceding microscopy observation. Mesosomes are of 2 types: Lateral mesosome: carries most of the membrane's function, such as electron transport, aerobic respiration and hydrolytic enzymes secretion (no organelles to carry out these functions). Septal mesosome: where nucleoid attaches for division of the cell, if this is absent no division occurs. Note that as the bacteria becomes more aerobic the mesosomes' number increases. | P a g e 12 It should be noted that energy production varies between aerobic and non-aerobic bacteria: Aerobic bacteria: The electron transport system consists of a system of electron carriers that sequentially transport electrons to the carrier with the next higher reduction potential i.e. to a stronger electron acceptor. This starts with an electron donor(Carbohydrates) and ends with O2 (strongest electron acceptor). During glycolysis and TCA cycle organic molecules donate electrons associated with hydrogen atom to NAD+ molecules so it becomes reduced (NADH, H+), these NADH act as electron donors to the electron transport system. Through a series of oxidation-reduction reactions electrons move in "Substrate Oxidoreductase complex" and generate energy for this latter to pump hydrogen outside the plasma membrane (periplasm)= Substrate Oxidation. Electrons are transported between the members of the transport chain till they reach oxygen and more hydrogen are pumped outside, and water molecules are produced too. Keep in mind that oxygen is the only terminal electron acceptor. The importance of this transport system is that: it generates ATP essential for bacterial cell function. Protons pumped outside re-enter the bacterial cell either alone or carrying another molecule (of same or different charge) Anaerobic bacteria: No O2 is present thus O2 is not the electron acceptor. Hence transport occurs in a reversible manner due to ATP hydrolysis followed by oxidation. | P a g e 13  Transport mechanisms of bacteria: Passive or Concentration Type of transport Description active gradient *Uses trans membrane integral Passive (no proteins. Facilitated diffusion *no nonpolar molecules can be energy with diffused, transferred substance needed) have the same concentration inside and outside Protein binding There is a periplasmic protein active against that differ between gram+ and transfer mechanism gram - bacteria Of several types: Chemo osmotic Uniport: one substance in one active against transport system. direction. Symport: two differently charged molecules in the same direction Antiport : molecules of same charge in different directions. Usually related with sugars transfer. A plasma membrane carrier becomes phosphorylated, grabs a sugar, and transports it Group translocation active against as a phosphorylated sugar(higher energy than simple sugar) Named:"Phosphotransferase System". | P a g e 14 C) Cell wall: In bacteria it is a peptidoglycan layer or a murein layer. All peptidoglycans are composed of a sugar backbone made of NAM: N-acetyl muramic acid and NAG: N-acetyl glucosamine linked by a beta 1-4 glyosidic bond. The length of this sugar chain varies between bacteria and may reach 65/66. To the muramic sugar there is a tetra peptide chain linked consisting of: 1st: L-Alanine 2nd: D-glutamate 3rd: depends on bacterial type: If gram -: DAP (diaminopimelic acid) If gram +: DAP or lysine or any other amino acid 4th: D-Alanine DAP: - unique to prokaryotes, it is a precursor of lysine aminoacid. - In gram negative bacteria a lipoprotein is bound to DAP. | P a g e 15 Linkage between tetra amino acid chains also differs between bacterial types: In gram negative: Gram negative bacteria consist of 1 peptidoglycan layer usually peptide bridges between the a.a chains within the same layer exist, between the amino end of the 3rd a.a of the first set and the carboxyl end of the 4th a.a of the second set. In gram positive: consist of almost 200 layers. Interpeptide bond named a pentaglycine bridge cross links parallel amino acid chains within the same layer. While peptide bridges cross link occurs between different layers in the same position mentioned above. Thus the cell wall becomes very tight and hard to break down. Also these contain teichoic acid (discussed below) | P a g e 16 Basic properties of the cell wall: 1. It gives osmotic protection: plasma membrane is delicate but cell wall is rigid that’s why it provides stability and protects the cell from lysis 2. It provides bacterial structure. 3. Has a role in cell division. 4. Site of many antigenic determinants (epitope): a site on the surface of an antigen molecule to which a single antibody molecule binds. Most obvious in gram positive (outer membrane of gram negative hinders this). 5. Primer for its own biosynthesis 6. It is an antiphagocytic structure which adds on bacterial virulence. 7. It is a mitogen: it activates lymphocyte mitosis nonspecifically (with no antigen), it triggers the mitogen protein kinase stimulating lymphocytes mitosis and thereby assessing the immune function of the host. E.g. on mitogens: Phytohemaglutin which is a structure derived from the beans. 8. Lack lipids and protein except for some bacteria such as mycobacterium tuberculosis. 9. Contains openings for transport (more prominent in gram negative bacteria). 10. Hydrophobic in nature 11. Non-selectively permeable for transport unlike the cytoplasmic membrane. Lysozymes (found in our tears, saliva...) break the beta glycosidic bond between NAM and NAG and thus are a natural defense mechanism | P a g e 17 D) Flagella: Not always present. But when present it causes motility and its arrangement will have significance on the bacteria type. They are proteins in nature thus are highly antigenic. Flagella is important because it helps in: Going toward specific nutrients Escaping dangerous structures Important virulence factor: some normal flora motile bacteria may escape their normal niche and thus become pathogenic. They are antigenic (antigen H). Types of flagella: 1. Atrichous: no flagella (not motile) 2. Monotrichous: one flagella at one end 3. Amphitrichous: one or more flagella at each end 4. Lophotrichous: two or more flagella at one end 5. Peritrichous: flagella surrounding the cell. E) Pili: Hair like appendages found peritrichously on the surface of many bacteria, and are involved in conjugation, adhesion and are antiphagocytic. Composed of protein subunits named pillin. They are antigenic, short and not spiral. We have 2 types: ordinary pili sex pili. | P a g e 18 F) Capsule: It is a well-organized layer, not easily washed off, and it can be the cause of various diseases not found in all bacteria. cannot be stained by gram stain and appear as a halo structure. it is made of polysaccharide usually but can be made of other molecules: e.g.: protein in bacillus anthracis. In the polysaccharide there is an antigenic part named K Antigen used to trigger immune response (usually represented as Vi). It comes in different forms: Capsule: tightly bound to the bacteria (doesn't stain well) Glycocalyx: made of polysaccharides that form fibrils with the bacterial cell and make a sort of meshwork. E.g. step. Mutants that form a meshwork in the gum trapping too many bacteria. Slime layer: a polysaccharide that is completely detached from the cell wall. E.g. pseudomonas aeruginosa that trap too many bacteria especially in cystic fibrosis patients, and we have staph aureus Function of capsule: 1. important for adhesion 2. Contain water which protects the bacteria against desiccation (dehydration) 3. Antiphagocytic (virulence factor) | P a g e 19 1.3-Gram positive vs. Gram negative These are two types of bacteria; we also have acid fast bacteria: Acid-fast organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids. These acids resist staining by ordinary methods such as a Gram stains. A) Gram positive bacteria: Cytoplasmic membrane, periplasmic space and a peptidoglycan layer (cell wall) some may be capsulated. Periplasmic space: A concentrated hydrophilic gel-like matrix that contains enzymes for disulphide bond formation, protein folding and trafficking, and small molecule transport. Other enzymes within are the detoxifying enzymes such as beta lactamase and alkalinephosphtase and penicillenase.. Other than enzymes it contains D-alanine and sugar units that function in osmoregulation and bacterial cell stabilization | P a g e 20 Teichoic acid (in the cell wall): It is a repetition of glycerol or ribitol to which are bound different types of amino acids.it is not a fixed structure i.e. it changes within different bacterial species and within the same bacteria depending on medium. We have two types of teichoic acid: 1. Wall teichoic acid: it is covalently linked to the cell wall, not present in all gram positive bacteria 2. Lipoteichoic acid: also called membrane teichoic acid, it is projected from the cytoplasmic membrane crossing the peptidoglycan layer to the outside. It is found in all gram positive bacteria.it is important in the pathogenesis of certain types of disease such as strep A (that causes the sore throat). Function (importance): 1. They are antigenic since they are exposed and contain proteins. 2. Help in bacterial adhesion to host's susceptible cells, and to each other increasing virulence (bacteria aggregated can form occlusions). 3. May also function in ions transfer (minor function) Note that aggregated bacteria are of different strains while colonies are from the same strain. | P a g e 21 B) Gram negative bacteria: Cytoplasmic membrane (called inner membrane); periplasmic space, cell wall and outer membrane (lipopolysaccharide). some also can have a capsule. No teichoic acid is present Outer Membrane-Lipopolysaccharide: it is a lipopolysaccharide, and it is unique for gram negative bacteria, it is an asymmetrical bilayer made of lipid A and a Polysaccharide portion The outer layer is also named an endotoxin because it may lead to a septic shock and death within 2-3 hours if not properly diagnosed (septic shock is very hard and needs an attentive dr.) Lipid A: It is the portion responsible for the toxicity of this membrane, and it is hydrophobic, it is made of: Two phosphorylated glucosamine units linked by a glycosidic bond. To these sugars we have acyl chains (fatty acids) attached, (it is believed that the optimal immune activating lipid A contains 6 acyl chains attached.) this fatty acid portion is bacteria-dependent i.e. it varies between bacterial types. One type of the fatty acid is called: beta- hydroxymyristic acid, a C14 fatty acid unique to bacteria. These fatty acids anchor the LPS to the bacterial membrane. | P a g e 22 Polysaccharide portion: Core polysaccharide linked to lipid A directly.it is made of an oligosaccharide (contains a small number of monosaccharide units). One of these sugar units is KDO=keto- deoxyoctulosonate which is unique to bacteria. This compartment also contains non-carbohydrate components such as phosphate, amino acid… Antigen O: also named O somatic antigen. A repetitive glycan polymer (monosaccharide polymer) where these sugars can be tri, tetra or pentasaccharide, linear or branched. It is attached to the core oligosaccharide. Its composition highly varies from strain to strain.it serves in: 1. Determining whether the LPS is smooth or rough: The absence or reduction of the O antigen renders the LPS rough so it becomes more hydrophobic and The presence of the O antigen thus easier access for makes the LPS smooth. hydrophobic antibiotics. 2. It serves as a site for antibodies' recognition (antigenic). {Although less antigenic than proteins.} 3. Important in serology: the type of antibody found in serology will indicate which O antigen we have and thus the specific bacteria present. (remember that each bacterial strain has a different O antigen) Somatic antigen: an antigen located in bacterial cell wall or LPS in contrast to the one in the flagella or in capsule. Serology: a diagnostic examination of the blood serum, especially with regard to the immune response to pathogens or introduced substances. Based on the results of the serology test we can identify the antibiogram. | P a g e 23 Outer membrane openings: In addition to this chemical composition, the LPS contain openings for transfer. These opening are width-dependent which means that large molecules cannot cross small width openings, therefore antibiotics of big size cannot cross and this is a form of bacterial innate resistance. 1. Porins: it is a trimeric protein (3 units of protein) which makes an opening for the transfer of many types of solute. Remember it is size dependent (no energy is required). 2. Lam B: it acts as a pore receptor for Lambda Bacteriophage (a virus that infect bacteria), and is also used for sugar transfer like maltose (not glucose transfer). 3. TSX: it acts as a pore receptor for T6 Bacteriophage, and is also used for nucleoside or amino acids transfer. 4. Omp A: it acts as a pore receptor for many different types of bacteriophages. And a very important function is that it is used for genetic material transfer (sex pili receptor) that is a sexual transfer between a donor and a recipient (conjugation of sex pilis). 5. Siderophore complex: serve to transport irons. 6. Ion transfer pores. all those pores are:.1 Protein in nature 2. Inducible; under certain conditions the bacteria will induce their production. | P a g e 24  Other properties of the LPS: 1. The site of many enzymes such as Trans peptidase enzyme. 2. The site where we have the Penicillin Binding Proteins: there exist at least 6 PBPs some of which are structural, others are enzymatic, but all are under chromosomal control. 3. They are heat stable due to interaction between its compartments. 4. They are endogenous pyrogenic: causes fever that is produced in our system 5. They are pyogenic: induce pus or abscess. 6. They induce platelets aggregation which leads to thrombosis and thus septic shock. 7. They induce hypoglycemia (symptom of septic shock) Oligosaccharide (LOS) vs. Polysaccharide (LPS) Some gram negative bacteria may have a short antigen O so the LPS become a LOS. The LOS is most prominent in Neisseria which is bacteria (of many types) that causes gonorrhea, meningitis… these LOS differ from LPS by: 1. Ability to change the antigenicity much quicker than LPS because they are shorter. That is: bacteria such as Neisseria replicate in the human system and upon replication it changes sugars of the Antigen O leading to antigenicity change. This causes no formation of new antibodies due to lack of time and the antibodies produced before will not recognize the new antigen and thus will not have a protective effect. 2. Ability of LOS to mimic many of our cellular antigens or properties. And thus the antibodies can fight the humans' own cells due to complementarity with LOS antigenicity. E.g.: neuraminic acid can be the antigen O of the LOS and at the same time it is found on the surface of our cells. | P a g e 25 Septic shock: What is a septic shock? Sepsis is the result of an infection, and causes drastic changes in the body. It can be very dangerous and potentially life-threatening. It occurs when chemicals that fight infection by triggering inflammatory reactions are released into the bloodstream. Doctors have identified three stages of sepsis: *Sepsis is when the infection reaches the bloodstream and causes inflammation in the body. *Severe sepsis is when the infection is severe enough to affect the function of your organs, such as the heart, brain, and kidneys. *Septic shock is when you experience a significant drop in blood pressure that can lead to respiratory or heart failure, stroke, failure of other organs, and death. It is thought that the inflammation resulting from sepsis causes tiny blood clots to form. This can block oxygen and nutrients from reaching vital organs. The inflammation occurs most often in older adults or those with a weakened immune system. But both sepsis and septic shock can happen to anyone. Septic shock is the most common cause of death in intensive care units in the United States. | P a g e 26 What are the symptoms of septic shock? Early symptoms of sepsis should not be ignored. These include: Fever usually higher than 38C, followed by a transient period of leucopenia and then leukocytosis low body temperature (hypothermia( fast heart rate rapid breathing, or more than 20 breaths per minute Severe sepsis is defined as sepsis with evidence of organ damage that usually affects the kidneys, heart, lungs, or brain. Symptoms of severe sepsis include: noticeably lower amounts of urine acute confusion dizziness severe problems breathing bluish discoloration of the digits or lips (cyanosis( People who are experiencing septic shock will experience the symptoms of severe sepsis, but they will also have very low blood pressure that doesn’t respond to fluid replacement. How does a septic shock develop? After getting infected with gram negative bacteria we have either of the 2 scenarios: 1. The release of endotoxin occurs only when the bacteria is died. This is the first case and it is much severe than the other one because the amounts of the LPS released in circulation are very high.it leads to a very quick sepsis. 2. The release occurs via living gram negative bacteria (they release their own endotoxin as they colonize). This is the second case and the amounts of endotoxin released are less compared to the case above. This case activates the inflammatory response (because it have a higher tendency of appearing on cell surface= antigenicity). | P a g e 27 Pathogenesis of sepsis: LPS released in blood, inflammatory response triggered, cytokines released in a massive amount (cytokine storm), 1. coagulation pathway activated: -LPS induce platelet aggregation through alternate complement pathway activation ----> LPS also activate coagulation reactions) through Hagemen factor (factor XII) activation followed by series of factors' transformation to proteolytic enzymes until prothrombin changes into thrombin (enzyme) thrombin cleaves fibrinogen into fibrin which form clots(fibrinogen has glycoprotein receptors on the platelets which help in their aggregation) plasminogen is cleaved to plasmin which leads to fibrinolysis and thus DIC (disseminated intravascular coagulation) thrombosis of capillaries (micro thrombi) hypo perfusion, hypoxia, hemorrhages (due to lack of platelets {all are aggregated} 2. in parallel: constriction of arterioles and venules peripheral Vasodilation (to compensate) increased vascular permeability decreased systematic resistance low Blood pressure(decreased cardiac output) stagnation in the peripheral capillaries hypo perfusion of organs ventricular dilation to compensate low blood pressure myocardial failure septic shock and death. Note that DIC and septic shock can be caused by gram positive bacteria but not through endotoxin. | P a g e 28 Lipoprotein: Another structure unique to gram negative bacteria It is the most abundant protein that we can find in gram negative bacteria.it binds the peptidoglycan layer at the third amino acid: DAP and is also bound to the LPS membrane rendering a stable structure. It is composed of two parts: lipid portion: Diglyceridthioether protein portion: it is made of amino acid motifs that are repeated linked to the lipid portion. These a.a motifs differ from bacterial strain to another. | P a g e 29 C) Other remarkable differences between gram positive and gram negative: 1. Site of hydrolytic enzymes secretion (plasma membrane enzymes): In gram negative they are In gram positive they are secreted in the periplasmic secreted outside the bacteria space This can give significance of the site of transportation. 2. Staining: In gram positive they stain purple because they have a In gram negative they stain thick cell wall capable of pink because they have a thin retaining crystal violet. (it also cell wall that can be dissolved retain the counter stain but it upon alcohol so they don't is not shown due to the retain purple stain. strong violet color) 3. Binary fission (discussion below) | P a g e 30 1.4-Spores & Binary fission A) Spores: Not all bacteria produce spores. Spores are produced in response to environmental nutrients depletion and remain intact until the environmental factors favor its vegetation that is its transformation into a vegetative (metabolically) active cell. Spore characteristics: 1. Can remain in environment for years 2. Metabolically inactive: Their DNA is not destroyed; they have all the genetic material as the bacterial cell. But they are inactive, inert 3. Not reproductive but they are stable because of the different layers that cover those spores. 4. Produced in response to nutrient limitation or extreme conditions. 5. Very resistant to desiccation, to different degrees of heat; they need higher temperatures, and are resistant to different chemicals: this is why they are used in biological wars. 6. Completely dehydrated Some of the spores forming bacterial species are: bacillus and clostridium. | P a g e 31 Structure of endospore: Core: containing DNA, RNA and ribosomes and enzymes in addition to dipiclonic acid. Dipiclonic acid functions in protecting DNA from chemicals present in environment. Layers: that covers the DNA to ensure endospore stability. 1. Spore wall: it is the innermost layer covering the DNA.it has the same composition of the peptidoglycan cell wall from which it is derived (with less water content). 2. Cortex: Spore cortex, is composed by peptidoglycan but it is thicker than the spore but not as compact as the cell wall. (Less bridges between the layers) 3. Spore coat: coat (the hardest layer to destroy in the spore), it is a Keratin like structure (protein present in hair, nails…) which is extremely resistant to all external factors so if the spore should be converted to a vegetative cell, the coat has to be destroyed (the cortex is easy to destroy) 4. Exosporium: the outermost layer, a thin layer that covers the spore, it’s Lipoprotein in nature. | P a g e 32 Sporulation: Endospore formation is usually triggered by a lack of nutrients, and usually occurs in gram-positive bacteria. it occurs at the end of the exponential phase of bacterial growth curve. I.e. at the end of the log phase. These spores are in a dehydrated or desiccated state means that they should excrete all the water. Before sporulation: Bacteria replicate their genetic material through an asymmetrical replication (unlike binary fission which is symmetrical=produces two identical daughter cells). This asymmetrical replication yields a nucleic acid with all bacterial parts destroyed except for plasma membrane and cell membrane which will undergo modifications (remember that they are the primers of their own synthesis). Steps and mechanism of sporulation: The bacteria sense the nutrient depletion and environmental conditions through its receptors it will adapt itself for spore formation that is by shutting some vegetative genetic information and activating other sporulation genes new enzymes (gene product) are produced some of which are: Dipicolnic acid synthetase: the enzyme responsible for Dipicolonic acid production that functions not only in protecting DNA but also in causing the cell dehydration which is vital for spore formation. Cell is ready for sporulation: DNA replicate and different layers start to form. | P a g e 33 The energy needed for sporulation is provided by Phosphoenol Pyruvate (PEP) which is a glycolysis intermediate that produces ATP upon its cleavage the source of energy for spores is glycolysis. The mother cell becomes dead after the spore formation. Note that: even if the conditions become favorable right after sporulation the spore cannot become a vegetative cell but to pass a certain period which varies depending on bacterial type (2 days- 2 week). Vegetation: when conditions become favorable (also sensed by bacterial spores) the genes of vegetation become active whereas those of sporulation become suppressed calcium diplicolinate will be destroyed and the cell will retain the water. A process named The Outgrowth occurs that is: the process whereby the newly formed vegetative cell completes its first round of division (binary fission). | P a g e 34  B) Bacterial division and Growth curve: Bacteria divide by an asexual mode of reproduction named binary fission. The rate of reproduction varies between different bacteria, ➔Mycobacterium tuberculosis needs 3 to 8 weeks to grow. ➔E. coli grows in 4 hours. In binary fission DNA replication and segregation occur simultaneously. Binary fission differs between gram positive and negative bacteria: Gram + Gram - the DNA attached to the septal mesosome will divide in both The slight difference is in a mechanism directions (directionally) and the called constriction: all the bacterial cell septum (cytoplasmic membrane) will is constricted from the middle so that it form. This septum will then elongate will be separated into 2 daughter forming a wall-like structure that bacterial cells. separate the bacterial cell into 2 identical cells. | P a g e 35 Growth curve: This growth curve is drawn upon the asexual reproduction phases to identify these different phases: On x-axis: time On y-axis: colony forming units CFU per ml of medium (which contains the bacteria). In microbiology, a colony-forming unit (CFU) is a unit used to estimate the number of viable bacteria or fungal cells in a sample. Viable is defined as the ability to multiply via binary fission under the controlled conditions. 1. Lag phase: When a microorganism is introduced into the fresh medium, it takes some time to adjust with the new environment. This phase is termed as Lag phase, in which cellular metabolism is accelerated, cells are increasing in size, but the bacteria are not able to replicate: growth rate is zero. NB: Growth rate of bacteria per ml per time is a coefficient and not a count of individual bacteria 2. Log phase: or exponential phase: with time, the population is increasing in amount per ml. The growth rate is constant. NB: Spores are produced at the end of this phase. 3. Stationary or plateau phase: in the majority of cases, there are around 109 bacterial cells or CFU/ml but growth rate is mostly zero. 4. Decline phase: when the nutrients get depleted in the medium, a gradual decrease in the growth is obtained until all the bacteria die. Certain toxic products are released due to the death and breakdown of the bacteria. But the rate never reaches a zero. So as a conclusion: the bacterial population jumps from 1 CFU/ml at the beginning of the curve to 109 CFU/ml at the stationary phase but keeps the same level during all of the stationary phase. | P a g e 36 | P a g e 37 1.5-Synthesis of bacterial components: It is important to discuss the synthesis of the different elements of the bacteria because at different stages, different antibiotics interfere. So that’s why a specific antibiotic is used for a particular type of bacteria. A) Peptidoglycan synthesis: Keep in mind that the peptidoglycan is the primer for its own biosynthesis, which means that in the upper layer we will have a fragment of the peptidoglycan that will be developed and used as a primer. The following diagrams summarize this process: | P a g e 38 | P a g e 39  B) Lipopolysaccharide synthesis: The lipopolysaccharide may follow the same procedure with different carriers. It has a lipid portion (which is a disaccharide unit with different fatty acids), to which are added different carbohydrates [KDO (core) + other polysaccharides] All of them are produced within the cytoplasm and are collected within the cytoplasm. There are other carriers which will transfer them to the outer layer of the inner membrane. A problem arises: the lipopolysaccharide is the outer layer of the outer membrane and it is a large molecule, very difficult to be transport through all the layers. Many studies propose that there are certain areas in the Gram - bacterial cell wall where the outer membrane and the inner membrane become contiguous (very close to each other), forming a pore-like opening which would help in the transfer of that lipopolysaccharide molecule. ➔Those empty areas within the bacterial cell are known as Bayer’s junctions. C) Lipoprotein synthesis: In the same way as peptidoglycan: It is produced in the cytoplasm It consists of a diglyceride thioether molecule to which are added different amino acids It is bound to the cell wall (to the DAP of the peptidoglycan) from one side, and inserted on the outer membrane on the other side. | P a g e 40 1.6-Growth requirements of bacteria: In order for bacteria to grow it should be provided by a medium that contains all of the necessary nutrients and elements. Special requirements are needed and they vary among bacteria. A) Nutrition: Bacterial growth requires the presence of many essential nutrients: carbon, nitrogen and phosphorous... some types of bacteria must consume pre-formed organic molecules to obtain energy, while other bacteria can generate their own energy from inorganic sources. Bacteria have the ability to store essential nutrients to overcome the depletion: 1. Carbon: Stored as insoluble granules in the form of "Poly beta hydrxobutiric acid "mostly, but can be stored in the form of starch and glycogen too. Carbon is needed for synthesize of nucleic acid, proteins… 2. Phosphate: Stored in the form of inorganic phosphate named "Volutin granules or Metachromatic granules". Theses Metachromatic granules help identifying the bacteria. | P a g e 41  B) Temperature: According to temperature differences, we can separate the bacteria into different categories: (note that here we are talking about environmental temperature not body temperature( Psychrophiles: they like to live in a temperature within 4 to 20 degrees Mesophiles: in which are included the bacteria that infect us. They live between 15 and 48 degrees. However, when they enter the human body, they will live at 37 degrees. Thermophiles: they live between 42 and 68 degrees Extreme thermophiles: they live in a temperature>68 degrees. They might even live in a temperature of 100 degrees Most of the bacteria are mesophilic. Eubactria Bacteria Archaebacteria Archaebacteria are environmental bacteria, which do not have a cell wall, and they can live in temperatures around hundreds of degrees because their proteins and enzymes are heat stable. N.B. Bacteria that are not extreme thermophiles will be destroyed when heated or boiled in normal water Example: Thermus aquaticus (archeo.) Note: Taq polymerase is derived from archae bacteria that is why it is heat resistant. | P a g e 42  C) Substrate: Carbon: usually produced from glucose degradation pathways (glycolysis, fermentation…). glucose is the first to be assimilated and the most easily assimilated compound in bacteria. Nitrogen: it should be of a specific form in order to be assimilated and that is either nitrites (NO2-) or nitrates(NO3-) which upon assimilation become ammonia (NH3) {an alkaline molecule}. Sulfur: it should be in the form of sulfide or sulfate salts in order to be assimilated. Assimilation of sulfur is detected by the blackening of the medium due to hydrogen sulfide (H2S) deposition. Sulfur is important for methionine, other sulfhydryl compounds and cysteine, etc… D) Ions: Phosphate: phosphorus is mostly taken from inorganic phosphate. (Ca2+, Mg2+, Na+, Cl-...) each one is present at certain concentration for the wellbeing of bacterial membranes. Because as we have hydrogen pump, we can have sodium potassium exchange. Iron in its ferric (Fe3+) form found in nature is insoluble. thus it needs a specific complex to transfer it to the bacterial cell, and that is the iron siderophore complex: it is an iron- chelating compound that transforms insoluble iron in a soluble form: ferrous (Fe2+) to the bacterial cell to cover its needs and resume its function. Therefore, bacteria secreting siderophores have increased virulence. | P a g e 43 E) Heat-shock (HSP) and cold-shock(CSP) proteins: HSP is used to protect and stabilize the heat-sensitive proteins when the bacteria are subjected to increased level of temperature (2 or 3 degrees) for a short period of time. After the heat is regulated to normal (decreases to its normal vale) HSP is destroyed due to lytic enzymes. CSP same as HSP but in case of temperature decrease. F) pH: pH of the medium is very important; we can see many bacteria that can grow at different pH value Acidic pH (1