1) Define the Human Microbiota - The Human Microbiota consists of all of the bacteria that are associated with

the human body - Relationship between Microbiota and Host is Mutualistic and/or Symbiotic in that both the host and microbiota benefit from the partnership. - Consists of microbial cells from all three kingdoms: Bacteria, Archaea and Eukarya - The number of microbial cells to human cells is ~ 10:1 - Microbial populations are distinct for each niche within the body (ie. Intestines houses a different population than on the skin) - Every individual may be unique in their Microbiota - Microbiota communicates with and can trigger responses in host - Microbiota is required for prostnatal development of systems (immune system and GI) - Microbiota communicate among themselves to promote a cooperative response - Supplies Host with Vitamins and other Cofactors - Destroys toxic agents - Digests substrates (polysaccharides) which the host cannot digest - Protects host by excluding certain disease causing (pathogenic) virulent microbes 2) Describe the Morphology and Anatomy of Bacteria - Bacteria are Prokaryotes and therefore do not have organelles (No mitochondria, No Nuclear Membrane, No Chorloplasts and No ER), which are found in Eukaryotes - Some bacteria are capable of Photosynthesis, Autotrophy (eat non-carbon materials), Sexual Mating and Communicating with each other. - Mitochondria and Chloroplasts developed from bacteria; Likely that Mitochondria developed from Rickettsia prowazekii. - Usual size is 0.2 to 80 um in length in Rods, and 0.5 um diameter for cocci - Cytoplasm is usually packed, causing an increased intracellular pressure, equivalent to ~4 atm. - 70% of cytoplasm is water - Cytoplasm also contains storage areas for lipids, carbohydrates and other essential nutrients Bacterial DNA - A Bacterial Genome consists of all DNA within the cell, this includes extrachromasomal plasmids, bacteriophage DNA or RNA and Episomes. - Most bacteria have a single, circular chromosome composed of a DNA duplex, which is folded and coiled and about 2mm in length (~1000x cell length). - Haploid - No Histone proteins. DNA structure is maintained by Polyamines such as Spermine and Spermidine. - Some bacteria have multiple linear chromosomes: Borrelia burgdorferi, Brucella abortus - Plasmids are Extrachromosomal that are small (smaller than chromosome) and usually circular, but can be linear. Plasmids are self-replicating. Morphology - Coccus = Spherical - Bacillus = Rod - Spirillum = Spiral shaped - Coccobacillus = Oval (mix of Coccus and Bacillus) - Fusiform Bacillus = stretched out oval - Virbrio = Boomarang shaped

- Spirochete = Corkscrew shaped Gram Positive vs Gram Negative Gram + cells stain purple in Gram Stain (due to Crystal Violet) Gram + cells have a Thick Peptidoglycan layer Gram + cells have only one lipid bilayer, the Cell Membrane

Gram cells stain Pink in Gram Stain (due to Gram Saffranin) Gram Cells have a Thin Peptidoglycan layer Gram Cells have two Lipid membrane, the Cell Membrane which is a lipid bilayer and an Outer Membrane which contains a single Lipid Layer facing inside the cell and a Lipopolysaccharide layer facing towards the exterior of the cell Gram + cells contain Teichoic Acid on their Gram Cells contain Lipopolysaccharide on outer layer of Peptidoglycan the exterior portion of their Outer Membrane Gram + cells have No Periplasmic space Gram Cells have a Periplasmic space between the Inner Cell Membrane and the Outer Membrane. This space contains the thin peptidoglycan layer and also contains specialized enzymes not found in Gram + Cells Note Mycoplasma do not contain a Peptidoglycan layer Peptidoglycan (Cell Wall) - Peptidoglycan is made up of a Dissaccharide: N-acetylglucosamin and Nacetylmuramic Acid - N-acetylmuramic Acid contains a Cross-Linking Tail, consisting of Alanine, Glutamic Acid and Lysine or Diaminopimelic Acid. This Tail is used to link with Nacetylmuramic Acid sugars of other layers in the Peptidoglycan - The multiple layers of Peptidoglycan are cross-linked with peptide bridges - Peptidoglycan protects the cell and prevents osmotic rupture of the cell and also provides the cell with its characteristic shape. Improper formation of Peptidoglycan layer is lethal to bacteria. Therefore, the Peptidoglycan synthesis system is the target of many Antibiotics. - Lysozyme is a natural enzyme in mammals that breaks the Beta-1,4 link in the Peptidoglycan Dissaccharide Teichoic Acids - Found in Gram + cells only - Attached to Peptidoglycan and anchored into cell membrane by fatty acids - Consist of Phosphodiester polymers of Ribitol or Glycerol with linked D-amino acids and sugars - Antigenic activates complement - may be used to attach some bacteria to specific hosts surfaces Outer Membrane - Gram Only - Acts as a Permeability barrier and confers innate antibiotic resistance, and protects the cell from Lysozyme and Complement attack.

- Contains Porin Channels and Transport Proteins that take up nutrients - Porins are Trimeric Proteins that can open and close to permit passage of solutes - Contains Lipopolysaccharide (LPS) on the outer surface. - LPS consists of 3 domains: 1) O-antigen Saccharide repeats, consisting of 1 to 40 repeats. The outer most part of the LPS which extends out from the cell. Also used to Identify Gram Negative Organisms (ex. E.coli 0157:H7 serotype has a unique O-antigen) 2) Core protein consists of an inner and outer core protein 3) Lipid A acts as an Endotoxin when released from the cell during lysis. Lipid A attaches the Core-protein and O-antigen to the Outer Membrane Cytoplasmic Membrane - Phospholipid Bilayer that does not contain any Cholesterol or Sterols (Bacteria do not make Sterols) - Contains the enzymes for Electron Transport, Lipid Synthesis, DNA Replication, Chemotaxis, Ribosomes and Protein Synthesis, etc Capsules - Usually composed of a Polysaccharide material, also called a Glycocalyx - Some may be composed of a Polypeptide (ex. Bacillus anthracis) - Poorly antigenic, and protects the cell from Phagocytosis, Detergents, Antibiotics and Antibodies - Allows the bacterium to stick to surfaces as well as helps in the formation of Biofilms - Most exposed host surfaces, such as the lungs, oropharynx, intestines and vagine are covered with normal flora biofilm embedded in their capsules. These normal floral biofilms prevent entrence of pathogens. Destruction of this barrier can lead to disease. Appendages - Pili (Fimbriae) found on some bacteria. Consists of Protein Subunits called Pilin, which form a hollow channel. Two types of Pili: 1) Somatic Pili a single cell may consist of hundreds of these. - May help a bacterium stick to surfaces due to Adhesins at the ends of the pili. - Allow for immune system escape by altering the pilin antigenic type - Allows for Surface Motility - Inhibits Complement attachment - Used to Deliver Toxins 2) Sex Pili (conjugal) Male cell has one or two conjugal pili that locates receptors on female cells - When a Male Sex Pili binds to a Female cell, the Sex Pili will undergo Depolymerization, and shorten, reeling in the female cell and allowing for DNA transfer to the Female. - Flagellum Used by many bacteria for motility. However flagella are not found on all bacteria. - Flagellum function in a cork-screw like manner propelling the bacterium

- Flagellum is propelled by a flow of ions either protons or sodium. Structure and function is almost identical to the ATPase, except it doesnt produce ATP, it spins the flagellum. - Rotates clockwise or counterclockwise, changing rotation direction allows the bacterium to turn and is the basis for Chemotaxis - May be arranged on the cell in Polar (at end of cell), Bipolar (on both ends), or Peritricous (all around the cell) - Mobility / Vigor of motility of bacteria can depend on the number of flagellum present - Strongly resembles a Type III secretion System, which is used to inject Toxins into the host cells. Instead of injecting toxins, the Flagellum produces new Flagellar Protein Subunits - Spirochetes contain specialized Flagellum which do not extend out of the cell, but rather spiral around the bacterium. These are called Axial Filaments, and impart a Screw-Like motility to the bacterium which allows it to penetrate viscous materials, such as mucus. Mycolic Acids A lipid material containing 60-90 carbons bound to the cell surface. - Found on specific bacteria Mycobacterium (which includes M. tuberculosis) - Makes the Mycobacterium more resistant to disinfectants and drying out than other bacteria (except spores are more resistant). - Stained with Acid-Fast staining. - Mycobacterium contain a Arabinogalactan layer between the Mycolic Acid layer and Peptidoglycan Archea Cell Walls - Contain L-Glycerol instead of D-Glycerol which is found in bacteria - Archea do not contain Peptidoglycan - The Archea have a surface layer of proteins - Currently, only a single Archea, Ignicoccus, has been found to have an Outer Membrane. 3) Describe the Growth of Bacteria in a Host - Bacteria do not engulf/phagocytose food particles. Rather, they release enzymes into the surrounding area that digest food particles, which can then be transported into the cell. - Membrane-associated transport systems concentrate solutes, both organic and inorganic (metals and ions), in the cytoplasm. - Transport systems include membrane-spanning, barrel shaped components with external receptors that are highly specific for the solutes they transport. - All bacteria associated with human and animal disease are Heterotrophic, meaning they oxidize organic compounds for energy and obtain carbon from organic compounds. - All heterotrophic organisms require water and some inorganic compounds (Phosphate, Sulfate, K, Mg) and trace elements (Fe, Co, Zn, Mo, Mn, Cu) - Oxygen requirements can differ between Aerobic, Anaerobic and Aerotolerant (Facultative anaerobes) - Some obtain Nitrogen from inorganic salts, others can fix nitrogen from the atmosphere (N2 NH3) - Nitrogen fixation from the atmosphere is a process that is exclusive to bacteria, without it life on earth would end.

- Temperatures which bacteria can withstand range from Mesophiles (35-44 degrees C), Psychrophiles (15-20 d. C), Thermophiles (45-80 d. C), Hyperthermophiles (>100 d. C). - Those that grow well in humans are Mesophiles. - Heterotrophs, those that get energy from organic compounds, do Substrate Level Phosphorylation (Fermentation) and/or Oxidative Phosphorylation. - Substrate Level Phosphorylation occurs with the conversion of Hexose to Pyruvate by taking a Phosphate from ATP and placing it on to the Hexose; this process uses a NAD and converts it to NADH. The concentration of NAD is limited in a cell, therefore, cells can do Fermentation to regenerate NAD by converting Pyruvate into Lactic Acid, CO2, Alcohols and other producs, which are then excreted. - Lactic Acid fermentation is the sole energy providing process in many Streptococci and is the likely cause of Disease by Streptococcus mutans, which causes dental caries. - Clostridium sp. (botulinum, tetani, and perfringens) also use Fermentation to produce energy, and produce various products, including H2 which is the gas in Gaseous Gangrene. Patients with Gaseous Gangrene can have bubbles of H2 under their skin that can be palpated. - Those species that are aerobic and are in the presence of Oxygen can use the NADH in the Electron Transport Chain. Furthermore, the Pyruvate produced in the Glycolitic cycle can be processed into FADH2, GTP and CO2 in the TCA cycle. FADH2 can be used in the Electron Transport Chain also. - Anaerobic species that are capable of doing Oxidative Phosphorylation use something other than Oxygen as the terminal electron acceptor of the Electron Transport Chain. Commonly NO3- is oxidized to NO2-. - The Electron Transport Chain is located in the Cytoplasmic Membrane, therefore Protons are pumped out of the cell, creating an electric potential. Protons flow back into the cell through an ATPase (ATP motor) which generates ATP for the bacterium. - Flagella use the same mechanism to turn, where Protons are pumped out of the cell creating an electric gradient. As Protons flow back in, they turn the flagellum. It takes 256 Protons to turn the flagellum 360 degrees. - Some marine bacteria use a Sodium (Na) flow to turn the flagellum. Specifically, Vibrio cholera uses a sodium driven flagellum in the environment, however when its infected a human it uses a different set of flagella that requires Protons. - Obligate Anaerobes Fermentation only or Anaerobic Respiration - Obligate Aerobes Aerobic Respiration - Facultative Forms Fermentation or Respiration - Aerotolerant Forms Fermentation, but often tolerate low atmospheric oxygen levels do not produce any - Most members of Genus Streptococcus fit the Aerotolerant category. They cytochromes and have no electron transport system.

DNA Replication - The circumference of DNA is about 1.5 mm and is therefore folded to fit in a 1 mm diameter cell - No visible condensation of DNA - Replication of the DNA begins at the Ori-C (Origin of Replication), and involves Helicase, Gyrase (Topoisomerase), Primase and DNA-dependent DNA Polymerases.

- Topoisomerases are the target for Quinolone Antibiotics. - Replication is Semi-conservative and Bidirectional and rapidly growing cells may have multiple replication forks - Leading Replication Strand will be in the 53 direction - Lagging Strand is in the 3 5 direction - Cell division occurs soon after replication of DNA is complete Cell Division - Cell division occurs by the formation of a Z-ring at the center of the cell by the FtsZ protein (a tubulin homolog). The Z-ring pinches off the cell into two equal cells. A protein called the Min Protein is responsible for locating the center of the cell. Min proteins form a coiled structure which cycles back and forth from both ends of the cell, in a manner of seconds, and in doing so locates the center of the cell. Mutations in the Min proteins result in the off-centering of the Z-ring and uneven separation of the cell during division, producing a Minicell which contains no DNA. Transport (Uptake) Systems - Cells contain numerous uptake processes. - Energy sources for transport come from the Proton Motive Force, ATP hydrolysis and Phosphate in Phosphoenolpyruvate -Ex. Antiporters and Symporters can use Protons that are pumped out (part of the proton motive force), to transport Na, Ca++ out of the cell, Proline into the cell - Transport systems may be very specific for the solute which they transport - Bacterial Iron Transport systems are key Virulence Factors in many bacteria Bacterial Growth Curve (Planktonic Growth) - Consists of a Lag phase, where bacterial growth does not occur and bacteria are in the process of upregulating the appropriate enzymes for growth. - Following the Lag phase is the Log phase, where growth is exponential - The Stationary phase occurs when nutrients and space become limited and competition ensues. Death rate of cells equals growth rate - Death phase occurs when nutrients become scarce and cannot support the bacteria. - The most preferred method of growth is in a biofilm. A biofilm is a microcosm of surface attached cells enclosed by a capsular matrix usually composed of polysaccharide. Biofilms are formed by attachment to biotic or abiotic surfaces by Pili, glycocalyx, LPS and other receptors. - Many human diseases may involve Biofilms: - Pseudomonas aeruginosa Cystic Fibrosis of lungs - Haemophilus influenza ear canal infections - Viscous clumps of Mycobacterium tuberculosis - Joint prostheses - Glue that binds struvite kidney stones - Dental plaque and other oral biofilms - Legionella pneumophila clumps wafted from air conditioners - Cells of biofilm are physiologically different from the planktonic (freefloating) cells. Gene Expression is different with cells in biofilms compared to

planktonic. Different types of bacteria may work together in a biofilm and may exchange genetic material with each other. - Polysaccharide matrix of a biofilm provides protection from UV and chemicals (antibodies, antibiotics), prevents dessication and osmotic shock. - Biofilms are organized communities of bacteria - Biofilms may be beneficial to host by excluding incoming pathogens, essential for development of GI system and Immune system. - Biofilms form from Planktonic cells that adhesins and are brought together by some environmental signal. The cells release a Acyl-HSL signal which begins the formation of the biofilm - Biofilms have specialized architecture that allows for water channels - 90% of biofilms on prostetic devices consist of Staphylococcus aureus and Staphylococcus epidermidis 4) Relevance to Disease - Disruption or imbalance of the host-microbiota can be caused by several conditions: - Antibiotic chemotherapy which damages select portions of the microbiota - Immunosuppression, either drug induced or disease induced - Infection with a virulent bacterium that overcomes exclusion by the microbiota and host-related defenses. - A fraction of a bacterial population will survive a stress like an antibiotic. Survivors of the antibiotic treatment were in a non-dividing state during the treatment and are called Persisters. Persisters that survive antibiotic treatment ARE NOT resistant to the antibiotic. Bacterial Spores produced by two gram-positive genera: Bacillus (pathogens are B. anthracis, B. cereus) - Spores are resistant to commonly used disinfectants and antiseptics. - Spores can be killed by Formaldehyde, Ethylene oxide or Steam under pressure (autoclave) - Spores can survive for an unknown length of time and have essentially no metabolic activity, but will begin germination almost immediately in favorable conditions. Endospore structure consists of a Exosporum which is the remaining membrane of the cell after formation of the endospore. Coat(s) layer which consists of Keratin-like protein with disulfide bonds, with no similarity to the peptidoglycan layer of a cell. The Coat(s) layer is impervious to many chemicals, UV, ionizing radiation. Below the Coat(s) layer is a Peptidoglycan layer and then a Inner membrane that resembles the Cytoplasmic membrane. - The core of the endospore contains DNA, protein synthesis apparatus (tRNA and ribosomes, but NO mRNA), enzymes for amino acid synthesis, energy producing enzymes, Calcium, Dipicolinic Acid (DPA). DPA and some small pore proteins cross-link to provide protection from heat, desiccation, chemicals and radiation. 5) How do pathogenic bacteria overcome host defenses? - Biofilm growth provides protection from antibiotics and antibodies. - Endospores allows bacteria to persist in a dormant state in a very protective capsule - Senescent / non-dividing cells are able to escape antibiotic treatments because most antibiotics require cells to be actively dividing for them to have an effect.

- Persistance of mutants some cells may acquire spontaneous mutations that allow them to be more resistant to the bodies defenses or antibiotics 6) What are the virulence mechanisms of pathogenic bacteria? - Genes that confer the Virulence phenotype on a bacterium occur on both chromosomes and plasmids; some plasmids harbor genes required for virulence. Iron Transport - Bacterial Iron Transport Systems are key virulence factors in many bacteria - ex. Heme Transport, Ferrous Iron Transport - Siderophore mediated iron transport Siderophores are iron chelating (binding) molecules that are excreted by the bacterium to capture iron. Secretion Systems - Secretion systems release digestive enzymes, toxins, DNA, wastes and deleterious agents like antibiotics - Secretion systems can be virulence factors - In Gram Negative bacteria, secreted substances must pass through two membranes. There are atleast six (numbered I through VI) distinct secretion processes. - Type I systems rapidly excrete Antibiotics, sometimes rapidly enough to confer resistance - Type III systems are found only in pathogens and are used to pump toxins and virulence factors, sometimes directly into the host. In short; How do microbes cause disease? - Disruption or imbalance of the host-microbiota relationship may cause infectious disease by: - Transfer of microbiotal cells to the Wrong, usually sterile, anatomic site. - Overgrowth of one member of the microbiota - Vulnerability to incoming pathogens