Bacterial Structures

Cytoplasmic Membrane 

Chromosome(nucleoid)

and

Plasmids

• no sterols
• high protein content (site of resp, cell wall and membrane synthesis
• chromosome has no chromatin (circular
• about 450 genes
• CpGs unmethylated
• small circ DNA molc (plasmids) that endow bacteria with adaptive properties

Bacterial Structures

Rudimentary ORganelles

• ribosomes: 70s, non specific membrane association, initiating AA=F-met (often F-met-lue-phe)
• inclusion bodies: nutrient/cofactor storage, decrease osmotic pressure

Bacterial Structures

Cell wall

• peptidoglycan (murein), teichoic acid and lipoteichoic acid
• protection, attachment and storage

Bacterial Structures

• LPS, phospholipids, proteins
• protection, storage/transport compartment

Bacterial Structures

• Pili (fimbriae): proteins, attachment, genetic exchange, (motility)
• Flagella and T3SS: proteins, motility and host alteration 

Gram Stain

• delineates the two major subdivisions of bacteria based on cell wall structures
• Crystal violet, than gram iodine, than decolorizer (alcohol or acetone), and last safranin red
• Gram positive: staph. aureus
• Gram negative: E. Coli  

Structural Basis for Gram Stain

• peptidoglycan thickness
• gram negative only has one or two layers of peptidoglycan
• Gram positive have several layers interwoven with lipoteichoic acid and teichoic acid

Gram Positive Cell Wall

• cell wall and cell membrane
• cell wall has teichoic acid, peptidoglycan (murein), and lipoteichoic acid
• also has proteins and carbohydrates

Gram + Cell Wall Components

Peptidoglycan

• thick 3D mesh
• high mechanical stability
• resists hydrophobic compounds
• resists Ab/complement mediated lysis
• resistant to proteolytic enzymes/alkali/bile salts
• lysozyme sensitive
• antibiotic target
• adjuvant for immune response

Peptidoglycan

Basic Chemical Structure

• sugar polymer with  peptides attached to the side of it
• based on Nacetyl glucasamine (NAG) and one derivitized with phosphophenyl pyruvate
• lysozyme can cleave the beta1,4 linkage that binds the NAG and NAM
• gram negative bacteria have diaminopimetic acid rather than lysine in the peptide side chain
• at right angles to surface of bacteria?
• transpeptidation forms the cross links

Peptidoglycan Synthesis:

Compartmentalization

• uses UTP for energy and one from phosphophenyl pyruvate to activate sugar (NAG and NAM)
• amino acid addition- 3 L amino acids added to third AA= lysine. A D-ala-D-ala dipeptide is than added
• attachment of NAG to NAM 
• addition of pentaglycine bringing the pentapeptide side chain (staph aureus)
• membrane lipid goes most of way through bilayer to create hing to flip molecule to outside of cell (undecaprenol phosphate membrane pivot)
• transpeptidation-crosslinking: penicillin binding protein(PBG) are responsible of tr anspeptidation (different ones also help with elongation, septum formation and shape)

Teichoic and Lipoteichoic Acids

structural features

• gram (+) only - some but not all
• polymers of glycerol phosphate or ribose phosphate decorated by various side chain R groups
• Teichoic acids are covalently attached to peptidoglycan
• lipoteichoic acids are anchored in the cytoplasmic membrane through a lipid tail
• synthesized by assembling components on lipid carriers as outlined for peptidoglycan

Teichoic and Lipoteichoic Acids

Functions

• structural Component of the cell wall
• sequesters divalent cations, Mg++, Ca++
• promotes adhesion
• determines serotype distinctions of bacteria (Staph or Strep
• virulence factor: activates/inhibits immune responses (endotoxin-like activity)

Gram Positive Cell Wall Components

Proteins and Carbohydrates

• serve various survival functions
• F Protein: of streptococcus binds to EC matrix protein fibronectin
• Protein A of staphylococcus aureus binds the Fc region of antibodies to prevent opsinization
• C polysaccharide of streptococcus: on seperate card

C Polysaccharide of Streptococcus

Gram Positive Cell wall component

• linked to peptidoglycan
• distinguishes streptococcus subgroups groups (serovars)
• precipitates C-reactive protein (helps evade innate immunity)

Gram Positive Bacterial Cell Wall

Key features

• thick peptidoglycan layer
• highly cross-linked
• contains teichoic and lipoteichoic acids
• components synthesized and assembled in a highly organized compartmented Fashion
• contains multiple virulence factors: maintains bact integrity in variable osmotic env; antigenic but can vary to avoid immune recog; provides unique resistance to host defenses; allows for colonization of specific env

Gram Negative Envelope

Key Features

• outer membrane-provides added protection from the extracellular environment: unique structure of lipids(LPS); porins;permeases, MOMPs
• periplasmic space (gel): a unique cellular compartment resulting from the addition of the outer membrane; contains synthetic and degradative enzymes. transport proteins
• thin peptidoglycan: one layer thick

Gram Negative Bacterial Envelope

Peptidoglycan

• thin- typically a single layer or two
• peptidoglycan cross=linking occurs via diaminopimelic acid (DAP) in the peptide side chain (instead of lysine for g+)

Gram Negative Bacterial Envelope

Outer Membrane

• Lipopolysaccharide (LPS) in outer leaf of the lipid bilayer
• proteins inserted through this membrane- porins, permeases and MOMPs
• occasional membrane contiguity with the cytoplasmic membrane

Lipopolysaccharide (LPS)

• structural element of the outer membrane
• barrier to hydrophobic compounds
• Virulence Factor!!:
• stimulates b cells and macrophages to overproduce IL-1, IL-6 and TNF-alpha (cytokines)
• inducer of septic shock: complement act, disseminated intravascular coag, neutrophil act and entry into tissues, fever induction, acute phase protein syn in liver, vasodilation with associated blood pressure drop, increased vascular permeability
• lipid A is component that causes most of the inflammation (some bacteria lack core polysach and less stimulating)

Lipopolysaccharide (LPS)

Endotoxin

• O-antigen: (galactose-mannose-rhamnose) repeated up to 40 times; made by extracellular prefabricated subunit extension
• Core-Sugars: complex polymer of hexose, heptoses and KDO that is synthesized within the cell
• Lipid A: disacch diphosphate with long chain fatty acid tails: form divalent cation-phosphodiester linkages, syn of lipid A occurs w/i cell and contains major endotoxic properties

LPS synthesis, assembly and transport

• o-antigen and core subunits are assembled within the cytoplasm
• than attached to lipid carriers, translocated to periplasmic leaflet, extended and combined to generate complete LPS
• complete LPS is transporter to the outer membrane via LptA, B, C, D, E, F and G proteins

Gram Negative Bacterial Envelope

Outer Membrane Proteins

• porins: hydrophillic channel for allowing free diffusion of nutrients to the cell. limited pore size 600-3000 MW, determinant of antibiotic sensitivity
• cell surface protein: major outer membrane protiens (MOMPs) of gonococcus provide antigenic variability and attachment functions

Gram Negative Bacterial Envelope

Periplasm

• periplasm constitutes the space btw the outer and cytoplasmic membranes where macromolecules can be extracellular but not lost from the cell
• this includes: nutrient binding and transport proteins, hydrolytic proteins, antibiotic resistance proteins

Acid Fast Bacterial Cell Envelope

Unique Properties

• resists gram staining due to the presence of lipid coat
• strong staining with acid fast stain (carbol-fuchsin, red), counterstain with methylene blue
• chemically resistant to disinfectants, detergents and common antibiotics
• bacteria are typically slow growing due to low nutrient permeability
• primarily associated with mycobacteria(TB and leprosy) but other related genera are also partially acid fast

Acid-Fast Bacteria Cell Wall

Basic Structure

• outer lipids (waxes)
• mycolic acid
• polysaccharides (arabinogalactan)
• peptidoglycan
• plasma membrane
• lipoarabinomannan (LAM)
• phosphatidylinositol mannoside (PIM)
• porins and peptides 

Acid Fast Envelope

Structural Components

• arabinogalactan-attached to murein
• mycolic acids: make up 60% of wall, wax D-glycolipid of 15-20 mycolic acids, cord factor dissacharide glycolipid + 2 mycolic acids (has long chain hydrocarbons, hydrophobic)
• other glycolipids: LAM and PIM
• polypeptides and porins- 15% of cell wall and acts as adhesins, enzymes and antigens

Bacterial Capsule

• sticky web of polysaccharide fiber (carbohydrate): glycocalyx, slime layer, biofilm; 
• allows colinization of difficult env (strep mutans on dental enamel), 
• immune evasion (group A strep hiding with hyaluronic acid), and olyglutamic acid used by B antracics
• Virulence factor is antiphagocytic, increasing size to prevent engulfment, bacteria to stick together and exted beyond limits of the cell
• capsule is also slippery to phagocytic cells, poorly antigenic/prevents Ab binding

Fimbriae= Pili

• virulence factor that allows for adherence (bacteria sex or host to bacteria)
• composed of pilin, a protein polymer whos variation is critical
• has genes composed of a constant region and variable region (undergoes switching and analogous to mammalian Ab gens)
• immune evasion- quick change
• colinization of unique env

Flagella

• Basal body- proton motor
• hook- attaches motor to the propeller
• filament (propeller) - flagellin antigenic
• proton motor - basal body: has membrane potential driven-256 H+/rev
• counterclockwise - run- bundle (more straight)
• Clockwise - tumble - splayed 
• can be monotrichous or peritrichous and most common in bacilli
• chemotaxis - alternating btw tumble and run leads to directed movement (requires receptors and memory, less than 3 sec)

Type III Secretion System

(T3SS; Injectosome)

• machinery related to flagella
• used to inject proteins and whatnot into host cells
• bacteria in gut have it 
• helps to colonize certain environments

Spores

Function

• gram (+) only
• adaption for prolonged survival under adverse conditions
• resistant to killing by heat, drying, freezing, toxic chemical and radiation
• life from mars?

Spores 

Structure

• core: includes chromosomes, minimal enzym, Ca2+, and dipicolinic acid (Ca chelator)
• Ca and dipicolinic acid stabilizes nucleic acids and enzymes that allow it to go from dormant to alive 
• inner membrane-cellular cytoplasmic membrane
• cortex-losely crosslinked peptidoglycan
• outer membrane-membrane facing in, due to spore formation
• tough protein coat - highly S-S cross linked protein forms major chemical barrier - keratin like
• exosporium - lipoprotein membrane
• sporangium - surrounding mother cell prior to lysis

Spore Cycle

Initiation

• switch to spore mRNA and protein syn
• turn off other mRNA syn
• post-translational modification of enzymes
• production of dipicolinic acid and Ca2+ sequestration
• excretion of antibiotics and toxins

Spore Cycle

Germination

• initiated by damage to the coat - by heat, water, trauma or aging
• requires energy for protein and DNA syn
• must go to completion once initated

Sites of Normal Flora Colonization

• normal flora is microbes that normally inhabit surfaces and sites on and w/i people
• outer layer of skin
• mouth
• nares
• upper respiratory tract
• GI tract
• urovaginal region

Benefits of normal Flora

• nutrition
• defense against infection with pathogens
• development and regulation of immune system
• many other influences

Sterile Sites and Fluids

• inner layers of skin
• lower respiratory tract
• bladder and kidney
• organs
• tissue
• blood
• urine (in bladder) 

Disease Production

Microbe Caused  

• inherent microbial properties (virulence)
• strain specific properties
• infectious dose
• site of entry

Disease Production

Host Caused

• immune defense
• immune pathogenesis
• genetics
• age
• nutrition
• general health (accident/surgery)
• luck

Virulence Properties

of

Successful Pathogens

• adherence
• invasion and dissemination or local colonization
• growth in host (knows its in host by temp)
• avoid or inactivate innate/immune clearance: inhibition or resistance to phagocytes, escapes complements, escape antibody detection
• resistance to antimicrobials

Virulence Mechanisms

EAT RICE

• E: Enzyems (hyaluronidase, hemolysin, streptokinase)
• A: Adherence(pilli)(gonorrhoeae and ecoli) 
• T:Toxins (exo, endo, super)
• R: Resistance to antibiotics
• I: Invasion
• C: Circulation to other sites
• E: Evasion of immune response

Colonization Basics

• adherence: streptococcus mutans: dental plaque, bacterial endocarditis, urethritis
• biofilm: strep mutans, psuedomonas 

Requirements for Growth

in Host

• carbon source
• specific metabolites (specific amino acids)
• iron and other minerals (diptheria toxin)
• +redox environment (anaerobe/aerobe)
• pH of environment (H. Pylori, Salmonella)
• Temperature 

Production of Toxic Molecules

• acids, gas, and degradative enzymes
• A-B toxins: cholera, botulism, tetanus (exotoxin) 
• endotoxin: LPS: Lipid A
• cell wall, flagella ect for endotoxins and like molecules
• superantigens (exotoxin)
• degradative enzymes (exotoxin)

A-B Toxins

• A for action/ B for Binding (the two subunits)
• exotoxin
•  ribosome target: diphteria, shiga toxin, pseudomonas exo A
• neuron target: tetanus and botulism
• GI adenylate cyclase target: cholera, e coli
• other targets through cyclase or G protein are pertussis and anthrax

Disruptive toxins

• staph exfoliative toxin
• cleaves linkages btw skin cells and sloughs off skin

Membrane Disrupting Toxins

• Alpha toxin: C perfringens: phospholipase
• leukocidin: s aureus: pore forming

Membrane Pore Forming Toxins

• pneumolysin: binds cholesterol and makes pores
• listeriolysin: binds cholesterol and makes pores

Enzyme Toxins

• C Difficile cytotoxin: depolymerizes actin
• C Speticum: gamma toxin: hyaluronidase beta toxin: DNAase

Endotoxin 

• lipid A from LPS and endotoxin like molecues (cell wall, flagella, ect)
• only gram negative bacteria
• shed from bacteria
• potent activator of TLR4: some varieties activate TLR2
• promotes cytokine storm (acutave phase and inflammatory cascades)
• good local activator and devasting systemic activation
• does not form a toxoid

Immune Escape Mechanisms

• antibody and phagocytes are the major anti-bacterial defenses
• capsule
• phagocyte
• antibody escape: antigenic shift, mimicry(m protein), intracellular growth, degrade antibody
• intracellular growth

Gram Postive Bacteria w/

Capsule

• strep pneumo
• s/ aureus
• s. pyogenes
• b. anthracis

Gram Negative Bacteria w/

Capsule

• neisseria meningitidis
• H. Influenza
• Salmonella
• E. Coli
• N. Gonorrhoeae

Antiphagocytic Abilities

• inhibit opsonization
• inhibit chemotaxis
• prevent phagocytosis
• kill phagocyte
• inhibit lysosome function
• neutralize toxic molecules
• resist action of lysosome inactivate block
• lyse lysosome and grow in cytoplasm

Immune Responses

• takes time
• are induced and antigen specific
• enhance and regulate innate responses and themselves: T cells produce cytokines, T cells bind and tell cells what to do (including apoptosis), B cell produces antibody

Innate Response "Actors" 

• neutrophils
• macrophages
• dendritic cells
• NK cells
• Weird T cells: NKT, Gamma Delta T cells

Soluble Components of Innate Response

• Antimicrobial peptides
• opsonins
• complement
• cytokines, interferons, chemokines

Mast Cell

• tissue resident in skin, mucosa, epithelium, and capillaries
• Cell surface receptors: FcR, C'R, neuropeptides, IL4 cytokineR ect
• action: initiator of inflammation
• cytoplasmic granules: acidic proteoglycans (basophillic staining)
• products: prostaglandins and leukotrienes, cytokins, chemokines, histamines

Neutrophils

•  predominant circulated WBC (50-70%)
• short lived
• major anti-bacterial cell
• phagocytic: cell surface receptors for opsonins(igM, IgG, C3b), anti bacterial enzymes and ROS
• extravasate into site of infection (adhesion molc)
• infection calls immature band froms from bone marrow for a left shift in blood count
• contribute to inflammation
• dead neutrophils become PUS, making net for microbes

Monocyte/Macrophages

• monocytes ar eblood resident precursors to macrophages and dendritic cells
• macrophages differ by tissue and function
• all are phagocytic
• M2 macrophages: status quo, tissue maintenance, wound healing, angiogenesis
• M1 macrophages: antimicrobial/inflammatory: enzymes, NO, ROS, Cytokine production (IL1, TNFa, IL12)

Other Names for Macrophages

• Blood: Macrophages
• Tissue: Histiocyte
• Lung: Alveolar Macrophage
• Liver: Kupffer Cell
• Brain: Microglial Cell
• Bone: Osteoclast

M2 Macrophages

• clean up, recycling, maintenance, status quo
• activated to promote wound healing
• may inhibit inflammation and related T cell Responses (Th1, Th17)

M1 Macrophages

• activated to kill phagocytized bacteria
• promotes inflammation and related T cell responses (Th1, Th17) 

Myeloid Dendritic Cells

• t cell activating/ cytokine producing DC
• phagocytic
• cytokine producer
• antigen processing/presenting cell
• directors of the immune response

T cell ACtivating/Cytokine Producing DCs

(true Dendritic Cells)

• myeloid dendritic cells
• Langerhans Cell: skin version of dendritic cell, contains langerin and birbeck granules
• plasmacytoid dendritic cells: make interferon alpha or IL12 when stimulated

B Cell Activating Dendritic Cell

• follicular dendritic cell
• sticky cell
• not of myeloid origin
• not a T cell APC
• cannot do anything with T cells like other dendritic cells

Natural Killer Cells and 

NK Like Cells (Innate lymphoid cells(ILS)

• large granular lymphocyte
• innate response to infection
• produce cytokines: interferon gamma (activating) and TGFbeta(regulatory) cytokines
• killing function: kills virus infected and stressed cells, and antibody decorated ones
• cell surface derminants: KIR, Lectins, Fc Receptors

NKT and Gamma Delta T Cells

• innate response
• responds to bacterial products or lipids (recognizes weird things but still t cell lineage)
• produces interferon gamma and other cytokines 

T Lymphocytes

• lymphocyte matures in thymus
• manipulators of immune response (cell-cell interactions/cytokine production)
• CD3, CD2 (sheep red blood cell), MHC 1, Chemokine rec
• TCR (antigen receptor) Alpha Beta or Gamma Delta
• CD4 expressing cells: cytokine production: Th1, Th17, Th2, Th9, Treg
• CD8 expressing cells: MHC1 directed killer cell, cytokine production (uses apoptosis pathways to kill)

B lymphocytes

• lymphocyte matures in bone marrow
• produces antibody
• antigen presenting cell
• cell surface determinants: surface immunoglobulin(Ig), MHC II, C3D receptor, etc

Memory and Plasma Cells

• memory cells: mature B or T cells, can be reactivated to restart immune response
• Plasma Cells: terminally differentiated B cells, Antibody factories

Primary Lymphoid Tissue 

• Growth and Training Venue
• Thymus
• bone marrow
• Liver, mucosa associated lymphoid tissue? 

Secondary Lymphoid Tissue

• antigenic sewage processing/action station
• lymph nodes
• spleen
• bone marrow
• mucosa associated lymphoid tissue
• skin associated lymphoid tissue

Lymph Nodes and Spleen

• purpose is the initiation and development of immune response
• optimized for presentation of antigen to lymphocytes
• B cells and T cells are attracted and concentrated to their own place
• B cell zones (no T in follicle or germinal center): follicles have FDCs that attract Bs. germinal centares are sites of B cell prolif
• follicles have dendritic cells in them
• T cell zones: paracortex (lymph) and periarteriolar lymphoid sheath (PALS) in spleen, also myeloid dendritic cells

White Pulp

• found in spleen
• has follicles
• B cell zone
• follicles are surrounded by a rim of lymphocytes and macrophages called the marginal zone 

Bacterial Temperature Requirements 

• psychrophiles: adapted for survival below 10-15C
• thermophiles: grow best at temperatures of 50C and above
• mesophiles: grow best at moderate temperatures (20-45C), includes essentially all human pathogens

Bacterial Atmospheric Requirements

• aerobe
• anaerobe
• facultative(aerobe/anaerobe) respires with O2 and ferments in its absence
• indifferent (aerotolerant anaerobe): ferments in the presence or absence of O2
• microaerophillic: grows best at low O2 but can grow without it also

Bacterial Uptake Transport Systems

• diffusion - H2O, O2, CO2, and NH3
• facilitated diffusion - concentration gradient dependent - glycerol
• active transport: ATP/H dependent-galactose, proton symport-lactose, siderophore mediated Fe, sodium-PO4 cotransport

How Bacterium sparate into 

2 daughter cells

• Spo proteins grab on to nucleic acid and move it towards either end of the cell
• FtsZ protein creates ring that begins to cleave cell in half
• FtsZ generates a cleavage furrow which together with Penicillin Binding Proteins(PBP) and autolysins results in membrane fusion and completion of cell wall synthesis

Bacterial Catabolism

• fermentation: organic compounds serve as electron donors and final electron acceptors (substrate lvl phosphorylation)
• Aerobic Respiration: molecular oxygen serves as final electron acceptor
• anaerobic respiration: inorganic compounds other than oxygen (nitrate and sulfate) serve as final electron acceptor (flora in back of mouth have this)
• phosynthesis
• ion/chemical gradients: extreme environments

Fermentation

• Glycolysis and other pathways are used for substrate level phosphorylation to generate ATP and pyruvate
• pyruvate is reduced to regenerate NAD consumed in glycolysis
• products are determined by bacteria specific fermentation enzymes (helpful in bacterial identification)

lac Operon

• controls genes that encode proteins for lactose transport and metabolism
• B-galactosidase(LacZ) cleaves lactose to glucose and galactose (responsible for production of inducer-allolactose
• galactoside permease (lacY)-transporter for lactose
• Balactoside transacetylase (lacA)
• allolactose prevents repressor protein from binding to the lac operon control site 

Tryptophan (trp) Operon **

• takes a lot of enzymes (big expense to make)
• has R (repressor region) that is inactive unless tryptophan is present
• if tryptophan is present it binds to inactive repressor which binds to R
• RNA folding in the presence of ribosomes and trp-charged tRNA results in RNA structures that can be recognized by transcription attenuator proteins that stop RNA synthesis

Quorum Sensing

• bacterial communication to deal with the presence of siblings
• high cell density produces QS signals that alter gene expression
• first described in bioluminescent bacteria in the 1970s
• common in pathogenic bacteria and regulates virulence genes
• gram neg use acyl homoserine lactones (AHLs)
• gram pos use peptides
• other autoinducers (Al-2) are used in both types
• Qs pathways converge with starvation-sensing pathways to regulate cell entry into stationary phase

Basic Mechanisms of Quorum Sensing

• AHLs are produced in the bacterium by the AHL synthase
• can diffuse from the cell to enter neighboring bacteria
• AHL binding to the receptor polypeptide leads to formation of active dimers
• receptor dimers bind to specific promotor sequence and activate transcription of sets of genes
• QS can affect up to 20% of bacterial genes, regulating such processes as genetic competence, virulence factor expression and sporulation

Bacterial Chromosome

facts 

• circular and covalently closed*
• double stranded DNA about 3-5mil bp(3 bil in humans)
• linear array of aprox 3k genes (23k in humans)
• haploid-no complementation*
• no histones or introns
• unmethylated CpGs
• operons
• gene islands: clusters of related operons are common and appear to be coordinately regulated

Plasmids

• autonomously replicating, extrachromosomal, circ double stranded DNA
• size range 1-400kb
• orgin of replication (ori) distinct from chromosome
• characterist copy number per cell determined by ori sequences
• conjugation plasmids contain info for self transfer
• contain genes for environmental adaption or survival
• no specific segregation during cell division*  

Plasmid Types

• durg resistance: carries resistance against one or more antibiotics
• bacteriociniogenic: codes for small protein that is bacteriocidal for susceptible strains
• fertility (sex factor): codes for proteins necessary for conjugation (includes F pilus)
• toxinogenic: codes for some bacterial toxins or surface antigens such as capsules or pili
• metabolic: codes for enzymes involved in catabolism
• cloning: constructed plasmid used for transferring specific genes into the cell-egrHuGH
• cryptic: encoded for functions unknown

Genetic Recombination

Jumping Genes

• insertion sequences (IS): contain genes for transposase and regulatory protiens (1kb), insertional mutagens, and regulate virulence factors (pillin and biofilm genes)
• Transposon (Tn element): IS that carries additional genes (drug resistance genes)

Major Mechanisms of

Genetic Exchange

• Transformation: a process of scavenging genes from dead cells (smooth(capsule) and rough strain expirement with mice)
• Transduction: gene transfer by bacterial viruses (bacteriophage)
• Conjugation: gene transfer through "sexual" contact

Bacterial Transformation

(genetic Exchange)

• competence is the ability to take up DNA
• Competence Factors: CFS encoded by chromosomal genes; include type 4 pili, autolysins, DNA translocases and secreted factors can induce competence in adjacent cells
• competence can be artificially induced by membrane perturbation w/ cations and temp shock
• entering DNA is eithe rdegraded, maintained as plasmid or incorporated by homologous recombination
• bacteria can discriminate foreign DNA via cell surface proteins and restriction endonucleases

Bacteriophage

• generalized transduction (genetic exchange)
• protein capsid
• Nucleic acid genome of either DNA or RNA but not both
• 6-1000 genes
• Virulent Phage(lytic): lyses bacterial cells upon synthesis of progeny phage
• Temperate phage (lysogenic): either replicates and lyses cell or enters a latent form (prophage) as a plasmid/integrating into host genome; later becomes induced to replicate and lyse cell

Generalized Transduction

(genetic Exchange)

• bacterial DNA can be packaged into the phage particle, this is called pseudotyping
• pseudotyping is the primary mech responsible for transduction-associated bacterial genetic exchange
• aprox 1 0f ever 1k phages is pseudovirion; DNA fragments can represent 1-2% of the bacterial chromosome
• Transduction typically requires recombination w/ chromosomal or plasmid DNA to perpetuate the genetic change

Specialized Transduction

(unique features)

• repressor protein: prevents phage expression
• Prophage DNA integrates: replicated with cell divison
• Phage DNA is excised: imprecision results in the acquisition of bacterial genes that are packaged
• The temperate Phage: facilitate bacterial gene transfer and integration into a new host genome
• medical significance: important in transferring antibiotic resistance genes in staph-VRSAs; corynebacterium diptherias-AB exotoxin is encoded by integrated phage

Bacterial Conjugation

Major Features

• sex pilus or adhesin/receptor mediates cell contact and transfer
• fertility plasmid(F) encodes genes fro sex pilus
• donor cells (F+) contain the F plasmid while F- does not
• high freq recombinant cells (Hfr) contain the F plasmid integrated into the chromosome
• excision of the F plasmid from some Hfr cells results in acquistion of a small number of chromosomal genes by the F plasmid (F')
• conjugation results in the transfer of resistance and virulence genes along with the F factor genes

Bacterial DNA Repair

• direct repair-pyrimidine dimers alkylated bases
• excision repair
• post replication repair
• SOS response- induction of 15 DNA repair genes
• error prone repair-fills in gaps with random sequences when no template exists
• homologous recombination

Endonucleases

• type I and III: cut distal to specific recognition sites (not highly useful)
• Type II: cut at a specific recognition site leaving blunt ends or overhangs (cohesive and non cohesive)

Staphylococcus Microbiology

• gram positive cocci that grow in clusters
• non-motile
• non-spore-forming
• facultative anaerobe
• colonies: white to golden/bronze
• catalase-postivite

Staph. Aureus Virulence Factors

• has capsules/biofil/adhesion factors: MSCRAMM (microbial surface components recognizing adhesive matrix molecules)
• toxins
• enzymes released
• multiple evasions of host protections

Staph aureus

Toxins

• pore-forming toxins (α, β, γ, δ)
• scalded skin syndrome (SSS)
• toxic shock syndrome (TSS)
• enterotoxins

Staph aureus

enzymes released

• hemolysins
• hyaluronidase
• nuclease
• lipase 
• protease
• plasminogen activator
• catalase

Staph. aureus

Evasion of host protections

• coagulase (clumping factors)
• fibronectin-binding protein (FNBP)
• surface and secreted protein A
• Leukocidin

MSCRAMM for S. aureus

• microbial surface components recognizing adhesive matrix molecules
• protein A and portein G: binds Fc of IgG
• clumping factor A (ClfA) binds fibrinogen
• coagulase: promotes conversion of fibrinogen to fibrin clorts
• fibronectin binding protein A (FnbpA and B)
• collagen binding protein (Cna) binds to C1q and blocks classical complement cascade

Staphylococcus Toxins

(also superantigens)

• exfoliatin(scalded skin syndrome, Bullous impetigo)L thing that sloshes off skin
• toxic shock syndrome toxin (TSST-1) (superantigens)
• Enterotoxins - A-F: also superantigens 

Mechanisms of Action of Staphylococcal 

Superantigens

• TSST-1, enterotoxins, exfoliatins A and B
• clamps MHC II molc of antigen presenting cells (macroph, b cells, dendritic cells) to T cell receptor of a large subset of T lymphocytes
• interaction causes prolific activation of T cell functions
• cytokine storm: release of IL-1, TNF, IFNgamma, ect
• leads to major systemic effects (fever, hypotension, skin lesions, shock, organ failure and death
• toxic shock syndrome

staph aureus

skin and superficial diseases 

• abscess (boils, furuncles)
• carbuncles - interconnected abscesses
• impetigo (bullous) (sloshing of skin)
• cellulitis

 Staph Aureus 

Systemic Diseases

• staph scalded skin syndrome (toxic mediated)
• endocarditis (colonization)
• pulmonary infections (colonization)
• osteomyelitis (colonization)
• septic arthritis (colonization and systemic inflammation)
• other 

Scalded Skin Syndrome

• caused by staph aureus
• exfoliatins and B are proteases which break down desmosomes releasing the grtanulosum and spinosum layers of skin
• causes skin to slough off
• its the same thing like having a burn

s. aureus

Food Poisoning

• requires contamination, sutable foods (high salt or sugar), time and temperature
• enterotoxin A-F
• incubation: 1-6 hours (short incubation time, especially for diarrhea)
• symptoms: nausea, vomiting, abdominal cramps, diarrhea
• recovery: 24 - 48 hours

S. epidermidis, and S. lugdunensis

• limited virulence: coagulase negative, produces biofilms, infections of damaged heart valve
• Diseases: opportunistic (nosocomial) infections of catheters, shunts, prosthetic devices
• those diseases cause bacteremia, endocarditis, and UTI

Staphylococcus

Epidemiology

• humans are major reservoir of s. aureus, carriers, 30% in exte3rnal nares
• colonize healthy persons: normal flora, colonize shortly after birth
• survives long time on dry surfaces
• spread person to person by aerosols (nosocomial or 2ndary after influenza/lethal pneumonia), contact and normal flora sites
• foreign body: splinter, catheter, prosthesis, etc
• Food poisoning: picnic foods: processed meats, custard filled pastries, potato salad, and icecream

People at risk

and risk factors for 

Staph infection

• relevant innate and immune deficiences
• infants
• individuals w/ poor hygeine
• cystic fibrosis
• surgery
• presence of foreign body (including surgical devices)
• loss of normal flora
• influenza A
• etc

Prevention of

Staph Aureus

• hygiene
• proper cleansing of wounds: germicidal soap: iodine, hexachlorophene
• no vaccine
• tetravalent vaccine in trials (pfizer): clumping factor, conjugated polysaccharide CP5, conjugated polysaccharide CP8, manganese transporter protein
• trick to vaccine is to stop staph before established by hitting those

Treatment for

staph aureus

• beta lactam resistance very comon in staph (penicillin)
• beta lactamase 
• beta lactamase resistant drugs (methicillin, nafcillin, oxacillin, dicloxacillin resistant (MRSA)
• those drugs are too bulky to bind and be broken by enzyme

Methicillin-Risistant Staphylococcus aureus 

(MRSA)

• community-acquired MRSA: highly virulent, contains plasmid with methicilin resistance and many other virulence factors, replaces penicillin sensitive petidoglycan synthetic enzyme(methicillin is resistant to beta lactamase)
• community accountws for 60% of skin infections in USA
• hospital acquired MRSA existed before

MRSA Enzyme

• acquisition of transposon like staph chromosome cassette mec (bacteriophage
• MecA encodes PBP2A and other antibiotic resistance factors and virulence factors, eg PVL a pore forming toxin
• PBP2a: binds beta lactams very poorly; combines transglycosylase and transpeptidase function to build the growing cell wall

Treatment of MRSA

• vancomycin (though vancomycin resistance does exist with thicker cell wall and enterococcus like replacement of D-ala D ala by D ala D-ser or other group
• trimethoprim-sulfamethoxazole
• daptomycin

Genus

Streptococcus 

overview

• gram positive (purple) cocci (round) bacteria
• occur on gram stain as chains or pairs
• catalase negative unlike staphylococci
• cultures positive w/i 24 hours on sheep RBC agar

Grouping Strep

• lancefield groupings-serology (Ab) (oldest)
• hemolytic patterns of bacterial colonies of the different species on sheep blood agar plates
• biochemical properties
• molecular techniques getting ready for prime time
• matrix assisted laser desorption ionization-time of lfight mass spectrometry (MALDI-TOF MS)

Hemolysis Grouping

Streptococci

• gamma: no homolysis, includes eterococci
• alpha: greening: partial hemolysis: includes viridans strept and streptococcus pneumoniae
• beta: total hemolysis
• includes groups A, B, C, F

Lancefield Typing 

for 

Grouping Streptococci

• groupoing of beta hemolytic strept
• serology: Ab to specific carbohydrates (Ag) present in cell walls
• differentiate groups A, B, C, F and G

Biochemical Testing

Grouping Streptococci

• divides the viridans strept into 5 groups
• S. mitis - includes S pneumo (most important
• S. anginosus
• S. mutans
• S. salivarius
• S. bovis

Group A Strep

Virulence Factors

• hyaluronic Capsue: similar to hyaluroic acid in mammalian membranes and poor immunogen
• C5a peptidase: this serine protease inactivates C5a
• LTA: initiate adherence to fatty acid binding site on host epithelial cells
• fibronectin: binding (F) protein-impt binding to respiratory epithelial cells
• M proteins: help bind to resp epithelial cells

M Protein

• important virulence factor for Group A Strep
• encoded by emm genes
• 80 types: class 1 share exposed antigens w/ human tissue and are involved in development of rhumatic fever and glomerulonephritis; class 2 do not
• aids adherence to epithelial cells (w/ F protein) pharyngeal colonization
• aids invasion into cell (along with f protein
• inhibits phagocytosis of the bacteria by binding fibrinogen and blocking binding of C3b to peptidoglycan
• Mlike proteins bind Fc of Ab, blocking activation of C'

Types of Group A Strep

Virulence Factors

• toxins
• hemolysins
• streptokinases
• Dnases

Group A Strep Spe Toxins

• Streptococcal pyrogenic exotoxins
• heat labile toxins; similar to that found in C. diptheriae
• superantigens
• SpeA, SpeB, SpeC, SpeF
• may rarely be found in GrpC and G strept
• mediate release of proinflammatory cytokines from macrophages and T helper cells
• clinical manifestations of necrotizing Fasciitis, rash of scarlet fever  

Group A Strep 

Hemolysins

• allow lysis of WBCs, platelets and RBCs (Hemolysis)
• Streptolysin O - heat labile exotoxin (inactivated at high temps), ASO titer-serologic test that helps identify GAS infection after Strept Throat
• Streptolysin S - heat, oxygen and serum stable, cardiogenic exotoxin

Group A Strep

Streptokinases A and B

• secreted, clot lysis
• lyse blood clots and allow spread of strep antistreptokinase - a serologic marker of GAS infection
• medical application: dissolve clots in coronary, cerebral, pulmonary and extremity arteries

Group A Strep

DNases

• deoxyribonucleases
• extracellular products of GAS
• 4 distinct enzymes: A, B, C, and D
• degrade DNA and potentiate spread of bacteria thru the tissue planes
• liquify pus
• can measure antibody (Ab) to Dnase B: elevated levels seen in post streptococcal glomerulonephritis

Group A Strep

Facts

• spread by respiratory droplets from clonized or infected person
• asymptomatic colonization of pharynx common (15%) and higher in crowded conditions
• invasivenesss of different strains (M protein)
• immuno compromised puts people at risk, eg lack of sleep
• skin infections can occur after breaks in skin: autoinfection in colonized person or skin contact with a person or colonized inanimate object

Group A Strep 

Diseases

• major cause of purulent pharyngitis (Strep throat)
• skin infections: cellulitis, impetigo, erysipelas, necrotizing fasciitis (flesh eating strep)
• less common: bacteremias, spetic and prosthetic joint infections, and meningitis
• sequelae of GAS diseases: autoimmune mech: Rhuematic fever and glomerulonephritis

Cellulitis

• laboratory evalouation unhelpful: nothing typically to culture unlcess pustules/wounds are present (uncommon)
• only 5% of Blood Culture are positive
• Group A strep is most common cause
• B hemolytic strep is more common cause in diabetics
• Grps C and G strep can cause it especially if venous stasis/lymphedema
• S. aureus typically see a focal purulent collection (Pus)

Necrotizing Fasciitis

• flesh eating strep or streptococcal gangrene
• extensive and rapidly spreading necrosis from site of typically trivial trauma to deeper tissue
• tissue rapidly evolve(1-3d): dusky to violaceous to bullous/hemorrhagic lesions of skin
• systemic toxicity: fever, severe pain
• myonecrosis (elevated CPK)

Scarlet Fever

• strep lecture
• strains produce erythrogenic toxins
• associated w/ pharyngitis and skin infections
• rash on 2nd day of illness: spares face/hands/feet(unlike syphilus); strawberry tongue, desquamation

Rheumatic Fever

• non-suppurative inflammatory lesions as a consequence of Group a Strep infection
• occurs 1-5wks post (lasts 3 mos)
• elevated ASO, anti DNase B
• Highest risk 5-15yrolds, F>M (0.9-3% of untx GAS pharyngitis)
• acute disease: fever, acute polyarthritis (75%), carditis (40%), chorea(movt disorder) (15%), subq nodules and erythema marginatum(smoke rings) (both less than 10%)
• sequelae: carditis: heart valvular insufficiency and/or stenosis

Rhematic Fever

Virulance Features

• rheumatogenic Group A strep
• distinct antigenic domain on M protein and superantigen pyrogenic exotxins evoked cytokine storm provoke a strong host autoimmune IgG response
• heavily encapsulated (mucoid colonies)
• fail to elaborate Alpha Lipoproteinase

Rheumatic Fever

Molecular Mimicry

• Group A strep
• most popular theory
• shared antigenic determinants of epitopes of streptococcal M proteins and human cardiac myosin, sacrolemmal membrane proteins, synovium and articular cartilage
• cross reaction with GAS cell membrane Ag and neurons of caudate and subthalamic nuclei (sydenham's chorea)

Post Streptococcal

Glomerulonephritis

• caused by nephrogenic strains of Group A Strep
• can occur 10 d post pharyngitis, 3 wks post skin infections
• immune complex mediated- deposits of immune complexes in subepithelium of glomeruli
• lab: test for  anti-DNase B

Diagnosis of GAS Infection

• ASO titers (antistreptolysin-o): helpful in diagnosis of rheumatic fever, glomerulonephritis; not seen in GAS skin infections since streptolysin O is inactivated by skin cholesterol
• Anti-DNase B: present if recenty pharyngeal or skin infection; strep associated glomerulonephritis

Group B streptococcus

• S. agalactiae - Group B Strep (GBS)
• baby strep: major cause of neonatal spesis as well as peurperal sepsis (chorioamnionitis
• diabetic strep: major cause of cellulitis and deep space foot bascesses in diabetics
• differentiated from GAS by lancefield typing as well as very small halo of beta hemolysis

streptococcus dysgalactiae

• subspecies equisimilis and a beta hemolytic strept
• typically lancefield groups C and G (large Colonies)
• act like GAS, can cauge pharyngitis +/- glomerulonephritis, esp in immunocompromised
• major cause of cellulitis post venectomy (vein removal)

Streptococcus constellatus

• Group F strep
• 40% are beta hemolytic
• usually react to lancefield group F antibody
• may cause purulent pharyngitis
•  youll miss it on Rapid strep screens

Streptococcus angiosus

• colonize mouth, colon and vagina
• mostly alpha but 12% are beta hemolytic
• can cause deep tissue abscesses including brain, liver, and spleen

Viridans streptococci

• show alpha hemolysis on sheep blood agar plates
• colonize mouth, respiratory, skin, and GI tract
• more than 30 species divided into 5 groups
• S. mitis: group includes S. pneumoniae: SBE, meningitis
• S. anginosus: incl s constellatus, s intermedius: abscess formers: livers brain and empyemas
• S. mutans: cause dental caries and mouth abscesses
• S. salivarius: causes SBE (bacterial endocarditis)
• S. bovis: biotype 1, S. gallolyticus closely assoc w/ colorectal cancer, also cause SBE

Streptococcus pneumoniae

Diseases

• major bacterial cause of community acquired pneumonia (CAP)
• sinusitis
• otitis media
• meningitis
• bacteremia
• less commonly: endocarditis 

Streptococcus pneumoniae

• aka pneumococcus
• encapsulated gram positive diplococci
• alpha hemolysis by pneumolysin production
• identified by Ab to the unique capsular antigens of over 90 different serotypes
• most common pathogenic strains contained in vaccines 

S. pneumoniae Virulence

• Capsule (polysaccharide for antiphagocytic protec)
• surface protein adhesins(PspA) for pharynx colloniz
• secretory IgA protease to ellude mucous trapping by IgA of resp tract
• pneumolysins: cell lysis, mediates inflammation (C' activation) and binds cholesterol of ciliated epithelial clells and phagocytic cells=pores
• phosphorylcholine(in bacterial cell wall): binds PAF on blood and tissue cells - entry into cell and passage into blood and CNS

Classic presentation of

Streptococcus pneumoniae

• most common bacterial cause of pneumonia
• sudden onset
• rapid deterioration
• lobar infiltration
• rusty sputum with gram positive diplococci on gram stain
• pneumococcal c polysaccharide Ag in adults can be used to diagnosis: Urine Ag (70% for bacteremic pneumococcal pneumonia and CSF Ag up to 100% for meningitis

S pneumo Risk Factors

• splenectomy
• Ig Deficiences
• hematologic cancer esp multiple Myeloma
• alcoholism
• transplant recipients
• HIV infected 
• Diabetes

Enterococci (Genus)

• live in gut
• most nonhemolytic, can be alpha, rarely beta
• catalase negative, bile insoluble, ptochin resistant PYR (L pyrrolidonyl arylamidase) test positive
• 40 species but most importnat are E. faecalis, faecium, gallinarum, and casselifavus

Corynebacterium

• gram positive rods, non-spore forming and small, non-motile
• pleomorphic: V and Y shapes and chinese letters under microscope
• about 60 different species: c diptheriae, jeikeium, minutissimum, archanobacterium hemolyticum and rhodoccocus equii 

Diptheria 

Pathogenesis and clinical manifistations

• respiratory or skin transmission
• after 2-4 days - toxin production
• toxin mediated tissue necrosis
• local inflammatory response
• formation of membrane
• toxin production expands.... 
• clinical manifistations: nasal, tonsillar, pharyngeal laryngeal, cutaneous, and other mucous membranes

Tonsilar, pharyngeal, laryngeal

Diptheria

• insidious process
• most resolve in 7-10 days
• membrane may extend-nose, trachea
• obstruction, swallowing difficulty
• respiratory arrest

Diptheria Toxin

• 60,000 mw protein
• 2 subunits A,B
• B subunit - cell receptor binding
• A subunit - enzymatically active
• inhibits elongation factor 2 - ADP ribosylation
• initiates DNA fragmentation and cytolysis
• corynebacteriophage: diptheria toxin- tox gene
• c diptheriae diptheria toxin repressor -dtxR, Fe + binds to promotor of tox gene to regulate toxin production
• so as long as theres high iron you are not going to make diptheria toxin

Diptheria Treatment

• equine antitoxin
• antimicrobial: erythromycin, penicillin G
• manage contacts
• tetanus shot has diptheria booster (antibodies)

Diptheria Vaccine

• toxoid (inactivated toxin)
• administered: well baby gets DTaP at 2, 4, 6 months of age
• boosters: DTaP: 15-18 mo, 4-6 years; Tdap: every 10 years

Listeria monocytogens

• present in stool of 5% healthy adults
• 250 deaths per year (elderly get lysteria because immune function diminishes)
• foods common source: refrigerator and deli
• lysteria likes cold temps (4 degrees)

lysteria monocytogenes

pathogenesis

• intracellular invasion: internalin, 60 kd protein (p60)
• able to grow intracellularly
• listeriolysin O is similar to hemolysin (allows to escape vacuoles to cytoplasm)
• invade adjacent cells by polymerize host actin filaments, a product of actA gene
• clinical manifestations are flu like illness, gastroenteritis, bacteremia and CNS infection

Listeria monocytogenes

CNS involvement

• goes from blood stream to CNS
• meningitis: meninges and brain, meningoencephalitis, brain stem encephalitis
• brain abscesses

Listeriosis

• pregnancy
• 30% of all cases of listeriosis
• mild impairment of cell mediated immunity
• usually 3rd trimester
• dissemination to the placenta
• also early and late onset neonatal listeriosis

Early onset neonatal listeriosis

• serotypes 1a, 1b
• may occur in utero
• maternal illness
• onset usually less than 24 hours
• sepsis (whole body inflamation) in 75%

Late Onset Neonatal Listeriosis

• serotype 4b
• not maternal illness (moms source of colonization but not sick)
• not in utero 
• onset 2-4 weeks
• healthy at birth
• meningitis in 90% of cases

Bordetella

• B. pertussis
• B. parapertussis
• B. bronchiseptica

Bordetella pertussis

• aerobic gram negative (red) rods
• cause acute respiratory tract infectino
• sole agent in epidemic pertussis
• exclusively a human pathogen

Pertussis

• highly contagious respiratory infection
• outbreaks first described in 16th century
• bordetella pertussis isolated in 1906
• estimated 295k deaths worldwide in 2002
• primarily a toxin mediated disease
• bacterial attach to cilia of resp epith cells enhances toxin effect
• result sin mucosal sloughing and reduced local clearance which causes obstruction of small airways
• mostly effects kids less than one year old (not strong immune system)
• lots of cases go unrecognized
• 84% of deaths less than 3 months
• immunity is not long lasting so adults can pass to kids (not as strong of effect on adults

Pertussis virulence factors

• important that multiple avenues
• pertussis toxin (PT)
• filamentous hemagglutinin (FHA)
• pertactin (PRN)
• fimbria (FIM)
• tracheal cytotoxin

Pertussis Clinical Features

• catarrhal stage: 1-2 weeks
• paroxysmal stage: 1-6 weeks (coughing phase)
• convalescence: weeks to months

Pertussis Prevention

• whole cell killed vaccine: routine in 1940s
• vaccine effective, but mild to moderate rx
• diptheria-tetanus-acellular pertussis (DTaP): effort to decrease reactogenicity (reactions), first offered in 1991, composed of 2, 3 or 4 antigens
• best way to treat pertussis is to avoid it

Pasteurellaceae

• haemophilus, pasteurella, actinobacillus
•  small gram negative coccobacillus (in btw rods and cones)
• non-spore forming
• nonmotile
• aerobic to facultative anaerobic

H. influenzae

microbiology

• pasteurellaceae?
• fastidious (complex nutritional req) grower: chocolate agar
• 2 groups encapsulated and nonencapsulated
• encapsulated (typeable): a-f types 
• serotype b is most common encapsulated and assoc w/ 95% of invasive disease and has pentose sugars
• nonencapsulated (nontypeable): its just natural and prob have it

H. influenzae 

epidemiology

• normal flora of nasopharynx
• rates of carriage - non-typable H. influenzae: 60-90% healthy young childen, and 35% in healthy adults
• rates of cariage: typeable H. influenzae: 5% among children, with half being type b
• transmitted by resp droplet
• type b was the most common before vaccination

H. influenzae type b

epidimiology

• children colonized in first 2-5 mo of life
• infection in first 2 months is rare due to maternal antibody
• peak incidence of invasive H. influenzae is 6-12 months of age
• exposure risk factors: young age, day care attendance, large growded households, school age siblings, and native american, alaskan inuiet populations

H. Influenzae Virulence Factors

and disease transmission

• capsule-PRP: resists complement and phagocytosis
• outer membrane component
• pili
• IgA proteases
• exogenous transmission: inhalation of infected droplets from active cases and carriers
• endogenous transmission:  

H. influenzae 

Invasive Disease

• gets in blood stream and can than go anywhere c 
• meningitis
• bacteremia
• epiglottitis
• pneumonia
• septic arthritis
• cellulitis

H. influenzae

Mucosal Disease

• gets in nasopharynx and just sits there
• otitis media and sinusitis are most common
• conjunctivitis
• bronchitis

H. influenzae treatment

• beta lactamase negative (70% of cases): use amoxicillin
• beta lactamase positive (30% of cases): use cephalosporin (2nd and 3rd gen)

H. influenza type b Vaccine

PRP-D

• uses protein rather than polysaccharide
• PRP polysacc. conjuagted to diptheria toxoid
• Tcell dependent response
• variable effectiveness in infants
• aproved for child greater than 18 months
• no longer used in USA

H influenza type b vaccine

HbOC, PRP-OMP

• PRP oligozaccharide conj to a nontoxic variant of diptheria toxin (HbOC) or outer membrane protein (OMP)
• t cell dependent response
• highly effective in children greater than 2 months
• administered at 2, 4, and 6 months of age with DTaP

Pasteurella

• gram negative coccobacilli
• found among mouth and GI flora
• common in dogs, cats, pigs, and variety of others
• P multocida is the most common isolate
• others include p canis, p pneumotropica, and p bettye

Pasteurella Multocida

• most often associated with animal bite wound infections
• 50% of cat bites wounds and 15% of dog bite wounds become infected-great majority p multocida
• related to mechanism of inoculation
• cellulitis is most common; osteomyelitis, meningitis, sepsis may also occur
• penicillin, amoxicillin, ampicillin, cephalosporins and fluoroquinolones can be used
• antimicriobial use: prophylaxis of animal bite wounds (face/hands) and treatment of infection
• wound debridement and drainage

Legionella

• greater than 20 known to infect humans
• L. pneumophilia has 16 dif serogroups
• L. pneumophila serogroup 1 causes 70-80% of disease: subtype recog by monoclonal ab - MAb2, and MAb2 is used in urine antigen screening

Legionella Microbiology and

epidemiology

• gram neg coccobacillus
• aerobic, non-spore forming
• requires cysteine and iron
• catalase positive
• gray white colonized with cut glass appearance and has distinct fatty acids in cell wall
• can survive wide range of env: includes tubs, showers and nebulizers, A/C
• elderlya nd immnunosuppresed are at highest risk
• infections can be epidemic or sporadic
• cases may be hospital acquired

Pontiac Fever

• legionella implicated
• slef limited febrile illness
• incubation 1-2 days and high attack rates
• contamnated aerosol exposure
• flu-like symptoms with no pneumonia
• spontaneous resolution

Legionnaires Disease

and clinical findings

• sporadic and epidemic
• incubation 2-10 days
• no person to person transmission
• .5-10% of lobar pnemonias (less than 1 % in children
• acute bacterial pneumonia(difficult to deffer from pneumococcus)
• accompanying flu like symptoms
• confusion
• GI findings- diarrhea and elevated LFTs
• mortality 15-20%

Legionella Diagnosis

and 

Treatment

• isolate organism on CYE agar
• DNA hybridization
• urine test is best and serology (acuve vs convalescent is used)
• macrolides used to treat: erythromycin and azithromycin
• fluoroquinolones also used: ciprofloxacin and levofloxacin

Legionella 

Prevention

• routine environmental cultures in hospitals and nursing homes
• chlorination of water systems
• thermal inactivation of water systems (140 degrees F)

Bartonella

Micro and epidem

•  gram negative, thin bacillus
• aerobic, fastidious, and motile
• slow grower - 2weeks to visible colonies
• chocolate agar or blood agar is best

Bartonella henselae

epidemiology

• cats worlwide, transmitted to them by fleas
• cats (kittins mostly) infected for months but appear healthy
• transmitted by bite, scratch or lick to humans
• 3-10 days you get primary papule at site of bite
• 1-3 weeks, regional lymphadenopathy, fever, malaise, myalgia, and anorexia
• 2-4 months lymphadenopathy disappears
• can involve CNS or other ograns (uncommon)

Bartonella

Clinical Manifestations

• lymphadenopathy (cerv or axillary)
• perinauds syndrome (conjunctivitis/preauricular node)
• fever undetermined origin
• hepatosplenic CSD
• encephalitis
• bacillary angiomatosis

Bartonella Diagnosis

and 

Treatment

• history of cats, lymphadenopathy 2 wks after contact
• negative studies for other causes
• positive serologies for B. Henselae'node biopsy
• azithromycin useful for lyphadenitis in 50% of cases
• 30% of nodes require drainage
• hepatosplenic CSD you treat with azithromycin

Bacillus

• contains 250 species: bacillus antracis (antrax) and b cereus (gastroenteritis and ramatic eye infections and actheter associated sepsis) are medically important
• bacteria of the family bacillaceae form endospores
• two clinically important spore formers are bacillus (aerobe) and clostridium (anaerobe)

Sporulation

• spore has inner membrane, two peptidoglycan layers and outer protein coat
• sporogenesis includes diplication of chromosome in core
• two outer layers are surrounded by the cortex
• cortex is surrounded by keratin like protein coat, that protects the spore
• protects genomic DNA from intense heat, radiation, dryness and attack by most enzymes and chemical agents
• mechanical stress, pH, heat, water, and the nturient (alanine) are required
• spore take up water, swell and shed its coat and produce vegetative cell

Bacillus anthracis

• Large gram positive rods arranged in single or pairs
• spore forming nonmotile nonhemolytic
• aerobes and facultative anaerobes
• D-glutamic acid capsule: inhibits phagocytosis of replicated cells (not plysaccharide)
• exotoxin: protective antigen (most immunogenic), edema factor (fluid accumulation), lethal factor (stimulats macrophages to release TNF-a, interleukin-1b, other proinflammatory cytokines

Cutaneous Anthrax

• caused by BAcillus anthracis
• exposure by inoculation of spores: 2-5 days papule
• vesicle filled with dark fluid-eschar and edema
• healing or massive local edema, toxemia, bactermia and death (20%) if untreated

Pulmonary Anthrax

• 1-5 days after inhalation of spore
• exposure: incubation period-days to months(usually days)
• first stage: non sprecific symptoms-mild fever, myalgia, malaise, non-productive cough
• second stage: begins abruptly 2-3 days after first stage: rapid sepsis, worsening fever, edema, enlargement of mediastinal lymph nodes, respiratory failure, meningitis in about 50% of patients, death due to shock in 24-36 hours

Gastrointestinal Anthrax

• ingest spores on raw or undercooked meat
• mesenteric adenopathy, hemorrhage, ascites production
• nausea, vomiting and diarrhea
• high mortality (90%)

Diagnosis of 

Bacillus anthracis

• organism is present in high concentrations in clinical specimens (microscopy typically positive) and grows readily in culture
• preliminary identification: gram positive, nonmotile rods and colonial morphology(nonhemolytic, adherent colonies)
• confirmation: demonstration of capsule, a positive direct fluorescent antibody test for cell wall polysaccharide or positive nucleic acid amplification assay

Anthrax Epidemiology

• reservoir: soil
• infected animals contaminate soil
• high mortality rate in animals (80%)
• humans infected when exposed to contaminated animals or products
• agriculture and industrial workers at risk
• routs of infection: inoculation, inhalation or ingestion
• cases are sporadic
• biological warfare/bioterrorism

Antrax Treatment

and

Prevention

• inhalation, gastrointestinal or bioterrorism assoc anthrax: ciprofloxacin or doxycycline, may be combined w/ one or two of others (rifampin, vancomycin, penicillin, impenem, clindamycin, clarithromycin
• naturally acquired cutaneous anthrax: amoxicillin
• animal vaccination is effective but human vaccines have limited usefulness
• vaccination of herds and people in endemic areas and proper disposal of infected animal

Bacillus Cereus

• gram positive (purple), spore forming, motile, catalase posive cells
• aerobic or facultative anaerobes
• straight or slightly curved, arranged alone or in short chains
• ubiquitous in soils throughout the world
• heat-stable and heat labile enterotoxins
• tissue destruction by cytotoxic enzymes (cerolysin and pospholipase C)
• risk factors: eating contaminated foods (rice, meat, vegies and suaces) penetraiting injuries(eye), recieve intravenous injectinos, immunocompromised

Bacillus cereus

Diseases

• Food poisoning: consuming contaminated food (rice, meat, vegies, sauces)
• bacteremia and endocarditis: most are due to central line related infections
• musculoskeletal infection: associated with trauma, IV drug use, immune compromise (gunshot, open fracture, post op, burns, cellulitis from heroin)
• CNS infection: meningoencephalitis, brain, abscess, associated w/ hematologic malignancy, neurosurgery
• ocular infection: endophthalmitis, keratitis
• bacillus bacteremia risk factors: IV drug use, hemodialysis, leukemia, IV catheters, pacemaker wires, skin or wound infection

Bacillus cereus Food Poisoning

Emetic Form

• rice is implicated food
• less than six hour incubation period
• symptoms: vomiting, nausea, abdominal cramps
• duration: 8-10 hours
• enterotoxin: heat stable (opposite of diarrheal form)

Bacillus cereus Food Poisoning

Diarrheal Form

• meat and vegatables implicated
• greater than six hour incubatin (mean of 9 hr)
• diarrhea, nausea, abdominal croms
• duration is 20-36 hours
• heat labile (opposite of emetic)

 Bacillus cereus

Panophthalmitis

• contaminated soil in direct contact with eye or penetrating trauma to eye
• symptoms: eye pain, fever and leukocytosis, marked vision deterioration, enucleation
• virulence factors: cerolysin (hemolysin), necrotic toxin (heat labile), phospholipase C (lecithinase)
• therapy: antibiotics and surgical intervention

Bacillus cereus

Diagnosis, treatment, epidemiology and prevention

• isolation of organism in implicated food product or in tissue specimen
• latex agglutination text, immunochromatographic test, PCR
• GI infection you treat symptoms
• ocular infections or other invasive infections: debridement, removal of foreign bodies, drug treatment with vancomycin, clindamycin, ciprofloxacin or gentamicin
• all infections originate from environmental source(soil)
• proper preparation of food, immediate consumption and proper refrigeration

Innate Immunity

Major Components

• physical and chemical barriers
• microbe pattern recognition receptors (PRRs)
• effector cells
• intra/intercellular signaling molecules: pro- and anti-inflammatory 

Physical and Chemical Barriers

Innate Immunity

• Skin: keratinocytes(oxidized crosslinked), and acid pH of sweat and sebaceous glands and hair follicles
• airway, gut, urinary tract: fluid flow, cough, peristalsis, ciliated epithelial mucous, macrophages, normal flora, defensins, low pH, bile enzymes
• Eyes and mucous membranes: tears, lysozyme, mucous traps and restricts microbe invasion, low pH, PGRPs, defensins

Epithelia in Innate Immunity

• physical barrier to infection: often ciliated and covered in mucus
• killing of microbes by locally produced antibiotics: α, β-defensins, cathelicidins, PGRPs
• killing of microbes and infected cells by intraepithelial lymphocytes: gamma-delta T cells, B1 B cells recognize and respond to commonly encountered microbial antigens (limited diversity)

Pattern Recognition Receptors (PRRs)

• proteins that bind/interact w/ common microbial structures (pathogen ass molc patterns;PAMPs) or with damaged cells (DAMPs)
• PAMPs are not present on host cells, but are essential for microbe survival (LPS, PG, dsRNA)
• PRRs are coded for by genes in germ line (do not undergo somatic recombination)
• recognition repertoire of PRRs is less diverse than adaptive immune receptors (antibodies and MHCs): need less genes though (10³ vs 10⁹ genes)

Pattern Recognition Receptors (PRRs)

Cell Associated Proteins

• toll-like receptors (TLRs)
• NLRs-NOD like receptors (recogn peptidoglycan)
• RLRs - RIG - like receptors (recogn double stranded RNA)
• mannose receptor
• scavenger receptor (recognize bacterial lipids

Pattern Recognition Receptors (PRRs)

Circulating effector proteins

• complement
• collectins-Mannose binding lectin, ficolin
• C-reactive protein-pnetraxin
• natural antibodies
• coagulation factors

Toll Like Receptors (TLRs)

Specificities and Location 

• Following on the plasma membrane
• TLR-1 and 2: bacterial lipopeptides
• TLR2: bacterial peptidoglycans
• TLR 4: LPS
• TLR 5: bacterial flagellin
• TLR2 and TLR 6: bacterial lipopeptidases
• Following on endosome
• TLR 3: dsRNA
• TLR 7 and TLR 8: ssRNA
• TLR 9: CpG DNA (unmethylated

Specificity of NOD like Receptors (NLRs)

• NOD 1 recognizes D-Glx and meso-Dap (gram neg bact peptoglycan
• NOD2, cryopyrin: D-Glx and rest of structure 
• also major component of inflammasome 
• inflammasome drives inflammatory processes

Circulating Effector Protein

Complement overview

• collection of 30 circ and membrane assoc proteins for microbial defense
• many components are proteolytic enzymes which act in an enzymatic cascade to form active molecules that kill microbes
• central component is C3 which is activated by proteolytic cleavage to C3b: C3b covalently attaches to microbes and can activate downstream complement
• coats(opsonizes) microbes for phagocyte recognition
• chemoattractant for neutrophils and monocytes (promotes inflammation)
• complement activation helps formation of Membrane attack complex (MAC)
• promotes antibody production

Circulating Effector Proteins

Collectins (Ctype Lectins)

• proteins with the ability to recognize specific types of sugars
• opsonize microbes for phagocytosis
• complement activation (lectin pathway) via lectin associated serine proteases
• mannose binding lectin: deficiency (4% of pop) increaes susceptibility to bacterial infection
• ficolin
• dectins: important for antifungal

Mannose Binding Lectin (MBL)

Collectin Properties

• collagen-like domain coupled to a Ca dependent lectin domain
• binds terminal mannose and fucose
• binds to C1q complement receptor on macrophages
• expressed by liver during acute phase

Collectin Properties

Ficolin and Dectin

• Ficolin: structure similar to MBL but contains a fibrinogen domain, binding specificty fo N-acetyl glucosamine (NAG)
• Dectin: transmembrane proteins w C-lectin domain extracellular, ITAM actiation domain cytoplasm can induce phagocytosis and respiratory burst

Circulating Effector Proteins

C-Reactive Protein

• belongs to the pentraxin family of plasma proteins
• an acute phase protein made by hepatocytes
• binds to: pneumococcal capsules, microbial phosphoryicholine and phosphatidylethanolamine on damaged cells
• opsonin that binds C1q and interacts with C1q receptors on phagocytes
• complement activation via the classical pathway

Dendritic Cells

• possess largest diversity of cell associated PRRs- engagement activates these
• found in skin, mucosal tissues where they phagocytose and process microbes fro antigen presentation
• activated cells release inflammatory cytokines
• important bridge btw the innate and acquired immune system
• traffic to lymph nodes to stimulate t cells

Neutrophils

(PMNs)

• characterized by multi-lobed nuclei and cytoplasmic granules
• produced in the bone marrow in response to cytokines GM-CSF and G-CSF
• 4k to 10k cells per ul blood
• first responder to infection 
• migrate to infection/damage, phagocytose and kill microbes or clear cell debris
• short lived - die within a few hours
• primary (azurophilic) and secondary (specific) granules
• primary has more enzymes in it

Phagocyte receptor classes

• those that bind to microbes directly: mannose receptor, scavenger receptor (Lipids) and CD14 lucine rich repeat receptor (LPS?)
• those that bind to opsonized microbes: ctype lectin rec, complement rec, Fc receptor, and receptor tyrosine kinase (RTKs) bind to apoptotic cells

Natural Killer Cells

• innate immune cells that recognize and kill virus-infected, damaged or transformed cells
• 10% of blood and peripheral lymphocytes
• kill via release of granule proteins that alter membrane permeability and induce apoptosis
• occurs via engagement of receptors that recognize surface markers expressed by stressed and infected cells
• enhanced by MO secreted IL-12
• active NK cells release IFNgamma that further activates macrophages
• NK killing prevented by engagement of inhibitory receptors known as killer cell immunoglobulin like Receptors
• NK cells also express Fc receptors to recognize cells coated with antibody 

Cytokines

(interleukins)

• simple polypeptide or glycoprotien that mediate cell-cell communication
• function to regulate the intensity and duration of the immune response
• typically affect cells of hematopoietic orign, but their cellular range can be broad
• bind receptors with high affinity and induce gene expression changes
• stimulate or inhibit cell proliferation, activation, differentiation, antibody secretion, cytokine expression
• production is typically transient, any constitutive production is either low or highly restricted
• their radius of action is local but is dependent on expression lvls
• primary drivers of inflam: IL-1, IL-6 and TNF

TNF actions

• low quantities: local inflammation, leukocyte activation and endothelial cells
• moderate quantities: systemic effects, brain causing fever, liver for acute phase proteins and leukocyte production in bone marrow
• high quantities: septic shock, low cardiac output, thrombus and low blood vessel resistance, and hypoglycemia (liver)

Chemokines

(chemotactic cytokines)

• lare family of small secreted proteins with potent leukocyte activation or chemoattractant properties
• higher the conc the more chemotaxis
• C chemokines, CC chemokines and CXC chemokines (CX3C chemokines-made by neurons)
• all GPCR that are named for what binds: signaling cascades, including processes involved in locomotino and altered integrin expression

Cytokines

STAT

• Source: producer cell
• Trigger: stimulus for production
• Action: consequences-response
• Target: cell (receptor)

Cytokine Receptor Cascades

• presence on cell determines response
• receptor hook up with one or more mediators inside cell
• pathways deliver response to nucleus: kinases (Jak-STAT, IRAK), IP3, Ca2 etc
• transcription factors determine new cell programs

Acute Phase Cytokines

(proinflammatory cytokines)

Tumor Necrosis Factor Alpha

(TNF alpha)

• produced by activated macrophages(M1) but also many other cell types
• promotes fever: endogenous pyrogen through prostaglandins
• promotes inflammation: prime and recruit PMNs, promote diapedesis, activate anr rucruit monocytes, and activate mast cells
• causes sepsis: promotes IL1 and 6 and chemokines, acute phase proteins from liver, IV thrombosis, vascular leakage and shock
• cachexia: catabolism of muscle and fat, loss of appetitie
• apoptosis(tumor necrosis)
• inhibits wound healing

TNF Receptors

• on most cells
• TNFR1 and TNFR2: family: LT-BR and Fas (killing) , CD40, OX40, RANK
• adaptor protein TRADD: death through activation of Caspase 8 (apoptosis)
• adaptor protein TRAF, RIP: kinase cascade: activation of NF-kB, AP-1 transcription factors, inflammation

Interleukin 1 (IL-1α, IL1β)

• produced mainly by activated macrophages, also neutrophils, epithelial and endothelial cells
• IL1alpha released by cell death or induced and released
• IL1beta cleaved to activate (inflammasome)
• promotes local inflammation (low conc): diapedesis, chemokine and cytokine release, inc ROS and iNOS and prostaglandin release
• promotes fever: endogenous pyrogen
• promotes sepsis:  increased acute phase proteins and promotes neutrophil and platelets
• contributes to chronic inflammation
• Typ 1 IL-1 receptor (TIR): domain also on TLRs and IL18 rec, response mediated by MyD88, IRAK-1, IRAK-4 and TRAF-6

Interleukin 6

(IL-6)

• produced by many cell types
• stimulates acute phase protein synthesis
• production of neutrophils in bone marrow
• growth of b cells
• antagonistic to regulatory T cells

Chemokines

• large family of proteins distinguished by position of cysteines
• receptors (CCR**): recruit and direct migration of cells, CCR5 is on certain T and myeloid cells and is a HIV co-receptor
• common names: CCL2, CCL3, CXCL8 etc
• activated T cells go to the place where the chemokines are made 

IL-12

• made by inate responses but directs the immune consequences
• dimer of p40-s-s-p35
• produced by dendritic cells and macrophages
• stimulated by TLR triggers
• activates NK cells
• directs T cells to Th1 response: promotes interferon gamma production, IL12 production is stimulated by IFN-gamma
• close cousin to IL-23

IL-23

• dimer of p40-S-S-p19
• produced by dendritic cells and macrophages
• stimulated by danger signals, including cell debris
• directs memory T cells to Th17 response: interleukin 17 production

Interferons

• type 1 interferons are primarily an early warning/activation part of the antiviral response with immunomodulatory activity
• type 1 interferons also act as cytokines to modulate antibacterial and other responses
• type 1 can be alpha or beta and primarily induced by viruses
• type 2 interferrons (IFNgamma) is produced by NK cells and Th1 CD4 Tcells as a cytokine
• type 2 are gamma and macrophage activation factor as part of the Th1 response

Antibody

Overview

• serum and secretory proteins
• first protection of neonate: courtesy of Mom and Ma nature
• soluble, spreads and seeps into spaces: helps clean up bloodstream and leaks out through openings or secreted (IgA and sometimes IgG)
• glops up function/targets for clearance or destruction of antigen: neutralizes toxins, prevents binding of microbes to receptors, targets for complement, phagocytosis or ADCC
• mixture of proteins( IgM, D, G, E, and A) randomly produced and get more specific for antigen as immune response progress
• membrane bound receptor and secreted from cell: acts as antigen receptor for B cell, secreted form has multifunctions
• one end binds to antigen other interacts with host cells or proteins for function

Definitions: Antibody-Antigen binding

• antigen: molecule w/ one or more sites recognized by antibody or T cell rec
• immunogen: substance (microbe, stuc, molec) that can elicit an immune response towards an antigen
• epitope: smallest structure that is recognized by an antibody or T cell receptor
• paratope: antigen binding site w/i the variable region of an antibody molecule
• affinity: intrinsic binding strength that exists btw a paratope and its epitope
• avidity: (affinity)n in which n is the number of arms immunoglobulin subunits has

Immunoglobulin Structure

• heavy and light chains
• light chain is kappa and lambda, has variable region 
• heavy chain (H) determines IgClasses and subclasses 
• hinge region: for widely spaced or closely spaced cell surface determinants
• hinge region differ in lengths in different isotypes and absent in IgM and IgE 
• pepsin and papain (at hinge region) are proteases that cleave immunoglobulin
• papain yields Fc (constant) and antigen binding region (Fab fragment, variable)
• Fc portion allows interaction with macrophages, PMNs, t cells, NK cells, mast cells for IgE and s. aureus protein A and B (way to cammaflouge

Definitions: regions defined by anti-antibodies

• isotype: constant region determinants that define each heavy chain class and subclass (same for everyone) 
• allotype: regions on the H or L chain that differ btw individuals (all o us are different
• idiotype: regions w/i variable/antigen binding region with diverse protein sequences (there are many idiots in the world) 

IgM

• first Ig produced following immunization: primary response, t cell independent (ABO blood type recognition)
• only class synthesized by fetus beginning at 5 months gestation: higher levels in congenital or perinatal infection
• serum half life of approx 5 days
• pentamer of dimers:  (10 antigent binding sites
• monomer: antigen receptor on naive b cell surface
• j chain holds together the pentamer (also used by IgA for secretion potential)
• most most effiecient for mediating complement fixation(CH3 domain) and most efficient agglutinating antibody (bacteria, virus, clumping of particulate and formation of precipitate

IgD

• T cell independent production
• not found in sign amounts and only 2.8 day halflife
• major surface component of many b cells and facilitates their activation

IgG

• four subclasses: IgG1, IgG2, IgG3, IgG4
• produced later following antigen challenge (T cell dependent
• most abundant Ig in serum: can leak from mucous epithelium, transported across placental and other membranes
• longest half life: 23 days for G1,2 and 4 and 7 days for 3
• predominant Ig in blood, lymph, CSF and peritoneal

IgG Functions

• blocking functions: neutralization of toxins and viruses (passive) and imobilization of bacteria
• Fc Mediated functions: opsonization(Fc portion bnids to phagocytic cell)
• Fc mediated: activation of complement (via CH2 domain with C1)
• Fc mediatedL antibody dependent cell mediated cytotoxicity (ADCC) (identifies cells for NK cells to kill)
• Fc mediated: placental receptor binding

IgA

• IgA1 and IgA2
• monomer and dimer (J chain) with halflife 5.5 days
• serum and secretory IgA
• serum IgA has no known biological function (Fc does not interact w/ cellular rec
• binds to polymeric immunoglobulin receptor (PIR) on epithelial cells to promote transcytosis (cleavage of PIR produces secretory component of IgA

Secretory IgA

• major Ig in external secretions (3-6 per day)
• exists as a dimer consisting of two four chain units linked by the same joining J chain
• has another attached protein, the secretory component (S)

Role of IgA

• role in mucosal infections: as first line of defense, can perform many IgG like functions, controls normal flora in GI and resp tracts 
• bactericidal activity (neutralization): against gram (-) organisms in the presence of lysozyme
• antiviral activity (neutralization): in secretions, sIgA is first line of defense
• passive immune protectin: found in breast milk

IgE