Mod 7

nomenclature of bacteria

genus + species

bacteria shapes

coccus, bacillus, coccobacillus, vibrio, spirochete, spirillum, phleomorphic

diplo-

bacteria: pair on 1 plane

strepto-

bacteria: chain on 1 plane

staphylo-

bacteria: grape cluster on 2+ plane

tetrads

bacteria: packet of 4 cells on 2+ plane

sarcina

bacteria: packet of 8 cells on2 + plane

bacteria cell membrane

no sterols

selective permeability and transport

secrete proteins

house enzymes & carrier molecules

e-t & oxidation phos

house receptor & protein for chemotactic & sensory transduction

bacteria cell wall

sei rigid, outside of membrane, in most bacteria

Maintains cell shape

Prevent osmotic lysis

Env protection

Anchor for external surfaces

PEPTIDOGLYCAN- Repeating disacchatide cross linked by tetra peptides NAG & NAM

ENZYMATIC CROSS LINKING of NAM &NAM to make sheet

Thickness of cell wall

Gram - thin

Gram + thick

Treatment for bacteria via cell wall

Secrete antimicrobial compounds: antibiotics- cell wall synthesis inhibitor (dep. on stage)

secrete of lysozyme: innate -destroy between NAG and NAM not crosslinking, in tears, saliva, mucus degrade glycan backbone of peptidoglycan, weakened integrity of cell wall

Staining

Smear -> heat fix -> crystal Violet (Stick to peptidoglycan) -> grams iodine (fuse CV to peptido)-> decolorizer (wash out background)-> saffron red (counterstain)

Differential stain: 2 stains to determine thickness of cell wall

ONLY REFERS TO BACTERIA

Gram → ID Org→ additional test →antibiotic therapy

Gram +

retain Crystal Violet →stain purple

Thick peptidoglycan layer + wall teichoic acid (WTA) + lipoteichoic acid (LTA) - elastic + structure

WTA & LTA g+

Adhesions - stick to target physically

First step to invasion, WTA/LTA bind to receptor physically, initiate endotoxin-like activity when released

On cell wall = no issue

Fuse -> release -> endotoxin -> inflammatory response (not as extensive as LPS)

WTA g+

anchored to peptidoglycan

LTA g+

anchored to cell membrane

Gram -

retain counter stain →stain red

Thin 1-2 pep + outer membrane (LPS, trimeric protein, lipoprotein) + periplasmic space

Outer membrane Gram -

external peptidoglycan layer, bilayer

1. outer layer like lipopolysaccharide (LPS) -end toxin, other proteins anchor LPS

contain ) antigen, core polysaccharide, lipid A

2. inner layer like cell membrane

LPS in gram -

O antigen, core polysaccharide, lipid A

O antigen in gram -

outer - long, linear, species & strain-specific, attachment site (inhibit phagocytosis), highly variable plus immunogenic (change often, antibody has to change every time - ID stains)

core polysaccharide g -

Branch 9-12 sugar, connect lipid A to O antigen

E coli O157: H7-classify surface antigen

Lipid A g-

Embedded in outer membrane

toxic activity: heat stable, B cell mitogen (B cell proliferate), production of cytokines and inflammatory mediators (overstimulate immune system), bacterial sepsis

Bacterial septic shock g-

LPS directly activate complement (alternative pathway - no antigen: antibody bind → spontaneous), bind to receptors → release cytokines and inflammatory mediators

endotoxin - bound to membrane = inactive

Infection -> antibiotics -> lyse bacteria -> lipid A released ->now toxic (antibiotic -> feel worse -> get better)

Lipid A nowhere to go -> stay in blood -> dilate capillary -> inflammation ->septic shock

Porins g-

Trimeric outer membrane protein channels - non-specific - water and small molecules across

Lipoprotein g-

Most abundant protein in gram -, stabilize outer membrane and anchor outer to peptidoglycan layer

Periplasmic space g-

 Between outer membrane and inner membrane, thin peptidoglycan layer, transport proteins, hydrolytic enzymes

Acid fast

have peptidoglycan- different structure can't gram stain

myoclonic acid layer

No gram stain ->  nothing come up -> miss diagnosis

mycolic acid layer + tetrameric proteins

stain: carbol fuchsin (1^st stain-pink) + methylene blue (counter stain)

Mycobacterium + Nocardia

Mycolic acid layer

Polymer of long chain fatty acid linked to peptidoglycan w/ arabinogalactan

“waxy coat”- resist desiccation, resist antibiotics, inhibit phagocytosis

prevent gram stain

Tetrameric proteins

Porins -in mycolic acid layer -passage of small hydrophilic molecules (different from gram - ones)

External structures of bacteria - virulence factors

Flagella, Pillay / fimbriae, glycocalyx

flagella

Filament - self-assembling helical flagellin with hollow core (bases for H antigen stereotyping - E coli 0157: H7 - type of flagella)

Hook - attach filament to cell surface, link to basal body

Basal body - anchor flagellum to cell wall and membrane

provide motility

Pili/ fimbriae

Pili - proteinaceous, hair-like structure with adhesion tips, promote adherence

Common Pili: dash fimbriae: pilin subunit + adhesion tips (lectins) -bind to sugar, peritrichous arrangement - adhesion + “twitching” motility

sex pili-conjugation Pili: some gram -, adherence to exchange DNA - bacteria conjugation, antibiotic resistance

Glycocalyx

Polysaccharide layer outside of cell wall, K antigen

Capsule: rigid, uniform density and thickness - closely surround cell-tight pack sugars

Slime layer: loose, non-uniform, diffuse-loose sugars

not necessary for survival - virulence factor: protect desiccation, barrier to toxic hydrophobic molecules, limit phagocytosis (block immune function), decrease PMN killing, decreased complement mediated cell lysis, induce abscess formation, increase adherence to cell /surfaces (sugar stick to target cell)

Internal structures of bacteria- virulence factors

Nucleoid region + plasmids, ribosomes, inclusion, and endospores

Nucleoid region + plasmid

Nucleoid: nuclear region - no nuclear membrane, DNA, RNA, protein - one single, circular chromosome, no introns, no histones

Plasmids - extra chromosomal DNA (not essential, take from environment) - acquired, Ancillary (non-essential) - antibiotic, antibiotic resistance, heavy metal resistance, toxin

Increase virulence: increase ability to cause damage and evade immune response

ribosome

rDNA + proteins, different than eukaryotic: 70S ( 30S + 50S subunit) - transcription + translation coupled

Target for protein synthesis inhibitor: selective toxicity: more toxic to microorganism than host

streptomycin (30S), chloramphenicol (50S), erythromycin (50S), tetracycline (tRNA + mRNA)

inclusion

Granules, vesicles, vacuoles w/in cytoplasm - storage deposit

Volutin granules - polymer of inorganic phosphate

Glycogen granules - polymer of a-D glucose

PHB granules - chain of b-hydroxybutyric acid

Endospore

resting stage- spore in Org- survive harsh environment conditions - endospore formation in nutrient depletion (resist to heat, chemicals, dehydration)

Endospore contain: 1 chromosome, decrease essential protein + plus ribosomes, peptidoglycan layer, increase concentration of calcium bound to dipicolinic acid, keratin spore coat

conditions favorable: endospore germinate -> 1 vegetative cell

Not reproductive function (won't recognize in test b/c resting -> think clear but not actually

Bacillus and Clostridium gram + make endospores - location and size varies -> ID

endospore ID

Bacillus cereus - central endospore

Bacillus subtilis - subterminal endospore

Clostridium tetani - terminal endospore

binary fission

1 bacterium → DNA replication with increased biomass (two of everything) →DNA separate + plasma membrane form + cross wall form → complete division → 2 new genetically identical cells

Measuring bacterial growth

Optical density (OD), colony - forming units (CFU), biomass

Optical density (OD)

Culture turbidity→ absorbance of light, more cells =more absorbance of light (big hydrophobic

viable +non-viable cells

Colony dash forming units (CFU)

Dilute culture +plate → each colony from one cell

only viable cells

Culture → dilute → plate → CFU cell X dilution factor → live cell

Biomass

 Sample wash, dried, weigh → dry weight

viable +non-viable cells

Grown in liquid → spin down → dry → weigh, more cells = heavier

Bacteria growth curve

1 bacterium → isolate colony on plate → inoculate liquid media →culture of identical organisms

4 phases: 1) lag 2) exponential 3) stationary 4) decline

lag phase

no cell division, increased biomass (get ready to divide) -depend on form and availability of nutrients and conditions of inoculum (space and nutrition)

log phase

Exponential growth, rapid cell division, doubling /generation time -rate of cell division, varies, pathogens make virulence factors, primary metabolites

Late log phase: transition to stationary, secondary metabolites, antibiotics and pigments (kill off competition to keep growing →limited by environment → culture own survival)

stationary phase

Energy and nutrients exhausted, resources renewed by cell death (peptides + nucleic acids), death =division, zero population growth (no change in CFU/ML, OD, biomass)

death/ declining phase

Insufficient resources, exponential death, determined by CFU/ mL(not OD or biomass) not as dramatic as log phase

culturing bacteria

Isolate Organism in pure culture -shriek plate with wire loop -help ID Organism, info about natural habitat and environment to find it, predict infection it causes

Homogenous → shriek out →colony - individual bacteria (can test if mixed infection - two colors in different colonies)

Media: liquid (broth) or semi solid (gel)-solid agar→ cell immobilized→ each cell own isolated colony

Types: define, complex, enrich, selective, differential

Defined media

Known quantities of each component

Defined carbon, nitrogen sources

Eliminate variability

Narrow growth range (excludes some that need specific environment)

More expensive

Complex

Components not defined (yeast extract)

no exact formula

broader growth range

Less expensive

Enriched

Complex plus growth factors

Fastidious organisms (picky eaters -will not grow without it)

Selective

Select against unwanted organism

Only certain organisms able to grow -inhibit others by some components (antibiotics -look for fungus)

Differential

 Differentiates between organisms

show visible changes (color of colony, formation of precipitate)

DOES NOT select for a specificity or inhibit growth (just tell apart-color/precipitates)

MacConkey agar

Peptone, lactose, bile salt (inhibit gram +), crystal Violet (inhibit gram +), neutral red -pH indicator (tell cell apart)

Differential and selective

Ferment lactose →color change

specific gram -, all the gram -grow →color tell apart

Fastidious organism

High maintenance, picky eater, “fussy” -need complex diet

Difficult to grow in vitro

Need growth factors and enriched media

Hemophilus/Neisseria-hemin + NAD-NEED chocolate agar (lysed RBC) not blood agar plate- cant lyse RBC but need it to grow

chocolate agar-lysed RBC-enriched medium (hemin + NAD)

Obligate organism

Without alternative - need these conditions to grow

Limited narrow range of environment

Facultative organism

has alternatives - grow in anything

Survive in different conditions

Environmental factors

Temperature, pH, oxygen, CO2, water availability / osmotic pressure

MIN - minimum value of parameters that supports growth

OPT - value of parameters that support optimum growth (best growth)

MAX - Max value of parameters that support growth (Max / Max growth)

Temperature

Psychrophiles (10C)< psychotrophs (28C)< mesophiles (37C)<thermophiles (60C)< hyperthermophiles (90C)

Most bacteria mesophiles optimum 30-37 C (range 12-42 C)

Temperature best growth rate - depend on bacteria

pH

Acidophile (1-4) < neutrophil (5.5-8.5)< alkaliphile (7.5-11.5)

most nucleophile -for human pathogen -normally PH7

oxygen

Obligate /strict aerobe- 02 required - Ros enzyme

Facultative anaerobic /aerobe- not required use if available - Ros enzyme

Obligate/strict anaerobe - O2 toxic – NO ROS enzymes

Requirement of O2 and ability to counteract Ros from metabolism

ROS enzymes: super oxide dismutase, catalase, peroxidase

CO2

Capnophiles dash require high CO2 level ( 3 to 5% )

Neisseria and hemophilia - chocolate Agar – lysis RBC and increase CO2

Special housing: candle jar, CO2 packet / incubator, automated system

Water availability /osmotic pressure

Halophiles: increase salt - obligate halophiles

Facultative / Halotolerant: skin →sweat →kill off a lot of bacteria

Osmophiles: increase osmolarity /sugar

Resistance to desiccation: spore formation: bacillus and clostridium & Cell wall modification: mycobacterium

Pathogenicity

Established cause for infection: isolate, pure culture

Koch’s postulate: ID cause of disease, flawed - inability to grow isolates in vitro

Kary Muilis- PCR: ID cause disease using single copy of DNA/RNA ( RNA/DNA -> melt-> replicate) no live bacteria just nucleic acid, taq polymerase -> can deal with high temperature

Colonization

Transient /permanently established microbial growth

No interference with normal bodily function (bacteria just there)

Infection

Transient /permanently established microbial growth

May manifest disease

Disease

Abnormal conditions of body structure and functions

Interaction =pathogenesis

Damage to host

Can occur w/o presence of microbe (bacteria doesn't need to be there)

toxins → intoxication (physiological changes)

clostridia & staph: toxin release → bacteria not there

sign

objective evidence of disease see by observer

fever, edema

symptoms

subjective evidence experienced by pt

some diseases have characteristics sign & symptoms

diff org→ similar sign & sym (similar pathogenicity mech)

same org→ diff sign & sym (different pathogenicity mech)

fever, sore gum

Stages of disease

Severity, duration, stage vary with pathogen, virulence, host susceptibility

5 stages: 1.incubation (preclinical), 2. prodromal (warning), 3. acute stage, 4. decline stage, 5. convalescent stage

Incubation (pre clinical)

Pathogen enter body, no sign and symptom, innate immune system not activated, need infectious dose, not contagious (not a lot of cells)

Prodromal (warning)

Nonspecific sign and symptom, activate innate immune system, pathogen # increase, asymptomatic carries -easily transmitted

Acute stage

Most severe time of illness, characteristic sign & symptom, activate acquired immune system, pathogen # stationary, easily transmitted

Acute disease: sign and symptoms develop rapidly, peak and decline

Chronic disease: sign in symptom persist, slow to move to decline phase

Decline stage

Illness apparent, sign and symptom decrease, immune system activity decrease, antibody formed (T reg→minimize immune response) pathogen cleared from host, contagious if carrier (not cleared completely, no sign or symptom, undetected amount but still there)

Convalescent stage

Patient return to full health, sign and symptom and, no immune system activity to pathogen, pathogen cleared, not contagious

Group of pathogens

Opportunistic: part of normal flora, no disease in normal setting, disease when in unprotected site (blood/tissue) by injury (burn), compromised immune system

Virulent: strict pathogens, always = disease, sign and symptoms because host inflammatory response or direct damage to or loss of tissue and organ

Mechanism of bacteria variance

Metabolites, invasins (spreading factors), adherence, toxin

Metabolites

Acid , gases, byproducts of metabolism from bacterial growth, directly damage host tissue

Streptococcus mutant → lactic acid →dental carries

Invasins (spreading factors)

protein /enzyme act locally to damage host cell, tissue matrix and intercellular spaces soften (allow better penetration) allow spread and replication of bacteria

Hyaluronidase: cell depolymerized by hyaluronic acid -connective tissue soften

Collagenase: breakdown collagen

Neuraminidase: degrade neuraminic acid -intracellular glue broken down

streptokinase and staphylokinase: fibrinolysis -breakdown blood clot

Adhesions

Find pathogen to receptor on host cell and tissue

Buying two carb moieties (glycoprotein)

Glycocalyx material: capsule and slime layers -> biofilm

Fimbriae/Pili: attached to specific host molecules (tissue tropism)

afimbrial adhesion: not fimbriae/pili, cell envelope

Toxin

Resemble enzymes, high activity and specificity, heat labile (deestroy with heat)

Exotoxin, Endotoxin, exo/enterotoxin, super antigen

 Exotoxin

Found in gram - and gram + cell, secrete into extracellular fluid (secrete from bacteria and bind to host)

Membrane active exotoxin: specific (protease, lipase, hemolysins)

Protease: destroy protein in host cell (cell membrane) IgA protease

Lipase: destroy lipids in cell membrane

Hemolysins: form pores in RBC and phagocytes

Endotoxin

Same as exotoxin, GI sign and symptoms - vomit, nausea, diarrhea (released when cleaved -in membrane /cell wall innert until released= issues)

Rotavirus NSP 4- infantile diarrhea

LPS of gram (- )not toxic until released

O antigen -polysaccharide -immunogenic

Lipid a -toxicity

Exo/enterotoxin: A-B exotoxin

Two domains A&B -B bind →a enters into cell, can form toxoids (inactive toxin)

A -enzymatic component -A attack cell →protein synthesis affected

B -B bind to toxin to host receptor

Super antigen:

Toxic, activate 40% T cells without additional antigen

Massive release of cytokines -lack of regulation →autoreactive lymphocytes

Virulence mechanism

Spread from primary site of infection: cytotoxic effects span larger area than focus of infection

Encapsulation: block phagocytosis (S. pneumoniae capsule-block phago)

Inactivation of antibody (S. pneumoniae secrete IgA degrading protease & S. aureus bind to Fc region of IgG→ cant produce further immune response)

Intracellular growth and escape detection (M. Tuberculosis-facultative intracellular bacterium phagocytose→ block phagolysosome rxn→ persist in phagosome)

Evasion by antigenic variation

Different mechanism, change epitope, present new antigens to immune system, initiate new cycle of disease (o antigen keep changing →no same antigen →disease over and over

Anti-genetic drift, anti-genetic shift, anti-genetic switching

Anti genetic drift

Accumulation of genetic mutations

Alter protein products

Lead to variability in population

Form antigenically distinct strands

DNA mutation build up over time -small mutations

E coli 0157: H7 -over 157 different O antigens -look different but can have something to neutralize it because something similar

Anti genetic shift

Influenza A

Reassortment of viral genes

New subtype of virus → flu pandemic

Giant complete reassortment of genome and swap out genes →hybrid virus never seen before, pig +bird +human →new strain

Anti genetic switching

Form genetic conversion / shuffling

Recombination w/in Group of genes

New surface antigen, no change in biological function

Pili, fimbriae, surface glycoproteins

pili of N. gonorrhea always present, but anti genetic structure changes

reassortment within group of genes, like B cell -> IgM -> IgG

Same virus - just rearrange -> different protein

fungi

Not bacteria- bigger but only small group pathogenic to humans

Unicellular (yeast) or multicellular (mold / mushroom), eukaryotic, heterotrophic

Primary or opportunistic - severity = host factors (immune system)

Commensals - part of human flora

Can make antimicrobial agent (penicillin) or toxic mycotoxins (fungus)

Any fungus that can grow at 37 C -> potential pathogen (HSB proteins - body temperature, two proteins, allow to live at 37 C, most survive in colder / hotter environment)

Fungal structure

Eukaryotic - reproduce asexually →spores (used to ID)

cell membrane: contains ergosterol - target for chemotherapies (look like our cholesterol →more like us then bacteria, toxicity issue)

Cell wall: rigid, chitin, glucan, mannan (unlike bacteria, sugar and carb →rigid)

Yeast: unicellular

Mold: multicellular- no chlorophyll- no photosynthesis, ID based on reproductive components

Mold

Filamentous and multicellular, ID by arrangement of reproductive structures

Fungal cell wall

Mannan, PLM, chitin, b- glucan

Mannan

Glycoprotein- large % of cell wall mass (protect and heavy)

PLM

Phospholipomannan- glycolipid linked to fungal survival of macrophages (protect from phagocytosis)

chitin

Polysaccharide- rigidity to cell wall

B glucan

Polysaccharide- rigidity to cell wall + pathogen- associated molecular pattern (PAMP) for fungi