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131 Cards in this Set

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Define the shapes and arrangements
Here they are
Examples of Cell wall associated proteins
Cell Wall Assoc Proteins
Describe Beta Lactam Structure
Beta Lactam Structure
How are pathogenic bacteria classified?
Classified by DNA Sequence (16s rRNA sequencing, PCR detection of specific markers), Antibody reactivity with specific antigens, and biochemical Tests (Gram stainig, Physical/Metabolic characteristics).
basic structure of the bacterial cell wall
Consists of peptidoglycan in alternating units of N-acetylglucosamine (NAG) and N-Acetylmuramic acid (NAM). Off of the NAM unit there is a variable chain of 3 AA's (depending on the bug), and then a D-ALA which is conserved and linked to the 3rd AA of the adjascent chain either directly or via a bridge.
Structural differences between gram + and Gram - bacteria
Gram staining divides pathogenic bacteria into two different classes on the basis of cell wall and membrane structure. • Gram-positive bacteria have a thick peptidoglycan layer and an inner (cytoplasmic) membrane. • Gram-negative have a outer membrane, thin peptidoglycan layer and an inner (cytoplasmic) membrane
Basic bacterial shapes and arrangements
basic bacterial shapes: cocci, bacillus, vibrio, fusiform (tapered ends), spirochete (e.g. Treponema), filamentous (e.g. Streptomyces), pleomorphic (e.g. Corynebacterium)…..basic bacterial arrangements: diplo, tetrad, sarcinae, strepto- , staphylo-
Types of Stable wall defective cells
• CWD (cell wall defective forms) = partial loss of the wall, • L-forms = complete loss of the cell wall
True or False - Bacteria can change morphology dependant on culture time
True: Rhodococus goes through a coci-rod morphology switch (cocci -> rod -> cocci)
None
Characteristics of Teichoic Acid
Acidic polysaccharide -glyceril or ribitol joined by phosphates. Covalently linked to muramic acid, can have AA's linked to it, gives typical negative charge to cell wall. It is more abundatn in gram + bacteria and can act as a barrier to penetraiton of negatively charged molecules.
2 flavors:
-Cell wall associated plain Teichoic acid
-Lipoteichoic (glycerol containing acidic polysaccharides attached to lipids) acid penetrates the cell wall and attaches to the inner leaflet of Gram + membrane
None
Sortase
The enzyme in bacteria that attach proteins to the cell wall.
Steps of Cell Wall Biosynthesis
"1. cytoplasmic steps (synthesis of peptidoglycan subunits) : NAM is synthesized from NAG and a pentapeptide chain is attached to NAM (AA1-AA2-AA3-DALA-DALA)…The DALA is Derived From LALA by racemase. 2. Cell Membrane Step: an undecaprenyl carrier transfers the NAG-NAM-pentapeptide subunit to the outer leaflet of the cytoplasmic (inner membrane). 3. Cell Wall Steps: Existing cell wall is cleaved by autolysins and the addition of new subunits is carried out by penicillin binding proteins (PBPs) via transglycosylation and transpeptidation.
Penicillin Binding Proteins
High Molecular Weight PBPs crosslink the 4th D-ALA with an adjascent subunit with or without and intermoleicular bridge. Low Molecular Weight PBP's cleave off the 5th D-ALA without crosslinking
Cell wall antibiotic resistance
Lack of cell wall – Mycoplasma; L-forms – genetic programmed loss of cell wall and remodeling of the membrane to become osmotically stable.; Cell wall synthesis inhibitory antibiotics require growing cells for activity
Antibiotics that inhibit cell wall synthesis
"Phosphomycin/Fosfomycin is a PEP analog that inhibits the synthesis of NAM from NAG.; D-cycloserine is an analog of D-Ala and inhibits the racemase reaction and addition of D-Ala to the AA1-AA2-AA3 chain Bacitracin binds undecaprenyl-PP and inhibits recycling of the carrier.
Vancomycin (glycopeptide antibiotic) binds D-Ala-D-Ala and sterically hinders PBPs from carrying out transpeptidation and transglycosylation reactions. B-lactams are structurally related to D-Ala and tightly bind PBPs inhibiting their transglycosylation and transpeptidation activity.
"
Describe the steps of gram staining
1. staining with crystal violet both Gram positive and Gram negative cells appear purple under oil-immersion light microscopy 2. treatment with iodine aggregates the crystal violet both Gram positive and Gram negative cells appear purple 3. washing with ethanol collapses peptidoglycan and extracts lipids from the outer membrane Gram positive cells appear purple; Gram negative cells are colorless as they get leaky 4. counterstaining with safarnin Gram positive cells appear purple; Gram negative cells appear red
Describe Bacterial Membranes
Bacterial membranes are made up primarily of phosphotidylethanolamine and phosphotidylglycerol.

The do not contain sterols with the exception of Mycoplasma that incorporate sterols from their environment into their membranes.
Gram + Membrane structure
• inner leaflet mainly phosphotidylethanolamine
• outer leaflet mainly phosphotidylglycerol
• lipoproteins
• lipoteichoic acid
• membrane proteins
Gram - Membrane structure
• both leaflets of the cytoplasmic (inner membrane) are made of a mix of phosphotidylglycerol and phosphotidylethanolamine
• inner leaflet of the outer membrane made of predominantly phosphotidylethanolamine
• outer leaflet of the outer membrane is composed of LPS (lipopolysaccharide
Components of LPS (AKA Endotoxin)
LPS is composed of:
• lipid A
• core glycolipid
• O-specific oligosaccharide subunit composed of repeating sugar subunits
Gram Negative transmembrane transport
the outer membrane of Gram negative bacteria contain porins for transport
• non-specific (water-filled channels)
• specific

A number of transporters are located in the cytoplasmic membrane of Gram negative and Gram positive bacteria.
The gram negative outer membrane is an additional barrier for antibiotics
Types of Transport
types of transport:
do not require energy
- free diffusion
- facilitated diffusion
require energy
- active transport
ATP
PMF (proton motive force)
- group translocation (PTS System)–
results in the phosphorylation of the transported substrate (there are many sugar transporters for each bacteria)
Differences between bacterial and eukaryotic cell walls
bacteria have:
Higher protein content
Phosphatidyl Choline is Rare
No Sterols (Mycoplasma grown in the presenceof sterols is the exception)
Polymixin
The only antibiotic agianst bacterial membranes that is widely used (as they are too similar to eukaryotic membranes).

Composed of a fatty acid tail and cyclic peptide head group

Disrupts bacterial membrane causing release of cytoplasmic components

Will destroy non-growing (non-replicating) cells• polymyxins have a higher affinity for LPS and phosphotidylethanolamine so are more effective against Gram negative bacteria (than gram +, Gram + being more affected than eukaryotic cells)

Often in topical solutions as skin regenerates.
Cationic Peptides
cationic peptides are a class of antibiotics that self-assemble to form pores in membranes (pass through the outer membrane and cell wall to form the pore in cytoplasmic membrane in both gram - and Gram + bacteria).
some new classes appear to be specific for bacterial membranes
Gram Negative transmembrane transport
Gram positive bacteria.
The gram negative outer membrane is an additional barrier for antibiotics
Types of Transport
types of transport:
do not require energy
- free diffusion
- facilitated diffusion
require energy
- active transport
ATP
PMF (proton motive force)
- group translocation (PTS System)–
results in the phosphorylation of the transported substrate (there are many sugar transporters for each bacteria)
Differences between bacterial and eukaryotic cell walls
bacteria have:
Higher protein content
Phosphatidyl Choline is Rare
No Sterols (Mycoplasma grown in the presenceof sterols is the exception)
Polymixin
The only antibiotic agianst bacterial membranes that is widely used (as they are too similar to eukaryotic membranes).

Composed of a fatty acid tail and cyclic peptide head group

Disrupts bacterial membrane causing release of cytoplasmic components

Will destroy non-growing (non-replicating) cells•

Often in topical solutions as skin regenerates.
Cationic Peptides
cationic peptides are a class of antibiotics that self-assemble to form pores in membranes (pass through the outer membrane and cell wall to form the pore in cytoplasmic membrane in both gram - and Gram + bacteria).
some new classes appear to be specific for bacterial membranes
Glycocalyx
glycocalyx – polymer layer outside the bacterial cell

composed of polysaccharides or polypeptides

Three Forms
1.capsule = associated with individual cells. Functions include: mediate adherence, protect from phagocytosis, protect from antibiotics, protect from desiccation

2.slime layer (matrix) = secreted layer embeds cells. Functions include: carbohydrate reservoir, matrix for biofilm formation

3.fibrils = thin tangled strands. Functions include possible signal transduction.
Bacterial Cell Attachment
Fimbriae
short flagella-like structures
not made of flagellin or involved in motility
protein
adherence

Pili
longer and thinner than fimbriae
adherence, protection, and DNA transfer(conjugation)
protein
Flagella
Composed of flagellin in long chains wrapped in a left handed helix. (self assembles

Uses the proton motive force to power bacterial motility.

3 arrangements
-monotrichous (polar)
-lophotrichous
-peritrichous
Function of bacterial motility
1. chemotaxis (towards nutrients or away from repellants)
2. eukaryotic cell invasion (may be basal body and not really motility)
3. biofilm formation

note- gliding and twitching motility are alternate forms of motility that do not depend on flagella

Motility can also beused diagnostically, detected directly or through use fo motility medium.
What is the Structure of DNA in bacteria
In order to fit in the bacterial cell DNA is supercoiled and compacted.
DNA Gyrase
DNA gyrases bind to DNA and catalyze strand cleavage and exchange to affect DNA supercoiling.

needed to make DNA accessible for transcription and replication

They retain a level of negative supercoiling necessary for chromosome activity and allow movement fo transcription and repliaiton machinery by adding negative supercoils ahead of the complexes.
Gyrases remove knots and fold/bend DNA
quinolones and floroquinolines bind the DNA gyrase:ATP complex thus blocking transcription and DNA replication
novobiocin inhibits DNA gyrase activity
Bacterial Cytoplasm
Bacterial cytoplasm does not contain organelles but does show some limited compartmentalization in the form of multi-component enzyme complexes.
ANTIBIOTICS-Mechanism of Action
1) ANTIMETABOLITES

2) INHIBITORS OF CELL WALL SYNTHESIS

3) AGENTS AFFECTING CELL MEMBRANES

4) INHIBITORS OF PROTEIN SYNTHESIS

5) INHIBITORS OF NUCLEIC ACID SYNTHESIS
Embden-Meyerhof-Parnas pathway
Bacteria use the Embden-Meyerhof-Parnas pathway to convert glucose to pyruvate.

Alternative to EMP is the Entner-Doudoroff pathway.

2 high energy phosphates are consumed:
ATP OR PEP donation of high energy phosphate PTS system – group translocation

4 ATP are produced.
NADH+H+ is produced and must be oxidized by either reduction of pyruvate (fermentation) or by being used in an electron transport system (respiration).
Energy/Carbon Sources
Glucose

A number of alternative sugars can be used by bacteria including:
• hexoses
• complex sugars
• citrate

Many of the intermediates are used for biosynthesis (anabolism) as well as energy. If pentoses or citrate are used,
gluconeogenesis can produce glycolytic intermediates needed for biosynthetic purposes.
Fermentation
Fermentation of pyruvate occurs when bacteria reduce pyruvate to recycle NADH+H+.

There are a number of end products that can be produced from the fermentation of sugars:
• acids
• alcohols
• gas

To deal with acid production bacteria can neutral products (formed by the neutralization of acid end-products), stop growing, alternate acid and alcohol production, or are acid tolerant
Fermentation Classification
Homolatics:
Produce Lactic Acid (Streptococci and some Lactobacilli)

Heterolactic:
Produce Lactic Acid, Ethanol, CO2, H+ (Lactobacillus)

Mixed Acid:
Produce Ethanol, 2,3-Butanediol, Sucinate, Lactate, Acetate, Formate, H2, CO2 (Enteric Bacteria including Escheria, Salmonella, Shigells, Klebsiella, Enterobacter). Note the neutral end products -ate's.

OR

Produce Butyric Acid, Butylalcohol, Acetone, Ethanol, H2, CO2
Voges-Proskauer Test
Voges Proskauer test determines if bacteria produce the neutral end products acetoin or 2,3-butandiol, and thus differentiates enteric fermenters.
Lab Diagnostics
Type of Sugar Used
Acid Production
Gas Production
Oxidation Vs. Fermentation
End Produce Identification
Ability to use Lactose
Ability to grow on citrate
Gas Production
Presence of cytochromes
Use of Thiolsulfate as a terminal electron acceptor
Use of nitrate as a final electron acceptor
The use of Fe-S proteins
Amonia source
Protein breakdown products
Respiration
Some bacteria can use NADH+H+ for respiration.

Bacteria that respire transform pyruvate into acetyl-CoA and further break it down to CO2.
This provides bacteria with:

NADPH for biosynthesis

additional molecules of NADH+H+ for use in generating a proton motive force

FADH+H+ that can also be used for the generation of a proton motive force

NOTE: both aerobic and anaerobic bacteria can respire.
Proton Motive Force
A proton motive force is produced by passing the electrons from NADH+H+ to a series of electron carriers that use the excess energy to pump protons (H+) outside the cell.

Carriers include:
• cytochromes
• quinones
• flavoproteins (Fe-S proteins)

The final electron acceptor can be O2 (aerobic respiration, oxidative phosphorylation) or other inorganic compounds (anaerobic respiration, UNLIKE EUKARYOTES).

This creates a membrane potential and a pH gradient. The re-internalization of the protons through ATPase is used to produce ATP.

NOTE: Aminoglycosides require oxidative phosphorylation (a PMF generated using oxygen as the final electron acceptor) for transport into the cell. The strength of the PMF depends on what is being used as a final electron acceptor and only oxygen provides a strong enough PMF to transport aminoglycosides due to their charge/size. Therefore, they require oxidative phosphorylation or aerobic respiration and NOT anaerobic respiration).
Bacterial Nitrogen Source
Bacteria can use four sources for NH3:
• arginine (from the environment, via arginine dehydrogenase)
• urea (via urease)
• tryptophan (from the environment via tryptophanase)
• N2 fixation
Bacterial Protein Degredation
Some bacteria will produce proteases to degrade proteins to produce free amino acids to supplement metabolism
• used in biosynthesis
• converted to amino acid precursors (cadaverine, putrescine)
• converted to pyruvate for use in the TCA cycle for energy and biosynthesis
Sugar test
types of sugars bacteria can metabolize and use as a carbon source are important

Possibilities include:
Glucose
Other Hexoses
Other Complex Sugars

The identity of each sugar the bacteria can use, and how well the bacteria grow on it are used.
Acid Production
detected via pH indicators/API strips and carbohydrate tubes. (note that both growth and acid production are required for a positive result). Fermentation often produces lactic acid of H+ itself.
Oxidation Vs. Fermentation
fermentation can our in the absence of O2, however not all bacteria can get enough energy via fermentation, so this test examines weather fermentaiton is sufficient, or if aerobic respiration is requries (no good for anaerobes). It uses a double zone tube with one area of oxygen exposure and one under that which is O2 free.
End-Product identification
Checks for Glucose Metabolism products
ONPG detection
Beta-galactose is formed from the breakdown of lactose during metabolim. Use of B-Gal requres B-galactosidase, ONPG assay measures its activity
Citrate
The ability of bacteria to grow with citrate as the sole C + E source. Use raises pH.
Gas production
trapping gas bubbles in a Durham tube inverted, or bubbles in the wells of API strips.
Oxidase Test
Determines if bacteria have cytochromes that can be used in respiration. It does not distinguish aerobic and anaerobic respiration.
Hydrogen Sulfide Test
The presence (+) or absence of H2S determines if bacteria can use thiosulfate a terminal electron acceptor indicating respiration, also however reveals the breakdown of sulfer amino acids (cysteine).
Nitrate Reductase Test
Presence of Nitrogen Reductase determines if nitrate can be used as a final electron acceptor indicating respiration.
Catalase Test
: The Fe-S proteins used for respiration can react with O2 to produce superoxides and hydrogen peroxide. Bacteria can neutralize hydrogen peroxide with catalase. The neutralization of hydrogen peroxide by catalase forms O2. The catalase test scores bacteria on the ability to produce bubbles of oxygen in the presence of hydrogen peroxide.
Urease Test
The presence of urease determines that urea can be used as an NH3 source.
The Indole Test
determines if tryptophan can be used as a NH3 source.
Argenine Dehydrogenase Test
The presence of arginnie dehydrogenase determines that arginine can be used as NH3 source.
Lysine Decarboxylase Test
The presecne of Lysine Decarboxylase determines that lysine can be broken down into cadaverine.
Ornithine Decarboxylase Test
The presence of Ornithine Decarboxylase determines whether ornithine can be converted to putrescine.
Other Energy Sources
Some bacteria have phospholipases which can liberate pyruvate and fatty acids, which to be converted to acetyl-CoA and fed into the TCA cycle.
Tween 10 Test
The addition of Tween10 to blood agar plates helps solublize phospholipids and increases growth of bacteria that can metabolize host phospholipids.
Bacterial folate requirements
One carbon transfers using TFA (tetrahydrofolic acid) are essential to the synthesis of DNA, RNA, and methionine-containing proteins.

Bacteria cannot transport folate so they synthesize TFA from PABA and pteridine.

Sulfonamides are a PABA analog that inhibit the TFA biosynthetic pathway by competing for Pteridine synthase (converts PABA and Pteridine to Dihydroperoicacid).

Trimethoprim is an antibiotic that binds dihydrofolate reductase thus inhibiting the TFA biosynthetic pathway.
Lab Diagnostic Options
API strips are series of metabolic tests run on a strip, the results of which are cumulitively examined to ID bacteria.

Flow charts are also used to evaulate what is known about a bacteria and to direct testing to come to the correct conclusion most rapidly (different charts for Gram + and Gram -).
Transcription
Bacterial transcription starts with a 4 part (2ABB') complex that cinbines with a sigma factor (endows specificity and allows binding to DNA to intiate the process) forming a closed complex at the promote site. The complex then opens, the sigma factor is lost, and transcription proceedes (coupled with translation as ribosomes start working on the nascent DNA quickly.
Rifampin
Rifampins bind to the RNA polymerase closed complex and prevent the initiation of transcription.
Ribosomes
The bacteria ribosome significantly differs (70s, consisting of 50s and 30s subunits) from the human ribosome. Therefore it is the target of numerous antibiotics.
Aminoglycosides
Aminoglycosides interact with the 16S rRNA in the 30S subunit of the ribosome, preventing the binding of f-Met (formyl-methionine), which is the 1st amino acid of most bacterial proteins.

They are bacteriocidal and affect Gram + and - facultatice aerobes and aerobes.

Aminoglycosides require oxidative phosphorylation for transport. This requires a proton motive force. Hence, they require a transmembrane potential. Possibly because the transporter is PMF-dependent. Most intestinal tract normal flora use fermentation or anaerobic respiration for energy. They have weak membrane potentials, and are thus safe from Aminoglycosides.
Tetracycline
Tetracyclines interact with the 16S rRNA in the 30S subunit and block the transfer of tRNAs into the acceptor site.

Since tetracyclines interfere with the codon:anticodon pairing, if the site is not entirely blocked, they can also cause misincorporation of amino acids.
Chloramphenicol/Lincosamides
Chloramphenicol and licosamides interact with the 50S subunit and inhibit the formation of peptide bonds.
Macrolides/Streptogramins
Macrolides interact with the 23S rRNA in the 50S subunit and cause the release of the growing peptide chain

Streptogramins block the P site. NOTE that the P site is on the 30 S ribosome, howeer the Streptogramins interact with the 50S subunit and inhibit sterically rather than directly.
New Antibiotics
Modification of Existing antibiotics (e.g. Azithromycin is an modified erythromycin.)

Novel Classes (e.g. Oxazolidinones They prevent the formation of the f-Met-mRNA-30S subunit ternary complex, and are the only new class sing the 1970's.)
Bacterial Protein Fate
After protein synthesis, proteins have two fates:
1. associate with chaperones (heat shock proteins) for proper folding
2. degradation by proteases or chaperones (if they are not properly folded)

Some proteins that persist are secreted. These proteins can be secreted into the environment orassociated with the membrane/cell wall.
Protein Secretion
Secreted proteins are targeted to secretion machinery by a signal sequence.



The signal sequence differs for each of the 4 types of secretion.

Type I, III and IV secretion have only been reported for Gram-negative bacteria, with the exception of a possible type III system in Streptococcus pyogenes.

Type II is in both Gram + and Gram -.
Type I protein Secretion
Type I transporters span the periplasmic space and export small molecules.

These transporters can act as non-specific pumps that can also pump antibiotics out the cell conferring intrinsic gram - antibiotic resistance.
Type II protein Secretion
Type II secretion is the general secretory pathway.

In both Gram-negative and Gram-positive bacteria, the nascent protein is passes from a chaperone complex to the membrane-bound transporter complex.

In Gram-negative bacteria, the nascent protein binds to a periplasmic chaperone where it is carried to an outer membrane porin.

In Gram-positive bacteria, protease inhibitors prevent the degradation of the protein by secreted or cell wall associated proteases.
Type III baterial Secretion
Type III secretion is important in the delivery of toxins to eukaryotic cells.

The proteins are synthesized in the cell, but are not secreted until the bacterial cell comes in contact with a eukaryotic cell.

The secreted proteins often include proteins that form a channel in the eukaryotic membrane for the "injection" of toxins directly into the eukaryotic cell.
Type IV bacterial Secretion
Type IV secretion allows for the secretion of :
self-assembling cellular structures (e.g. pilin)
DNA (conjugation)
toxins

Note: Some researchers divide Group IV into:
Group IV – DNA transport
Group V – toxin and self-assembling structures
Cell Wall Associated Proteins
Cell wall proteins contain a sorting signal that associates the protein with a sortase that in turn covalently links the cell wall protein to peptidoglycan.
Phases of Bacterial Growth
There are 4 phases of bacterial growth in laboratory conditions:
1. lag = before cells start to grow
2. log = exponential growth reflecting binary fission of bacteria. Doubling time (generation time) of the culture is the tie it takes for hte pop to double in size (divide once)
3. stationary = no net gain in cell numbers (survival or balanced slow growth and death due to competition for resources, contact inhibition, etc...) persistence = ability to survive in stationary phase (often associated with asymptomatic carriage)
4. death = decrease in cell numbers
Culture Measurments
Increases in cell mass can be determined by optical density measurements.

Direct counts (using a hemocytometer) can be used to determine the number of bacteria in a sample (live and dead).

Colony forming units per millileter (CFU/ml) is used to measure the number of live/culturable bacteria in a sample. Dilutions are made and plated. Assuming each colony arose from an individual bacterium, the number of colonies on a plate can be used to calculate the number of bacteria per mL. However, since some bacteria can form chains, a colony can also be formed by more than one bacteria. Therefore, the term "colony forming unit" CFU is used.
Bacterial Iron
Iron is very limited in the environment. Therefore, bacteria use siderophores to obtain Fe for synthesis of Fe-containing proteins. These siderophores can be of human or bacterial origin.

Bacteria produce hemolysins to lyse red blood cells for iron scavenging.
Blood Agar Plates
Bacteria produce hemolysins to lyse red blood cells for iron scavenging.

Blood agar plates (BAP) are used to determine if bacteria produce hemolysins. There are 3 reactions:

Gamma-hemolysis = no detectable lysis

Alpha-hemolysis = partial hemolysis forming a green zone around the bacteria

Beta-hemolysis = complete lysis of the bacteria forming a clear zone around the colonies
Growth Media
Growth media can be:
defined- All components are present in known amounts
enriched- Has components to help bacterial growth (e.g. blood)
differential- Allows differentiation of bacteria
selective- Allows growth of only certina bacteria
A single medium can have multiple characteristics (e.g. differential and selective).

MacConkey's Agar is selective (contains bile salts) and differential (lactose fermentors form red colonies).

Mannitol Salt Agar is selective (7.5% NaCl = halotolerant) and differential (mannitol fermenters produce acid).
Bacterial Growth Requirements
pH – most pathogens are neutrophiles or acidophiles
salt concentration – most pathogens are non-halophillic or halotolerant
temperature – most pathogens are mesophils
oxygen-
aerobes – require oxygen to grow
anaerobes – do NOT require oxygen to grow
obligate anaerobes – oxygen is toxic
aerotolerant – do not use oxygen to grow but tolerate it, fermentative
facultative – respires with oxygen, ferments in its absence
microaerophillic – prefer an atomsphere of increased CO2 (low oxygen)
Fastidious Bacteria
To be cultivated in the lab fastidious bacteria have special nutritional requirements which must be met.

The dependence of Hemophillus on factor X (hemin) and factor V (NAD) distinguish H. influenzae (requires both X and V) and H. parainfluenzae (requires only V). A plate with 3 discs can be used, one with V, one with X, and one with XV.
Colony Morphology
Colony morphology is used to classify clinical isolates in the laboratory on the basis of.
size
shape
color
consistency

Ex...Bacillus Anthracis produces white flat irregularly round colonies that stand on end like egg whites when looped up.
Stationary Phase Persistance
Pathogens adhere and grow extracellularly or intracellularly.

after log phase, they need alternate lifestyles to growing rapidly

After cells have reached a critical density (quorum sensing), extracellular bacteria can:
• spread or invade and become quiescent
• remain localized produce toxin
• form a biofilm

Intracellular bacteria can:
• spread cell to cell
• enter a latent/persistent state
Bacterial Sensing/Signaling
Bacteria can sense their environment. Sensing leads to altered activity of response regulators that control gene transcription, translation or protein/mRNA stability. Signals can affect response regulator activity by directly binding to the response regulator or phosphorylation of the response regulator (2 component signaling system with histidine kinase)


Bacteria can sense other bacteria of the same or different species by cell-cell communication. Homoserine lactones (HSL) or peptides are secreted and sensed by surrounding bacteria by direct diffusion into the cell, active transport into cell, orinteraction with a 2-component system. Cell-cell signaling is used to induce plasmid transfer, and for quorum sensing

As bacteria grow, they use quorum sensing to determine when they have reached a critical concentration (population density). A diffusible signal is produced at a constant rate. When the signal reaches a certain threshold the bacteria are in high cell density and respond accordingly. For instance Quorum sensing can cause a switch from cell-associated virulence factors to secreted virulence factors that allow for bacterial spread. e.g. the Agr regulator from Staphylococcus aureus.
Biofilm
Biofilm formation occurs on surfaces. After formation the bacteria are in a different physiologcal state that can make them antibiotic resistant. The physical formation of the matrix protects the bacteria from phagocytosis (as well as antibiotics?). Biofilm formation often occurs when the bacteria is contact with an abiotic surface such as an implant or prosthetic. As antibiotics may not be effective, prosthetic/implant removal/rotation (as in vocal implants) may be indicated.

Normal flora can also form biofilms. Dental Plaque has been established to be a biofilm. GI tract normal flora is thought to exist as a biofilm.
Exceptions of the Growth Phase System
Some bacteria do not actively grow, but remain localized and secrete toxins. Example Corynebacterium diphtheria.

Some intracellular bacteria grow by spreading from cell to cell. Example Listeria monocytogenes moving by actin polymerization in the tail.

Some intracellular bacteria are held quiescent by the immune system. Example Mycobacterium tuberculosis that is held in macrophages forming an abscess.

Extracellular bacteria can persist in the host. Persisting bacteria often undergo phenotypic changes or become dormant.

After killing the host, some bacteria can survive for long periods of time in the soil due to sporulation. Sporulation is a developmental process that occurs in high density/low nutrient conditions. It results in the formation of a endospore which is very environmentally resillient. Example Bacillus anthracis.
Normal Flora
The skin, colon, conjunctiva, respiratory tract, anterior urethra and vagina all contain normal flora.

They are considered beneficial and even necessary to compete with pathogens, create environments unsuitable for other bacteria, production of essential nutrients (e.g. vitamins), prime the immune system, develop certain tissues and as probiotics (transients).

Classification:
resident = established
transient = remain for short periods of time
carrier = normal flora with pathogenic potential – can be transient or resident. Ex-carrier of Staph Aureus are either persistant (10-35%, Intermittant (20-75%), or non-carriers (5-70%). Note that the bacteria are not pathogenic to the carrier as it is part of their normal flora.
Charcteristics of a Good Antibiotic
Spectrum - balance between rapis an daccurate diagnosis and need for Tx.

Does not affect normal flora

ability to penetrate baterial cells/eukaryotic cells

low bacterial resistance

low toxicity for the host

nonallergenic with minimal side effects

bility to reach the site of infection (Administered IV/orally, Chemical stability, Serum stability).

Inexpensive and easy to produce.
Sources/Uses of antibiotics
Antibiotics can be synthetic, natural (primarily produced by Actinomyces, Bacillus spp. and fungi), or semi-synthetic (modified natural antibiotics)

Antibiotics are used for the Tx of human disease as well as production of food (Tx of animals and food presercation_
Antibiotic effects.
When an antibiotic is added to bacteria there are 3 possible outcomes:
1. Bacteria are resistant and continue to grow.
2. Bacterial growth stops but the bacteria are not killed and growth will resume after removal of the antibiotic (bacteriostatic). Killing of the bacteria depends on the immune system.
3. Bacteria are killed (bactericidal).

NOTE that some of these are different depending on the concentraiton of antibiotic used.

The minimum inhibitory concentration (MIC) of an antibiotic is the lowest concentration at which bacterial growth is inhibited. Both bacteriostatic and bactericidal antibiotics have MICs.

The minimal bactericidal concentration (MBC) is the lowest concentration at which bacteria are killed. It is determined by removing an aliquot of bacteria from the MIC assay and determining if the bacteria will resume growth in the absence of antibiotics. Only bactericidal antibiotics have an MBC.
Disk Diffusion assay
Disk diffusion assays assess how large of a zone of clearing surrounds an antibiotic-impregnated disk:
small zone = resistant
large zone = sensitive
The exact size of resistant zones varies depending on the antibiotic. Recently stripts with a defined gradient of antibiotic concentrations have become used rather than a disk.

Resistance is determined by the achievable clinical dose
Sensitive = inhibited or killed by 1/4 of the achievable clinical dose
Intermediate = inhibited or killed by 1/2 the achievable dose
Resistant = cannot be killed by an achievable dose
antibiotic resistance mechanisms
There are a number of antibiotic resistance mechanisms. The 3 major categories are:
1. natural- (intrinsic) These are properties that make the bacteria more resistant. (Gram-negative outer membrane, efflux pumps (Type I secretion), proteases that destroy drugs
2. acquired- These are acquired on mobile genetic elements from other bacteria.
3. mutational- These arise from mutation of the chromosome.
Efflux Pumps in terms of antibiotic resistance.
Efflux pumps remove the drug from the bacteria.

They can be non-specific or specific.

They can be intrinsic, acquired, or mutational (derepression of pump expression). Most antibiotics have at least one pump that will remove them.
Beta Lactamase
Gram-negative and Gram-positive bacteria produce B-lactamases that will inactivate penicillin by cleaving the B-lactam ring.

B-lactamase inhibitors can be co-administered with penicillin. These have a reduced rate of cleavage causing the beta-lactamase to be less efficient

We now have B-lactamase resistant forms of B-lactam antibiotics. Emerging B-lactamases destroy resistant forms (ESBL) Extended Spetrum Beta-Lactamases
Other Antibiotic modification enzymes
Fosomycin is cleaved by fosfomycinases

Aminoglycosides are inactivated by modification by the bacteria. There are 3 basic modifications that will inactivate Aminoglycosides:
1.N-acetylation
2.O-phosphorylation
3.O-adenylation

Chloramphenicol can be inactivated by acetylation (acetyl transferase)
Target Site Modification
Target site modifications will prevent antibiotic action. These include mutation to specific targets that are either acquired or mutational.

Ex. MRSA (methicillin resistant S. aureus)
mecA gene encodes a new beta-lactam resistance PBP (PBP2a)
S. pnuemoniae PBP2X – low level resistance PBP2B – low and high level resistance.

MLS (macrolide-lincosamide-streptogramin) strains have a erthromycin methyltransferase gene that methylates 23S rRNA prevent interaction of macrolides and lincosamides/clindamycin with the 23S rRNA and the 50S ribosomal subunit.

Vancomycin resistance is acquired and encoded by 7 genes. These genes change the D-Ala-D-Ala pentapeptide to D-Ala-D-Ser or D-Ala-D-Lac. There are 3 types of Vancomycin resistance operons: VanA, VanB, VanC.
Metabolic Bypass
There are either modification or complete substitution of Dihydropteroic acid synthetase and Dihydrofolate reductase allowing for the subersion of sulfonamides and trimethoprim (THF)
Summary of Antibiotic Resistance
Answer
Reduction of antibiotic resistance
Prudent use of antibiotics is essential to prevent the further spread and reduce resistance development. These practices include:
•appropriate antibiotic use – diagnosis versus rapid treatment, viral versus bacterial disease
•public education – understanding viral versus bacterial susceptibility, using full antibiotic regime
•selective removal, control or restriction of certain antibiotic classes – new antibiotics used only for resistant bacterial, use of antibiotics in cyclic patterns
•use of combination therapies to reduce likelihood of resistance emergence
•use of physical means to prevent spread of resistance organisms – careful washing of hands between patients
Cell Wall Synthesis (Cytoplasmic Steps)
Fosfomycin (Phosphomycin)

Target: UDP-N-acetylglucosamine-3-enolpyruvylether synthetase (NAM synthesis from NAG)(bactericidal)
Vulnerable to: Drug Inactivation, Reduced Transport

CYCLOSERINE (D-CYCLOSERINE)

Target: Attachment of the pentapeptide chain. Bactericidal


Vulnerable to: Target alteration
Cell Wall Synthesis (Membrane Steps)
BACITRACIN

Target: Undecaprenyl-P-P carrier (prevents recycling) Bactericidal
Cell Wall Synthesis (Wall Steps)
Vancomycin (glycopeptide)

Target: PCN binding protin (prevents transglycosylation) Bactericidal

Vulnerable to: Drug Efflux, Target alteration (5th D-ALA to D-Ser or D-Lac)


B-lactams (penicillin, cephalosporin, carbapenems, monobactams)

Target: PBP (prevents crosslinking) Bactericidal

Vulnerable to Drug inactivation bia B-Lactimases, Reduced transport, Drug Efflux, and Target Alteration
Folic Acid Synthesis
Sulfonamides

Target: PABA analog. bactericidal

Vulnerable to: drug efflux, target alteration (Bypass)


Trimethoprim

Target: dihydrofolate reductase competitive inhibitor. bactericidal

Vulnerable to: Drug efflux, Target alteration
Cell Membrane
Polymyxin

Target: disrupts cell membrane bactericidal
DNA Replication
Quinolones/Floroquinones

Target: DNA gyrase-ATP complex bactericidal

Vulnerable to: reduced Transport, drug efflux, target alteration


Novobiocin

Target: DNA gyrase
Transcription
Rifampicin, Rifamycin, Rifampin

Target: DNA-dependent RNA polymerase. bactericidal

Vulnerable to Drug Efflux, Target Alteration
Protein Synthesis
Aminoglycosides

target: 30S subunit (initiation).bactericidal

Vulnerable to: Drug Inactivation, Drug Efflux, Target Alteration


Tetracyclines

Target: 30S subunit (loading acceptor site). bacteriostatic

Vulnerable to: Drug Efflux, Target Alteration


Lincosamides

target: 50S subunit (inhibit peptide bond formation). bactericidal

Vulnerable to: Drug Efflux, Target Alteration


Chloramphenicol

Target: 50S subunit (inhibit peptide bond formation). bactericidal

Vulnerable to: Drug Inactivation, Drug Efflux, Target Alteration


Macrolides

target: 50S subunit (release peptide chain). bacteriostatic

Vulnerable to: Drug Inactivation, Drug Efflux, Target Alteration


Streptogramins

target: 50S subunit (block P site). bacteriostatic

Vulnerable to: Drug Inactivation, Drug Efflux, Target Alteration
Blood Agar
Addition of defibrinated blood to nutrient agar enhances the growth of some bacteria such as streptococci. In addition to distinctive colonies, it provides an indicator for hemolysis (Alpha, Beta, Gamma).
MackConkey Agar
Selective indicator for Gram -rods, especially family Enterobacteriaceae. The Peptone base medium contains bile salts, crystal violet, lactose, and neutral red (Ph Indicator). The bile salts and crystal violet inhibit gram positive bacteria and the more fastidious gram -'ves like Neisseria an dpasteruella. Only those that ferment lactose produce the red colony, often with a distinctive morphology.
Gram Positive Cocci for Identification
Staphylococcus

S. Aureus is a primary casue of furuncles, abscesses, carbuncles and many other infetions (esp osteomyelitis). Local infection may lead to septicemia.

S. Epidermiditis is one of the main causes of prostetic valve endocarditis. Can also lead to infections around an indwelling catheter, and can complicate hip replacements.

S. Saprophyticus looks like epidermiditis, distinguised by biochemical tests. May cause UTI in young women.

For Staph the telltale exams are the catalase test (can they produce bubbles from H2O2, staph are +). The Coagulase test will detect the enzyme which will clot plasma (diff Aureus + and epidermiditus)


Streptococcus

Pyogenes (group A), agalactiae (group B), and Enterococcus are medically important. They are differentiated based on hemolytic properties. Group A strep are susceptable to A discs (impregnated with bacitracin). Strep Pneumococcus is susceptable to P discs impregnated with optochinin. The PYR test, if +, indicates enterococcus or some staph and group A.
Reactions for Gram + Cocci
summary of reactions for Gram + Cocci
Gram - Coci diagnostic tests.
Neisseria Gonorrhoeae and Meningitidis.

Oxidase Test - if the organism has cytochromes it will turn the reagent blue/purple as Neisseria, pseudomonas, and haemophilus.

CTA sugars- Cysteine Tryptose agar has sugar and phenol red, the one who can oxidize this sugar turns the indicator yellow (distinguish between gonorrhoea and meningitidis

X and V tests- haemophillus requires one or both to grow, and some require enriched CO2 as well.

lactose fermentation is also used to distinguish as all lactose fermenters produce at least some acid. Also, salmonella and proteus produce H2S.
General Gram - Biochemical Tests
Citrate- can the bug use citrate as a carbon source

Urease- teste for the hydrolysis of urea to H20, Co2, and NH3

H2S test- Iron salts in the medium turn black in the presence of H2S.

Ornithine decarboxylase- bacteria ferment the glucose reducing pH and providing optimal conditions for ornithine decarboxylation causing the pH to rise again.

Indole- bacteria that produce tryptophanase deaminate it to aminoa, pyruvic acid and indole. The latter forms a red complex in the media.
Glucose and Lactose Fermentation
Glucose Lactose
E. Coli + +
Klebsiella + +
Enterbacter + +
Proteus + -
shigella + -
salmonella + -
S. Typhi + -
Pseudomonas - NP
Gram - Rod Id Flow chart
Does it grow on MacConkey's?

No but grows on chocolate, Haemophilus requires X and V

Yes, then does it ferment Lactose?

No- Salmonells, Shigells, proteus, Pseudomonas

Yes- E. Coli, Enterobacter, Klebsiella.

The final differentiation is examined via H2S on Hektoen, and Green pigment on T. Soy.
Taxonomy
Protozoa- unicellular eukaryotes

Metazoa- Multicellular eukaryotes including platyhelminthes (flatworms) and nematodes (roundworms)
Protozoa
Ameba:
Entameba histolytica

Flagellates:
Giardia
Trichomonas
Leishmania
Trypanosoma

Sporozoa:
Plasmodium (malaria)
(pneumocystis)
Cryptosporidium
Toxoplasma
Platyhelminthes (flatworms)
Trematodes (fluke):
Shistosome

Cestodes (tape worms, segments ribbon like body)
Nematodes (roundworms)
Intestinal:
Trichuris
Enterobius
Ancylostoma
Ascaris

Tissue:
Trincinella
Filiarias
Onchocerca
Larva Migrans
Entamoeba histolytica
Amoebic Dysentery, associated with poor sanitation. Humans are the principal host but dogs, cats, and rodents may be infected.

Life Cycle: Mature cysts are ingested and excystate in the small intestine. Trophozoites multiply in large intestine, and are passed in feces. Transmission can also occur through fecal exposure during sezual contact (in which case cysts and also trophozoites could prove infective).

Trophozoite Form- amoeboid with a vesicular nucleus, small central karyosome, no mitochondria, possibly with ingested erythrocytes and inclusion bodies.

Cyst Form- Spherical with a refractile wall, 1-4 nuclei, and cytoplasm that contains dark staining chromatoidal bodies. Can survive days to weeks in the extermal environment.

pathology- Trophozoites invade the intestinal mucosa causing appendicitis perforation, stricture granuloma, and pseudo-polyps. The resulting ulcers are flask shaped.

Disease: Amoebic Dysentery- asymptomatic carriers pass cysts. Acute disease includes frequent dysentery wtih necrotic mucosa and abd pain. Chronic disease- recurrent episodes of dysentery with blood and mucus, intervening Gi disturbanes and constipation. Systemi infection leads to liver dysfunction, pneumonitis, and encephalitis.

Dx- symptoms Hx, Epidemiology with Lab confirmation (cysts andheme in the stool). Distinct from Shigella dysentery due to lack of high fever and PMN leukocytes. Antibody positive in 75% of cases.

Tx/Immunology- Does not correlate with Ab response, yet steroids exacerbate disease. Cell mediated immunityis important. asymptomatic infections may or may not require meds (Lodoquino). Symptomatic Chronic disease and extraintestinal disease require metronidazole.
Giardia lamblia
Giardiasis/Lambliasis/Bever Fever (Flagellate). Not uncommon in Pa, the most frequent protozoan intestinal disease in the US. Common cause of disease associated wtih breakdown of water purification, outdoorsmanship, Travel to endemic area (Russia, Rocky mountains, Etc), and day care centers.

Life Cycle: Ingestion of cysts in contaminated water or food leading to excystatin in the small intestine. Trophozoites attach to the mucosa via a ventral sucking disk, Encystation occurs in the colon.

The Trophozoite form is half-pear shaped with 8 flagella, 2 axostyles in b/l symmetry, 2 anteroir large suction discs, 2 nuclei, and 2 parabasal bodies.

Cyst Form- Ellipsoidal with a smooth well-defined wall. They are hardy and can survive several months in cold water.

Pathology- early symptoms include flatulance, abd. distension, nausea, foul smelling bulky explosie often watery diarrhea, stools containing excess lipids but rarely blood or necrotic tissue. In the chronic phase there is malabsorption (a result of flattening of the mucosal surface and a giardia covering), B12 and disaccharidase deficiency, and lactose intolerant.

Dx- Hx of symptoms, epiemiology, lack of mucus and blood, no increas in PMN's in stool, no fever. The lab will ID cysts inteh stool and trophozoited in duodenal content.

Immunity/Tx: Iga offers incomplete protection. Infections to cause some lasting immunity, however relapses occur due to antigenic variation. Increased incidence in immunodeficiency.
Trichomonas Vaginalis
Flagellate, STD with a world wide ditrubution

Life Cycle: colonized the vagina and urethra/prostate of men. Initial infection via sexual contact and then ping pond re-transmission occurs. Trophozoites live in close contact with the urogenial epitheliam causing contact dependant damageto the epithelium. Reproduction is via binary fission (favored by low acidity). The organism does not encyst and infection may persist for years. No non-human reservoir.