Describe the structure of peptidoglycan
Composed of repeating dissaccharides with proteins attached.
1: NAM: N-acetylmuramic aicd
2. NAG: N-acetylglucosamine
Each muramic acid has a pentapeptide attached which contains both D- and L-amino acids, with a D-ala end.
How are peptidoglycans assembled? 4 steps
- Disaccharide and peptide units are assembled in cytosol
- Exported across plasma membrane
- Disaccharides are assembled into long chains
- Chains cross linked by peptide bonds between side chain and this goes in both directions.
How does penicillin compromise the cell wall.
The enzyme that does the crosslinking, penicillin binds irreversibly (covalent bond) to the active site. The new peptidoglycan formed has no cross links, the structural integrity is compromised and cell lyses.
This is because penicillin is a structural analogue of D-ala-D-ala.
Summarize Gram positive and Gram negative bacteria.
Poor Negative Grandma takes pepper out perfectly
Gram positive - violet stain (TPTPGC)
- thick peptidoglycan-20-50 layers
- teichoic acid: polymers of sugar alcohols linked by phosphate groups, they are the major antigen that sets off TLRs
- protein fibrillae (adhere to mammalian cells and extracellular matrix)
- Group carbohydate: linked to peptidoglycan and that is useful for identification Ex. streptococcus
Gram negative - red stain
- thin peptidoglycan-1-3 layers
- outer membrane (composed mostly of LPS)
- periplasm between the outer and inner membrane containing: peptidoglycans to outer membranes via lipoproteins, also contains enzymes, solute carrier proteins
- porins found in outer membrane
What are the steps of gram staining?
- You heat fix bacterium to slide, caramelize the attached carbohydrates a bit to make it sticky
- You stain with Gentian or Crystal violet
- Add Iodine ‘Mordant’ which complexes with the violent and sits just inside the cell wall
- Use alcohol wash and the complex will escape from the thin wall, Gram negative, making it colorless
- Add Safranin which makes Gram negative stain red and Gram Positive turns violet
Describe LPS three components
- O-antigen: terminal polymer of 3-5 sugars (10-50 copies): helps species evade immune system by altering their antigenic structure or keep the MAC at bay with their length
- Core - phosphorylated oligosaccharide - the cores strongly attach adjacent LPS via divalent cations
- neutrophils use defensins in cytoplasmic granules that are highly cationic which disrupts the outer membrane, sensitizing phagocytosed bacteria to toxic metabolites
- Lipid A- disaccharide + 6-8 saturated fatty acids
- the endotoxin
- monocytes and other cells have receptors for lipid A, they will secrete cytokines in the presence.
Describe the consequences of endotoxins (LPS)
Receptors for endotoxins are found on monocytes (TLRs)
-LPS binding leads to synthesis and release of inflammatory cytokines
-a small localized amount is defensive
=-too much can cause over production of cytokines which dilate blood vessels, drop in blood pressure, spetic or endotoxic shock and death.
Endotoxin can also activate the blood clotting mechanism and cause DIC. Disseminated Intravascular Coagulation ultimately leading to hemmorhage.
What are the three bacterial appendages found in all.
smooth vs random tumbling
- Conjugation pili - transfers DNA between cells
- Flagella aka the H antigen- locomotion
-basal body rotates and its driven by H+ inflow
-counterclockwise rotation = smooth
-clockwise rotation = random tumbling
“think of a rewinding clock in movies when they go in the past”
- Pili aka fimbrae - adhesion
Describe bacterial spores
Bacterial spores are produced when nutrients get low and are specialized for enduring unfavorable conditions. They are resistant to drying, radiation, boiling water temperature (food poisoning) and disinfectants.
Spores do not grow or divide, when it encounters favorable conditions, it will exit the spore wall and resume growth.
Useful for identification as only some Gram positive (thick wall) bacteria produce them.
There are medial spore and terminal spore (tennis racket)
Need to use autoclave to actually kill.
-do not stain becasue of thick walls
Describe the two modes of transduction
- A virus can contain bacterial DNA in place of viral DNA and this bacterial DNA inserts into the host DNA. This is however very rare. Only 1/1000 virus particle will contain bacterial DNA. Insertion will only occur if there is homology and this piece of bacterial DNA is only 1/100 of the bacterial chromosome.
- Via proviruses which have inserted its DNA into the host bacterial DNA. When the host cel chromosome is damaged and the cell is likely to die, these proviruses bail. In some rare cases, a bacterial gene is excised as well. This gene will be part of the proviruses formed by them.
Describe flagellar phase variation between H1 and H2 in salmonella
There is an enzyme hin (H inversion) which recognizes two inverted repeats and inverts DNA between repeats. It inverts a portion of DNA which contains the promoter to the H2 gene and the rH1 repressor gene.
So in this case H2 is being expressed and H1 repressed.
When the promoter is flipped it goes on into oblivion so H2 is no longer expressed nor is the H1 repressor, so H1 flagella is expressed .
Describe phase variation of pili in Neisseria
So a major form of defense against Neiserria attaching to mucous membrane is IgA which coats the pili and blocks adhesion. So producing new antigenic forms of pili will escape this immunity.
Neiserria has one copy of gene encoding pilin protein and many incomplete silent copies.
It then can undergo recombination trading blocks of DNA sequences to form a new antigenic version of PilE.
Describe phase variation of PII in Neisseria
Repeats of CTCTT occur at specific position within the coding sequence of the PII gene. Strand slippage continuously produces changes in the copy number of repeat. However only multiples of 3’s will produce a PII.
PII is an outer membrane protein important for adhesins. Neisseria is constantly varying between production and non production of PII, lack of adhesion is compensated by lack of immunologic recognition.
List the 7 biochemical mechanisms of drug resistance
- Decreased levels of drug - decreased entry and increased efflux
- Inactivation of drug-bacteria produces enzymes that inactivate the drug
- If drug is an antimetabolite, Increased levels of normal metabolite antagonizes enzyme via competition
- The target the drug is directed toward has increased expression
- Lowered affinity of drug receptor
- Lowered activity of the pathway the drug is meant to target - so if the normal mechanism that the drug is mean’t to inhibit isn’t even active, then the drug isn’t effective
* 7. decreased conversion of a drug to its active metabolite
Describe vancomycin action and resistance
Vancomycin only kill gram POSITIVE bacteria but it kills the superbugs and is standard for MRSA.
Vancomycin like penicillin binds to the D-ala-D-ala moiety leading to its Cell wall destruction
VanHAX (hack): operon generates a D-ala-D-lactate moiety, leading to peptidoglycan which can form a matrix but can’t bind vancomycin
What is an API strip?
You put bacteria into different wells and each well test a different thing. You add up results to get a score, compare it to a database and identify.
This is largely automated now.
What is MALDI-TOF? Matrix assisted laser desorption ionization-time of flight spectrometry.
an ionization technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation.
MALDI methodology is a three-step process. First, the sample is mixed with a suitable matrix material and applied to a metal plate. Second, a pulsed laser irradiates the sample, triggering ablation and desorption of the sample and matrix material. Finally, the analyte molecules are ionized by being protonated or deprotonated in the hot plume of ablated gases, and can then be accelerated into whichever mass spectrometer is used to analyse them. Time of flight is measured and in the end you get a measure of charge and size
You get an ID in 5 minutes, cost is like 25-50 cents, accuracy is over 95%
What are examples of antimicrobial susceptibility tests?
- The broth dilution - broth containing lowering dilutions of antibiotics. When there is no growth that is you MIC minimum inhibitory concentration.
- Disk diffusion: measure the zone of inhibition which will give you a linear correlation to the MIC
- Etest- use a strip of varying concentrations of antibiotics on a lawn of bacteria. Measure where the clearing starts
What are the 30S inhibitors?
Aminoglycosides (“mycins”)- streptomycin, gentamicin, tobramycin, Amikacin, Neomycin, Kanamycin
-all exhibit nephrotoxicity
Tetracyclines (“cycline”) Tetracycline, demeclocycline, minocyclin, doxyclcine, oxytetracycline, tigecycline
What are the 50S inhibitors?
- MacROlides (romycins) - Erythromycin, Clarithromycin, Azithromycin, Telithromycin
- Lincosamide- clindamycin
- Streptogramins- quinupristin/dalfopristin
- Oxazolidinone - Linezolid
A patient develops fever, pneumonia, severe headache and hepatitis. He has been around farm animals. The organism was found to be gram negative. What is this patient suffering from?
A patient has been in contact with livestock. He suffers from fever, anorexia and chills which develop into an undulating fever. What does this patient have?
Microbes are found to be gram negative coccibacilli
Patient has diarrhea, headache, fconfusion fever and an atypical pneumonia with unilobar infiltrate. The patient has been swimming in a warm lake.
The microbe was found to be gram negative but better visualized with a silver stain and oxidase positive. An urine antigen test identified the organism. What was it?
Largely asymptomatic, conjunctivitis leading to scarring and blindness.
This women has cervicitis, urethritis, and PID which lead to infertility.
She also had lymphogranuloma venereum LGV - inguinal nodes
What is the agent?
Agent is an obligate intracellular. Its extracellular form is sporelike. It stains gram indeterminate.
Child is 10 years old.
He had mild upper respiratory tract infections but can cause pneumonia or bronchitis. What is the agent?
A bird keeper comes down ill with pneumonia.
The bacteria stained is gram indeterminate.
THe patient started off with a fever, headache and sore throat which later developed a cough as well. What is the agent?
A 20 year old military personnel comes down with bronchitis and pneumonia.
However it was noted that there was patchy infiltrate in the X ray yet the patient seemed alot better than what the X ray suggests.
The microbe responsible could not be cultured and also contained no stain.
A sample of the patient’s RBCs lead to agglutination at 4 degrees celcius.
He was sent out with Z pack because I had figured it out! IT WAS!??
It was a lovely May. Patient has fever, headache and mylagia. She also has neutropenia and thrombocytopenia.
Morulae was discovered in neutrophils.
What is the agent?
Human Granulocytic Anaplasmosis (HGA)
Patient has fever, headache and mylagia. She also has leukopenia and thrombocytopenia.
Patient has a maculopapular rash: made of both flat and raised skin lesions. The name is a blend of the words “macule,” which are flat discolored skin lesions, and “papule,” which are small raised bump
Human Monocytic Ehrlichiosis (HME), Ehrlichia chaffeensis
A person has been around sheep and other livestock in a barn. The patient has a fever but no rash. He also has pneumonia and a headache along with hepatitis.
The microbe was gram negative .
Q fever, Coxiella burnetii.
Person develops immediate cellulitis and later on osteomyelitis. Organism is gram negative, oxidase positive, catalase positive. The treatment was fine with just penicilin.
Patient has severe headache (unremitting), fever, myalgia and the rash began in the trunk but spread to extremities. The rash does spare the face, palms and soles.
He was bit by a lice and scratched a lot.
Microbe is obligate intracellular and does not stain
Patient suffers from headache, severe fever, myalgia and rash which is petechial and began 3 days after the fever began and started around ankles and spread toward trunk. The rash was even on palms of hands and soles of feet.
Microbe is obligate intracellular and does not Gram stain
A man in NYC who is a dogowner comes down with fever and generalized rash. He also has multiple dark eschars throughout his body. What is the microbe?
Its gram indeterminate and obligate intracellular
What are the 7 nontuberculoid mycobacteria and their distinguishing features
- M. kansasii: photochromogen, causes a chronic pulmonary infection that resembles Mtb. Tap water is major reservoir.
- M. marinum: photochromogen, cutaneous infections following exposure to salt, freshwater after trauma. “swimming pool granuloma”. Most common in southern coastal states: Grows in cold water.
- M. gordonae: grows where the light don’t shine and where it is. Scotochromogen, pigmentation in dark and light. Very commonly recovered. Colonizes respiratory tract. Long, wide branching beaded acid fast bacili.
- M. xenopi: scotochromogen: causes chronic pulmonary disease for those with existing COPD. Grows best at hot temps
- Mycobacterium avium complex (MAC): includes 3 members, and its found in the environment. It is the only non-chromogenic.
Important for immunocompromised peopel
- M.abscessus- rapid growing mycobacteria, post trauma wound infections- Grows best at lower temps (30 degrees like marinum). Short, nonbranching Acid fast bacilli in clusters, appear as gram positive on stain. The opposite of gordanae
Often drug resistant
- M. leprae; reservoir is armadillo, spread from person to person via anaesthetic skin lesions. It can be lepromatous or tuberculoid.
Bacteria with resistance genes carried by temperate bacteriophages can be transferred to genetically distinct isolates. What is probably required for transfer?
Excision of the bacteriophage from the bacterial chromosome
New virus formation.
A DNA fragment transferred into a bacteria will be lost from the recipient cell soon if this doesn’t happen
Homologous recombination between the transferred fragment and the bacterial chromosome
A strain acquires a virulence factor after being exposed to a cell free extract. What produced this genetic change?
Epidemic cholera is vibrio cholerae O1, what does the O1 refer to.
The carbohydrate chain of LPS
What is Bactec
BACTEC is a relatively fast way to test for M. tuberculosis. It relies on that M. tuberculosis can metabolized long chain fatty acids. A sealed vial of labeled C-16 is placed in a vile with antibiotics to suppress contaminants. If mycobacteria are present, CO2 is detected. This can be used to determine antibiotic sensitivity. Use one as a control and then try different drugs.
Why could a PPD test produce a false negative
If the person has impaired cell mediated immunity: elderly, immunocompromised.