Unit 4b Flashcards
Selective toxicity
i. Fundamental feature of abx therapy
ii. Abx effect exerted selectively on microbe and NOT the host
iii. Use biochemical differences between pathogen and target
Common biochemical differences between pathogen and target (5)
- Folate metabolism (intracellular synthesis) required in bacteria
- Protein synthesis in bacteria uses 30S and 50S ribosome
- Nucleic acid synthesis in bacteria uses DNA gyrase (not topoisomerase) and structurally distinct RNA polymerase
- Cell wall synthesis (peptidoglycan component unique)
- Fungal cell membrane (ergosterol in fungal membrane, cholesterol in humans)
Antibiotic spectrum and the 3 types
What abx can be used for what
- Narrow
- Broad
- Extended
Narrow antibiotic spectrum
effective against gram+ OR gram-
- Often most effective on susceptible organism
- Less disturbance of host flora
- Switch to narrow spectrum coverage as soon as possible
- Target definitive therapy to known pathogens and antimicrobial susceptibility test results
Extended antibiotic spectrum
effective against gram+ AND gram-
Broad antibiotic spectrum
effective against gram+, gram-, and atypical organisms
- Sacrifice efficacy for greater scope of activity for initial empiric coverage
- More likely to cause superinfection
- Should be used to treat acute, severe infections
Why are bacteriocidals preferred in severe infections? (3)
- Act more quickly, often irreversible with sustained effect
- Can compensate for patients with an impaired host defense
- Required for treatment of infections in immune sanctuaries (e.g. CNS-endocarditis infections)
Natural resistance and three examples
microbes lack a susceptible target for drug action
- Fungal cell walls - do not contain peptidoglycans
- Mycoplasma - do not have cell walls at all
- Pseudomonas - drug cannot penetrate outer membrane
Escape resistence (3)
microbes are sensitive and abx reaches target, but…
- Organism “escapes” due to availability of purines, thymidine, serine, methionine released from purulent infections
- Failure to “lyse” due to lack of osmotic pressure difference
- Important role of surgical drainage
Acquired resistance and two examples
selective pressure produces successive generations with biochemical traits that minimize drug actions
1.Mutational (chromosomal) resistance - proper dosing and duration of abx therapy prevents this
- Plasmid mediated resistance → multiple drug resistance
- Can emerge during a single course of treatment
Selective distribution of clindamycin
bone
Selective distribution of macrolides
pulmonary cells
Selective distribution of tetracyclines
gingival crevicular fluid and sebum
Tetracycline toxicity in gingival crevicular fluid
bind Ca2+ in bone and teeth → abnormal bone growth and tooth discoloration
Selective distribution of nitrofurantoin
urine
Selective distribution of aminoglycosides
potential for toxicity, accumulation in inner ear and renal brush border
Mnemonic for drugs eliminated by nonrenal mechanisms
DQ Crimes:
i. Doxycycline
ii. Quinolones (IS RENAL but CYP450 inhibitor)
iii. Clindamycin (avoid in liver disease)
iv. Rifampin (inducer of CYP450, potential hepatotoxicity)
v. Isoniazid (genetic polymorphisms, potential for hepatotoxicity)
vi. Metronidazole (drug-drug interaction with alcohol due to inhibition of aldehyde metabolism) - Antabuse reaction
- Avoid in liver disease
vii. Erythromycin-like (Azi-Clas-Ery): (drug-drug interactions due to inhibition of CYP450 (not Azi)
vii. Sulfonamides (metabolized to more lipid-soluble compound → increased risk for renal crystalluria)
persistent suppression of bacterial growth after limited exposure to some antibacterial drugs is called…
post-antibiotic effect
5 mechanisms of resistance to antibiotics
- altered targets or receptors to which abx cannot bind
- enzymatic destruction of abx
- Bypass pathway- alternative resistant metabolic pathway
- Decreased entry
- Increased efflux
4 examples of altered targets ot receptors conferring resistance to abx
- Penicillin binding proteins: B-lactam antibiotics (penicillins, cephalosporins, carbapenems)
- DNA gyrase (Fluoroquinolones)
- Peptidoglycan side chain (Vancomycin)
- 50S ribosome methylation (Erythromycin, Clindamycin)
3 examples of enzymatic destruction of abx
- B-lactamase (B-lactams - penicillins, cephalosporins)
- Acetyl-phospho-adenylyl transferases (aminoglycosides)
- Acetyltransferase (Chloramphenicol)
Example of bypass pathway
Overproduction of PABA or thymidine nucleotides (sulfonamides)
3 antibiotics affected by decreased entry
- B-lactam abx
- Fluoroquinolones
- Aminoglycosides
3 abx affected by increased efflux
- tetracyclines
- macrolides
- fluoroquinolones
bacteria develop resistance to tetracyclines by which primary mechanism?
Increased drug efflux → MDR gene
Which of the following is the primary mechanism of B-lactam antibiotic resistance with Strep pneumoniae?
Modulation of drug target
Bacteriostatic or bactericidal?
Cell wall synthesis inhibitors
typically bactericidal
Bacteriostatic or bactericidal?:
protein synthesis inhibitors
Exception?
typically bacteriostatic
Exceptions: aminoglycocidal which binds irreversibly
Bacteriostatic or bactericidal?:
Inhibitors of DNA synthesis
typically bactericidal
Bacteriostatic or bactericidal?:
Drugs that interfere with cell membrane function
typically bactericidal
Present in Gram+, Gram-, or both?
1) Peptidoglycan
2) Teichoic acids
3) Lipopolysaccharide (LPS)
4) Cytoplasmic membrane
5) Outer membrane
6) Periplasmic space
7) Porin proteins
1) Peptidoglycan - BOTH
2) Teichoic acids - GRAM+
3) Lipopolysaccharide (LPS) - GRAM-
4) Cytoplasmic membrane - BOTH
5) Outer membrane - GRAM-
6) Periplasmic space - GRAM-
7) Porin proteins - GRAM-
Lipid A
- component of outer membrane in Gram- organisms
- inner portion of LPS
- Toxinogenic (endotoxin)
3 parts of LPS
1) Lipid A
2) Core oligosaccharide
3) Outer oligosaccharide
Core oligosaccharides
attach to lipid A
-not significant clinically
Outer oligosaccharide
“O chain”
- Varies from strain to strain
- Target for recognition by abs
The E. Coli O157:H7 serotype
What do the “O” and “H” antigens refer to?
O = oligosaccharide antigen located in LPS
H = flagellar antigen
Coagulase
Which are coag + staph?
Coagulase promotes deposition of fibrin and walls off S. aureus
S. aureus
MacConkey Agar is selective for __________ and differential for ________
only grows gram - bacteria (bile salts and dye crystal violet)
ability to ferment lactose (lac+/lac-)
red/pink –> lac +
colorless –> lac -
Klebsiella pneumoniae are gram ______ _______ that are lac _______
negative rods
lac +
_______ and _______ are lactose fermentors
E. coli and Klebsiella pneumoniae
Gram stain is determined by the quality/character of _________
peptidoglycan
N. gonorrhoeae can express different antigenic forms of pili. This is due to the process of…
Genetic recombination
Bacteria taking up naked DNA or DNA fragments and integrating them into the chromosome = ________
transformation
Acquisition and expression of ditheria toxin in C. diptheriae is a result of…
Lysogenic conversion
Why is it difficult to become immune to:
1) Strep. pyogenes
2) Sprep. pneumoniae
3) N. Gonorrhoeae
1) Strep. pyogenes - M antigen with many serotypes
2) Sprep. pneumoniae - polysaccharide capsule with many different capsular antigenic types
3) N. Gonorrhoeae - pilus with intra/interstrain genetic variation prevents you from becoming immune
Which bacteria?
Production of Coagulase (helps to wall off infection by forming fibrin capsule)
Staph. aureus
Which bacteria?
Production of Glycocalyx
Coag-negative staph (Staph epidermidis)
Which bacteria?
Antiphagocytic capsule
Strep pneumoniae
Which bacteria?
Production of Dextrans (enhance adherence to damaged heart valves and teeth)
Strep viridans
Which bacteria?
Production of Toxins that transfer glucose from UDP-glucose to Rho family GTPases, altering the cytoskeleton of enterocytes
C. diff
Which bacteria?
Gram-negative rod with intrinsic resistance to penicillin, ceftriaxone (3rd gen cephalosporin) and erythromycin
Pseudomonas aeuriginosa
Which bacteria?
Gram-negative anaerobe frequently associated with abdominal infections and part of normal flora of colon.
Bacteriodes fragilis
Which bacteria?
Production of M protein (anti-phagocytic)
What is mechanism of action of M protein?
Strep pyogenes
M protein = virulence factor associated with antiphagocytic activity due to inhibitory effects on complement
Which bacteria?
Antigenically distinct pili allows repeated infection with different strains
N. Gonnorhoeae
Which bacteria?
Lack of cell wall precludes use of beta-lactam agents
Mycoplasma
Which bacteria?
Obligate intracellular pathogen
Chlamydia
Diptheria toxin acts by…
irreversibly inhibiting protein synthesis by inactivating EF-2 through ADP ribosylation
Blood agar plates are differential to detect ________
hemolysis
Hektoen agar is selective for _________ and differential for __________
gram - bacteria (does not allow growth of gram + bacteria)
lac + (lac + –> yellow-orange color)
Salmonella and pseudomonas are lac _______ while E. coli is lac ________
lac -
lac +
What structure on the Salmonella is responsible for reacting with the different antibodies during serotyping
oligosaccharide on LPS
How are flagella stained?
Flagella are too small to be seen directly by light microscopy.
Flagella stains use a mordant that precipitates on the flagella and increases their size so that they can be observed by light microscopy in the stained preparations.
How are capsules stained?
Capsules do not stain by procedures such as the Gram stain or acid-fast stain.
negative stains:
-(e.g., India ink, in which case the capsule appears as a clear zone between the bacterium and the background stain)
positive stains:
interact directly with capsular material (such as CuSO4)
How are endospores stained?
In Gram stains: developing endospores visualized as unstained regions within stained mother cells
positively stained:
-vigorous staining procedures allow stains to enter spores, followed by decolorizing procedures that remove the stain from vegetative bacteria but not from the stained spores
What are the major rules for specimen collection (6)
1) Collect before abx started
2) Use capped syringes containing aspirates, fluid, or tissue
3) Swabs are poor way to recover infecting agents
4) DO NOT obtain fluid cultures from drains/catheters
5) More is better than less
6) Deeper specimens preferred to superficial samples
Empiric antibiotic use takes what into context (4)
1) Clinical Context of infection: Intra-abdominal infections, Hospital acquired pneumonia, Severe community acquired pneumonia, Meningitis, Infection in immunocompromised patient
2) Exposure history / travel history
3) What organisms are most likely involved given exposure and clinical context
4) Are there antibiotic resistant organisms I might need to consider?
Antibiogram
sensitivity profile for organisms isolated in last year
3 methods to determine antibiotic susceptibility
1) Broth dilution
2) Disk diffusion
3) E test
Broth dilution
- Serial dilutions in liquid medium
- Visualize tubes for evidence of bacterial growth
- Lowest concentration of abx that prevents visible bacterial growth → Minimum Inhibitory Concentration (MIC)
- Measures MIC
Disk diffusion
- Spread isolate suspension on agar plate
- Disks with defined concentrations of abx placed on surface
- Diameter of clearing around the disk is measured
- Measure mm zone size
E test
- Strip with concentration gradient of abx on agar plate
- Find area of no clearing around strip (ellipse)
- Where ellipse meets the strip = MIC
- Measures MIC
MIC
Minimum inhibitory concentration
- Used to determine bacterial susceptibility
- MICs of different agents for a particular organism are NOT directly comparable
Caveats of MIC
1) NOT adjusted for site of infections
2) NOT adjusted for number of organisms in an infection
3) NOT adjusted to conditions in the host
4) NOT adjusted to reflect patient’s host defenses
MBC
Minimum bactericidal concentration
Lowest concentration of the antibiotic that kills 99.9% of the original inoculum
NOT used in determining drug susceptibility
MBC = MIC –>
MBC»_space;> MIC –>
MBC = MIC –> Bactericidal
MBC»_space;> MIC –> Bacteriostatic
Susceptibility
max serum concentration of drug must exceed the MIC of bug
Cp > MIC = break point
Appropriate abx selection based on…
1) Drug distribution (local abx concentration at site of infection must be > Cp)
2) Local environments
3) Host factors
Host factors to consider when selecting an abx (7)
1) History of previous adverse rxn to abx
2) Renal/liver function
3) Age
- Don’t use sulfonamides in neonates
- Don’t use tetracyclines (bone and teeth) in kids
4) Genetic/metabolic factors
5) Pregnancy
6) Drug interactions
7) Immune status
In immunocompromised patients you should use…
a bactericidal abx
For certain infections like meningitis or endocarditis you want to use…
bactericidal abx
Intrinsic resistance
occurs “just because” of natural properties of bacteria
What kinds of drugs are intrinsically resistant to Vancomycin?
Gram - bacteria (can’t cross outer membrane)
E. coli has intrinsic resistance to ________ but is sensitive to ________ due to…
Penicillin
Ampicillin/Amoxicillin
porin channel doesn’t allow penicillin in, but allows ampicillin in
Mycoplasma is intrinsically resistant to…
B-lactams
No cell wall
________ has high intrinsic resistance
Pseudomonas
Acquired resistance
- develops by genetic mutation or acquisition of new genes
- Mobile genetic elements (plasmids, transposons, bacteriophages) spread resistance from cell to cell
Antibiotic tolerance
-organisms that test susceptible to a drug in the lab, but not killed/inhibited by it in the patient
EX) Biofilms, Metabolic bypass, Anaerobic growth, Stationary phase
Categories of antibiotic resistance (3)
1) Inactivate or modify the drug
2) Alter antibacterial target
3) Reduce ability of drug to get to target (porin channels, efflux pumps)
Porins
outer membrane of GRAM-NEG bacteria
- Hydrophilic channels
- Allow selective uptake of essential nutrients (and hydrophilic abx)
- Change in configuration/number of porin channels → affect uptake of abx
EX) ampicillin vs. penicillin in E. Coli
Efflux pumps
- bacterial cell membranes, eliminate substances from bacterial cytoplasm
- Get rid of toxic substances and abx
- GRAM-POS and GRAM-NEG
- Adversely affects uptake of abx
- Can result in multi-drug resistance or specific
Structure of peptidoglycan
two alternating sugars (N-acetylglucosamine and N-acetylmuramic acid) cross-linked via peptide bridge
D-alanine terminal AAs - last D-alanine cleaved off during cross linking
Penicillin Binding proteins perform the _________ reaction in order to ________
transpeptidase/transglycosylase
cross-link peptidoglycans of cell wall
B-lactam antibiotics work by…
Irreversibly binding and inactivating transpeptidase reaction of PBPs → inhibit cross-linking and peptidoglycan synthesis
Resistance to B-lactam abx can be acquired via what 2 mechanisms
1) B-lactamases (change the drug)
2) altered PBPs (change the target)
B-lactamases
enzymes that inactivate B-lactam abx by splitting B-lactam ring → protects activity of PBP (change drug)
- In both Gram- and Gram+ organisms
- Encoded by chromosomal genes or plasmids
4 types of B-lactamases
1) Narrow spectrum, “Penicillinases”
2) ESBL
3) ampC
4) Carbapenemase
“Narrow-Spectrum” Beta Lactamases
-Hydrolyze penicillin-type antibiotics = Penicillinases
Not effective against cephalosporins or carbapenems
In gram+ and gram- organisms
VERY common form of resistance
Can be inhibited by B-lactamase inhibitors
3 common bacteria that have narrow-spectrum B-lactamases
S. aureus
E. Coli
Klebsiella pneumoniae
Staph aureus and BLA plasmid
BLA plasmid that gives resistance to penicillin/ampicillin
ALL STAPH have this!!
E. coli and TEM-1 plasmid (penicillinase)
→ ampicillin resistance
20-30% of E. Coli have this
(intrinsically penicillin resistant because gram-)
Klebsiella pneumoniae and SHV-1 chromosome (penicillinase)
→ ampicillin resistance
ALL Klebs have this!!
(intrinsically penicillin resistant because gram-)
Extended Spectrum B-Lactamases (ESBLs)
1) effective against what abx?
2) Found on plasmid or chromosome?
3) What bacteria is it common in?
4) can it be inhibited by B-lactamase inhibitors?
5) treat with what?
1) attacks cephalosporins and penicillins
2) PLASMID only
3) Gram-neg rods (E. Coli, Klebsiella pneumoniae)
4) YES - inhibited by B-lactamase inhibitors
5) Treat with Carbapenem
Ecoli with ESBL will have resistance to what?
resistant to ampicillin, penicillin, and Cephalosporins [Cephalexin (1st gen), Ceftriaxone (3rd gen)]
Remains sensitive to carbapenems
ampC-encoded B-lactamase
1) effective against what abx?
2) Found on plasmid or chromosome?
3) What bacteria is it common in?
4) can it be inhibited by B-lactamase inhibitors?
5) treat with what?
1) hydrolyzes penicillins and cephalosporins
2) CHROMOSOME ONLY
3) Gram-neg rods (Enterobacter, Pseudomonas –> LAC-)
4) NOT inhibited by B-lactamase inhibitors
5) Treat with Carbapenems
Enterobacter with ampC
Inducible –> resistant to…
Constitutive –> resistant to…
Inducible –> resistant to ampicillin and cefazolin (1st gen cephalosporin)
Constitutive –> resistant to ampicillin and cefazolin, ceftriaxone (3rd gen), and pip/tazo
Carbapenemases (KPC and NDM-1)
1) effective against what abx?
2) Found on plasmid or chromosome?
3) What bacteria is it common in?
4) can it be inhibited by B-lactamase inhibitors?
5) treat with what?
1) degrades penicillins, cephalosporins, AND carbapenems
2) PLASMIDS ONLY
3) Gram-neg rods (Klebsiella pneumoniae, E. Coli)
4) NOT inhibited by B-lactamase inhibitors
5) TX ? not any good ones
ampC can be expressed two ways:
inducible
constitutive
Inducible expression of ampC
ONLY induced by Ampicillin and Cefazolin
- Inducer = peptidoglycan breakdown product
- When ampicillin and cefazolin they act on peptidoglycan that causes much more inducer to be made
→ Inducer binds ampC → amp C expression
Constitutive expression of ampC
- Mutation in inducer → increase concentration of inducer → activates ampC → ampC expressed all the time
- Can get from inducible → constitutive expression via mutation
- Mutational events can change inducible → constitutive expression of ampC during therapy!
PBP alteration can be through 2 ways
1) Mutation of existing genes (Mosaic PBPs)
2) Acquisition of new PBP genes (mecA)
mecA
1) Encodes for what?
2) In what bugs
3) confers resistance to what abx
1) New gene that encodes for a new low affinity PBP (PBP2a)
2) Staphylococci
3) all B-lactams (penicillin, ampicillin, cephalosporins, carbapenems)
MRSA has what gene?
mecA
→ resistant to penicillin, ampicillin, cephalosporins, carbapenems
Mosaic PBPs
1) how does it happen?
2) in what bugs?
3) confers resistance to what abx
1) Genes encoding PBP become “mosaics” due to swapping with other bacteria DNA → change sequence of PBP
2) Strep pneumoniae and Neisseria gonorrhoeae
3) Reduces affinity for B-lactam abx - NOT ALL OR NONE phenomenon like it is in staphylococci
Mechanism of Vancomycin
binds peptidoglycan precursor molecule by binding to terminal D-alanine-D-alanine portion of the five-member peptide chain
Inhibits incorporation of precursor into peptidoglycan → cell death
“Stage 2” inhibitor
How can you get vancomycin resistance?
Modifying the target: plasmid acquisition changing terminal peptide
-Precursor peptidoglycan molecules altered so they terminate as D-ala-D-lactate → can’t bind vanco
- Carried on PLASMID
- VRE = Enterococci
Mechanism of macrolides
inhibit bacterial protein synthesis by binding 23S domain of 50S subunit of the bacterial ribosome → prevent peptide chain elongation
How can you get resistance to macrolides (3)
1) Modifying the drug (very rare)
2) Modifying the target (very common) - ERM
3) Prevent drug-target interaction with increased efflux pump
Efflux pumps for macrolides confer resistance to ________ but remain sensitive to _______
macrolides
clindamycin
erm
Modifies Macrolide drug target:
enzyme that dimethylates 23S domain → prevents macrolide binding to bacterial ribosome
Erm regulation can be inducible OR constitutive
erm expression is induced by __________
Constitutive expression of erm –> resistance to…
Inducible expression of erm –> resistance to…
MACROLIDES ONLY
Constitutive expression of erm –> resistance to clindamycin AND macrolides
Inducible expression of erm –> resistance to macrolides, sensitive to clindamycin
erm D-test tests what?
Used when your bacteria tests resistant to macrolide and susceptible to clindamycin
- Must differentiate if this is because it has inducible erm or efflux pump mode of resistance
- risk giving clindamycin and it turns out bug has inducible erm that transformed to constitutive erm and then that sucks
- Disc with erythromycin placed near disc containing clindamycin
D-test results:
Inducible erm if…
Efflux system if…
Inducible erm system → erythromycin will induce resistance to clindamycin (zones of clearing present around Erythromycin)
-D test positive → report resistance to erythromycin and clindamycin
Efflux system → erythromycin resistant, clindamycin sensitive
no blunting/clearing zone
What is the classic mechanism you get resistance to aminoglycosides?
Modifying the drug
Enzymes covalently modify aminoglycosides on transmissible plasmids
1) N-Acetylation
2) O-Nuecleotidylation
3) O-phosphorylation
Inactivates drug
Mechanism of quinolones
target bacterial enzymes DNA gyrase and DNA topoisomerase
How can you usually get resistance to quinolones
Modify the target
point mutations in subunits of DNA gyrase/topoisomerase at quinolone-resistance-determining region (QRDR)
Stepwise accumulation of mutations
Enzyme-DNA complex less sensitive to inhibition by reducing affinity for quinolone
