Introduction to Infectious Diseases Part 2 Flashcards
Gram stain used to
differentiate bacteria
Gram positive bacteria
appear purple due to thick peptidoglycan cell wall
Gram negative bacteria
appear red/pink due to thin peptidoglycan cell wall
Atypical bacteria
do not stain using gram-stain
Acid-fast bacilli
resistant to acids/ethanol based decolorization procedures
ex. mycobacterium species
Gram positive - cocci anaerobic
anaerobic: peptococcus peptostreptococcus
Gram positive - cocci aerobic - clusters
clusters (catalase +) –> coagulase (+) —> staphylococcus aureus
clusters (catalase +) –> coagulase (-) –> CoNS (staphylococcus epidermidis)
Gram positive - cocci aerobic - pairs/chains
pairs/chains (catalase -) –> alpha-hemolysis –> streptococcus pneumoniase, viridans streptococci
pairs/chains (catalase -) –> beta-hemolysis –> streptococcus pyogens (group A), streptococcus afalactiae (group B)
pairs/chains (catalase -) –> gamma-hemolysis (nonhemolytic) –> enterococcus faecium, enterococcus faecalis
Gram positive - bacilli anaerobic
anaerobic –> spore forming –> clostridium spp, clostridioides difficile
anaerobic –> non-spore forming –> cutibacterium, actinomyces
Gram positive - bacilli aerobic
aerobic –> spore forming –> bacillus spp
aerobic –> non-spore forming –> corynebacterium, lactobacillus spp, listeria monocytogenes
Hemolysis patterns
alpha, beta, gamma
Gram positive morphology
most medically important pathogens are cocci rather than bacilli
gram positive bacilli should be interpreted within clinical context
Gram positive colony clustering
staphylococcus form clusters
streptococci and enterococci appear in pairs or chains
Gram positive biochemistry testing
catalase test: staphylococci from streptococci
coagulase test: staphylococcus aureus from coagulase-negative staphylococcus
Gram positive agar appearance
oral flora: alpha-hemolytic
skin, pharnyx, genitourinary: beta-hemolytic
gastrointestinal: gamma-hemolytic
Gram negative - aerobic
cocci –> neisseria spp, moraxella catarrhalis
coccobacilli –> haemophilus
Gram negative - anaerobic
cocci –> veillonella spp
bacilli –> bacteroides spp, fusobacterium spp, prevotella spp
Gram negative - aerobic bacilli
aerobic bacilli –> enterobacterales –> lactose fermenters (oxidase negative) –> citrobacter spp, enterobacter spp, E. coli, klebsiella spp
aerobic bacilli –> enterobacterales –> non-lactose fermenters –> morganella morganii, proteus spp, providencia spp, salmonella spp, serratia marcescens, shigella spp
Gram negative - aerobic bacilli (non enterobacterales)
aerobic bacilli –> lactose-fermenters (oxidase positive) –> aeromonas hydrophila, pasteurella multocida, vibrio cholerae
aerobic bacilli –> non-lactose fermenters –> pseudomonas spp, acinetobacter spp, alcaligenes spp, burkholderia cepacia, stenotrophomonas maltophilia
aerobic bacilli –> fastidious –> campylobacter, helicobacter, bartonella, HACEK organisms
Gram negative atypical
chlamydia pneumoniae, chlamydia trachomatis, legionella pneumophila, mycoplasma pneumoniae
Gram negative spirochetes
treponema pallidum, borrelia burgdorferi
Gram negative morphology
bacilli predominate pathogen
Gram negative lactose fermentation
helps identify enterobacterales from non-fermenting gram negative rods
oxidase test helps distinguish between enteric vs non-enteric lactose fermenters
Gram negative fastidious organisms
slow growers, require special supplemental media
Gram positive and gram negative cell overview
gram positive: thick peptidogylcan wall, semi-permeable membrane, beta-lactamases located in extracellular space
gram negative: thin peptidoglycan wall, contain porins to let drugs through, beta-lactamases in periplasmic space, contain lipopolysaccharides (endotoxins)
Bacterial structure composed of
cytoplasmic membrane, peptidoglycan layer, outer membrane, periplasmic space
Cytoplasmic membrane
acts as selective barrier
certain drugs must pass through to reach target site
Peptidoglycan layer (cell wall)
GP: thick, GN: thin
permeability barrier for large molecules
PBPs: proteins essential for cell-wall synthesis
Outer membrane (gram-negative)
lipopolysaccharides: mediator of immune response and sepsis
porins: hydrophilic chanels the permit diffusion of essential nutrients and small hydrophilic molecules
Periplasmic space
compartment between cell membrane and cell wall (GP) or between cell membrane and outer membrane (GN)
vital for bacterial protein secretion, folding, quality control; acts as reservoir for virulence factors
Penicillin binding proteins (PBPs)
these are enzymes vital for cell wall synthesis, cell shape, and structural integrity - transpeptidases, carboxypeptidases, endopeptidases
differ from one bacterial species to another
binding to PBPs 1A, 1B, 2, and 3 result in bactericidal effect
transpeptidase most important PBP - catalyzes the final cross linking in the peptidoglycan structure
Intrinsic resistance
always resistant to given antibiotic (naturally resistant)
MOA: absence of target site, bacterial cell impermeability
ex. cephalosporins vs enterococci; beta-lactams vs mycoplasma
Acquired resistance
initially susceptible but develop resistance due to some mechanism
MOA: mutation in bacterial DNA (spontaneously vs selective pressure); asquisition of new DNA (chromosomal or extrachromosomal plasmid)
ex. stable depression of AmpC; acquisition of KPC gene in GNRs
Acquired resistance - plasmids
self-replicating, extrachromosomal DNA; transferable between organisms
Acquired resistance - transposons
genetic elements capable of translocating from one location to another; move from plasmid to chromosome or vice versa
Acquired resistance - phages
viruses that can transfer DNA from organism to organism
Acquired resistance - conjugation
direct contact or mating via sex pili
most common
Acquired resistance - transduction
transfer of genes between bacteria by bacteriophage
Acquired resistance - transformation
transfer or uptake of “free floating” DNA from the environment
Mechanisms of antibiotic resistance
4 different mechanisms:
1. altered cell wall protein/decreased porin production
2. efflux pump (pump it out)
3. drug-inactivating enzyme
4. modified drug target
don’t let it in, pump it out, chew it up, change it up
Enzymatic inactivation: beta-lactamase
enzymes that hydrolyze beta-lactam ring by splitting amide bond: inactivates drugs
two classification systems: ambler class, bush-jacoby medeiros
Two types of beta-lactamase
serine beta-lactamase: serine residue at active site
metallo-beta lactamases: zinc residue at active site
Serin beta-lactamase MOA
serine active site creates acyl-enzyme complex through acylation –> through de-acylation, we open up the beta-lactam ring –> opening it up inactivates the beta-lactam antibiotic
restores beta-lactamase
Metallo-beta-lactamase MOA
open up the ring and inactivate the beta-lactam antibiotic
Beta-lactamase types
extended spectrum beta lactamases (ESBL)
serine carbapenemases
metallo-beta-lactamases
cephalosporinases
OXA-type
Extended spectrum beta lactamases (ESBL) enzyme example
CTX-M-15
hyrdolyze narrow and extended spectrum beta-lactam antibiotics
(bacteria that carry these enzymes are resistant to these antibiotics)
Serine carbapenemases enzyme example
KPC-1, KPC-2, KPC-3
hydrolyze carbapenems, cephalosporins, and penicillins
Metallo-beta-lactamases enzyme example
NDM-1
hydrolyze carbapenems
Cephalosporinases enzyme example
Amp-C
inducible
OXA-type enzyme example
OXA-48
hydrolyze oxacillin, oxyimino beta-lactams, and carbapenems
Ambler class A: ESBLs
Plasmid-mediated enzymes that hydrolyze most penicillins, cephalosporins, and monobactams - Do not inactivate non-beta-lactam agents (eg. ciprofloxacin, doxycycline, gentamicin); Organisms with ESBL genes often harbor additional resistance genes
CTX-M enzyme most common: Most prevalent in Escherichia coli, Klebsiella pneumoniae/oxytoca, and Proteus mirabilis; Ceftriaxone non-susceptibility
Treatment of choice: Carbapenems (meropenem, imipenem, doripenem, ertapenem) Piperacillin/tazobactam an option for urinary source only
Ambler class A: carbapenemase
Most frequent cause of Carbapenem-resistant Enterobacterales (CRE) - Resistance to whole beta-lactam class
Klebsiella pneumonia carbapenemase (KPC) - Plasmid-mediated enzyme; KPC-2 & KPC-3 most common variants; Found in: K. pneumoniae, K. oxytoca, E.coli, E. cloacae, E. aerogenes, P. mirabilis
Treatment options:
◦ β-lactam: ceftazidime/avibactam, meropenem/vaborbactam,
imipenem/cilastatin/relebactam
◦ Non β-lactam: Plazomicin, eravacycline, omadacycline
Ambler class B: metallo-beta-lactamases
Confer resistance to all β-lactams except monobactams (aztreonam) - Harbor additional antibiotic-resistance genes to other antimicrobial classes
Examples: New Delhi MBL (NDM) - Present in P. aeruginosa, Acinetobacter spp, and Enterobacterales
Treatment options: Limited!; Not inhibited by any current β-lactamase inhibitors; Cefiderocol; aztreonam + ceftazidime/avibactam
Ambler class D: OXA-type
Large heterogenous group often accompanied by other beta-lactamase classes (e.g., co-expression of ESBLs and AmpC)
Primarily found in Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa and some Enterobacterales, such as Klebsiella pneumonia
Treatment options: Extremely limited; Cefiderocol; Sulbactam/durlobactam
Ambler class C: AmpC
Three different mechanisms:
◦ 1) inducible via chromosomally encoded AmpC genes
◦ 2) Non-inducible chromosomal resistance via mutations (rare)
◦ 3) Plasmid-mediated resistance
Not inhibited by older β-lactamase inhibitors (clavulanic acid, tazobactam, sulbactam); Inhibited by newer β-lactamase inhibitors: avibactam, vaborbactam, relebactam
Found in: Hafnia alvei, Enterobacter cloacae, Citrobacter freundii, Klebsiella aerogenes, Yersinia enterocolitica (HECK-YES); Also in Serratia marcescens, Morganella morganii, Aeromonas hydrophilaà HECK-YES Ma’aM
Referred to as AmpC or inducible organisms
AmpC induction mechanism
Transient elevation in enzyme production in the presence of certain beta-lactam agents
Initially, gene for beta-lactamase production is repressed –> inducer –> gene derepressed –>
increased beta-lactamase production
Remove inducer –> gene repressed –> beta-lactamase production back to low level
Genetic mutation –> gene derepressed –> stable derepression –> high level beta-lactamase production continuously
Different beta-lactams induce AmpC beta-lactamases to varying degrees
AmpC inducers
ceftriaxone is a weak inducer, with high susceptibility to ampC hydrolysis
cefepime is a weak inducer, with low susceptibility to ampC hydrolysis
carbapenems (imipenem, meropenem, ertapenem) are strong inducers, with low susceptibility to ampC hydrolysis
cefepime is 1st line –> carbapenems
Selection and treatment of stably derepressed mutants
Treatment: Cefepime(1st-line), Carbapenems, Non-β-lactams (Fluoroquinolones, trimethoprim/sulfamethoxazole, tetracyclines)
Enzymatic inactivation: aminoglycoide-modifying enzymes
Most common method of aminoglycoside resistance
3 mechanisms: Acetylation, Nucleotidylation, Phosphorylation
Modify aminoglycoside structure by transferring the indicated chemical group to a specific side chain –> impairs cellular uptake and/or binding to ribosome
Nomenclature based on chemical group transferred and site of transfer
Bifunctional enzyme –> acetylation and phosphorylation of aminoglycoside
Seen in Enterococci –> high level of gentamicin resistance
Altered target site: cell wall precursor
Mechanism of vancomycin resistance in Enterococci species
Vancomycin binds to D-Alanine-D-Alanine terminus of peptidoglycan precursors: Inhibits cell wall synthesis
Resistance alters D-Ala-D-Ala to D-Ala-D-Lac or D-Ala-D-Ser: Mediated by VanA or VanB gene –> most common; Produces vancomycin-resistant enterococcus (VRE)
Treatment: Daptomycin or linezolid
Altered target site: penicillin binding proteins (PBPs)
Alterations in PBPs leads to β-lactam resistance
Due to decreased affinity of PBPs for antibiotic or change in amount of PBP produced by bacteria - Addition of β-lactamase inhibitor is ineffective in restoring activity of β-lactam antibiotic
Methicillin-resistant Staphylococcus aureus (MRSA): Resistance due to expression of mecA gene (mecA + = PBP2A + = MRSA)
◦ Encodes for PBP2A –> low affinity for beta-lactam antibioticsà resistance to β-lactam class with 2 exceptions (see below)
Treatment: Ceftaroline, Ceftobiprole; vancomycin, daptomycin, linezolid
Streptococcus pneumoniae: Alteration in PBP confers penicillin and cephalosporin resistance
Altered target site: ribosomal target
Responsible for macrolide resistance in S. pneumoniae: ermB gene –> cross resistance with clindamycin
Aminoglycoside resistance in Gram negatives Clindamycin resistance
Altered target site: DNA gyrase/topoisomerase IV
Responsible for Fluoroquinolone (ciprofloxacin, levofloxacin) resistance in Gram-negative and S. pneumoniae
Efflux pumps
actively transport antibiotics OUT of periplasmic space: overexpression can lead to high-level of resistance; Efflux is important for a range of antibiotic classes
Important resistance mechanism for P. aeruginosa against carbapenems & S. pneumoniae against macrolide antibiotics
Porin channels
are hydrophilic diffusion channels
Rate of antibiotic diffusion depends on porin & antibiotic physiochemical characteristics - smaller, more hydrophilic antibiotics pass easier than larger, hydrophobic antibiotics
Mutations result in loss of specific porins –> leads to antibiotic resistance - Most commonly seen with Enterobacterales and carbapenem-resistant P. aeruginosa
Cidal vs static
In vitro terms not based on linkage to any predictive ability in vivo
Not always a clear distinction: some bactericidal drugs may be static against certain organism and vice versa
Bactericidal agents still preferred for certain infections (meningitis, endocarditis)
Important to optimize dose based on patient factors, site of infection, and organism
Concentration dependent
Exert effect when concentrations well above organism’s MIC: ↑Cmax/MIC = greater killing –> correlates with increased AUC
Some agents, such as fluoroquinolones and aminoglycosides exhibit PAE: Gram-positive and Gram-negative bacteria
Fluoroquinolones (Levofloxacin, Ciprofloxacin): Concentration-dependent bactericidal activity –> fAUC0-24/MIC
Aminoglycosides (Gentamicin, Tobramycin, Amikacin): Concentration-dependent bactericidal activity –> Cmax/MIC; Optimal dosing achieved through TDM and use of high dose extended interval
Time-dependent
All β-lactam antibiotics (penicillin, cephalosporin, carbapenem, monobactam)
Time that free drug concentrations remain above MIC correlates with clinical and
microbiological outcomes (fT>MIC)
fT>MIC Penicillin: 50%
fT>MIC Cephalosporin: 60-70%
fT>MIC Carbapenem: 40%
Antibacterial properties: Not rapidly bactericidal; Time-dependent bactericidal activity; Little to no PAE
Beta-lactam dosing optimization
Maximize fT>MIC (as a % of dosing interval): Gram-negatives: Carbapenems: ≥40%; Penicillins: ≥50%; Cephalosporins: ≥ 60%; Gram-positive: ≥40-50%
Strategies to maximize fT>MIC: Increase dose, same interval (1g Q8h vs. 2g q8h); Same dose, shorter interval (1g Q12h vs. 1g Q6h); Continuous infusion: Stability issues; need dedicated IV line
Prolonged infusions: Infuse dose over 3-4 hours; Provides longer T>MIC than traditional infusions
AUC/MUC dependent (vancomycin)
Time-dependent bactericidal activity; very long PAE for Gram-positive organisms
PD Target: AUC0-24/MIC
Goal AUC0-24/MIC ≅ 400-600
Prolonged, elevated AUC0-24/MIC ≥ 600-700 mg*h/L is a risk factor for nephrotoxicity
Dosing is patient-specific and achieved through TDM using Bayesian programs
Aminoglycosides PK/PD
concentration dependent
predictive PK/PD parameter: peak/MIC, AUC/MIC
cidal
Beta-lactams PK/PD
time-dependent
predictive PK/PD parameter: T>MIC
cidal
Daptomycin PK/PD
concentration-dependent
predictive PK/PD parameter: AUC/MIC, peak/MIC
cidal
Fluoroquinolones PK/PD
concentration dependent
predictive PK/PD parameter: AUC0-24/MIC
cidal
Vancomycin PK/PD
time dependent
predictive PK/PD parameter: AUC0-24/MIC
cidal (slowly)
