Antibiotics and Antimicrobial Resistance MDR and Antimicrobials in Sepsis MDR

Gram-Positive Bacteria Structure

Thick cell wall composed primarily of peptidoglycan

• This retains the crystal violet dye used in Gram staining, giving it a purple color

Gram-Negative Bacteria Structure

Thinner peptdoglycan layer surrounded by an outer membrane containing lipopolysaccharides

• Do not retain the crystal violet dye and appear pink during Gram staining

Bacterial Capsule

Prevents phagocytosis but good target for vaccines

Bacteria Slime Layer

Part of what allows bacteria to form biofilms

Bacteria Plasmid

Responsible for genes encoding virulence and resistance

Bacteria Fimbriae

Allow adherence

Bacterial Pilus

Aids in transmittance of resistance factors

Bacteria Flagella

Allow swimming

Antibiotics Included in the Beta-Lactam Class

Penicillins

Cephalosporins

Carbapenems

Beta-Lactams Mechanism of Action

Inhibit bacterial cell wall synthesis by binding to and inhibiting enzymes called penicillin-binding proteins (PBPs)

• Weakens the cell wall, fluid flows into the cell, leads to bacterial lysis

Beta-Lactam Spectrum of Activity

Highly effective against gram-positive bacteria

Varying activities against gram-negative bacteria due to differences in the permeability of their outer membrane

Mechanism of Action of Cephalosporins

Target enzymes called penicillin-binding proteins (PBPs) that are responsible for the cross-liking of peptidoglycan strands during cell wall synthesis

How do beta-lactamases work?

Break the B-lactam ring, rendering the antibiotic ineffective

Spectrum of First-Generation Cephalosporins

e.g. cefazolin, cephalexin

Good activity against gram-positive bacteria, including Staphylococcus aureus and Streptococcus pneumoniae

Have some activity against certain Gram-negative bacteria such as E. coli and Proteus mirabilis

Spectrum of Second-Generation Cephalosporings

e.g. cefuroxime, cefoxitin

Enhanced activity against Gram-negative bacteria compared to first-generation cephalosporins

Effective against a broader range of Gram-negative organisms such as Haemophilus influenze, Neisseria gonorrhoease, and some Enterobacteriaceae

Spectrum of Third-Generation Cephalosporins

e.g. ceftriaxone, ceftazidime

Even broader activity against Gram-negative bacteria

Spectrum of Fourth-Generation Cephalosporins (e.g. cefepime)

Extended spectrum of activity similar to third-generation cephalosporins against Gram-negative bacteria

Spectrum of Fifth-Generation Cephalosporins

e.g. ceftaroline, ceftobiprole

Broad-spectrum activity against both Gram-positive and Gram-negative bacteria, including MRSA

Specifically developed to target multidrug-resistant organisms

Aminopenicillins MOA

Work by targeting an enzyme called penicillin binding protein (PBP) and interfering with this cross-linking process, preventing the formation of a stable cell wall

• Leads to weakening and eventual lysis of the bacterial cell, rendering it unable to survive and replicate

Interference with cell wall synthesis makes the bacteria more susceptible to the effects of osmotic pressure, further contributing to their destruction

Spectrum of Aminopenicillins

More effective against gram-positive bacteria, which have a thinner peptidoglycan layer in their cell walls

Active against some gram-negative bacteria, but their effectiveness is limited due to the presence of an outer membrane that acts as an additional barrier

Often administered with beta-lactamase inhibitor to maintain efficacy in the presence of beta-lactamase and expand the spectrum against gram negative bacteria

Vancomycin MOA

Specifically targets the synthesis of peptidoglycan by binding to the precursor molecules called lipid II

• Once it binds to lipid II, it undergoes a conformational change that strengthens its interaction with the peptidoglycan precursor

• By binding to lipid II, vancomycin prevents the transglycosylase enzyme from adding new sugar subunits to the growing peptidoglycan chain, inhibiting cell wall synthesis

Also interferes with the transpeptidation step, which involves cross-linking the peptidoglycan chains through peptide bonds

• Without proper cross-linking, the cell wall loses its integrity and becomes weak, eventually leading to cell lysis and bacterial death

Vancomycin Spectrum

Primarily targets Gram-positive bacteria due to its large molecular size and limited ability to penetrate the outer membrane of Gram-negative bacteria

Aminoglycosides MOA

Interfere with bacterial protein synthesis by binding to the 30 subunit of the bacterial ribosome, epcifically to the 16S rRNA, and interact with proteins associated with the ribosome

• This inhibits initiation and elongation of protein synthesis, leading to misreading of the genetic code and production of faulty proteins

Interfere with the fidelity of the ribosome, causing the premature termination of protein synthesis

• The ribosome stops the translation process before the entire protein is synthesized, resulting in production of incomplete and non-functional proteins

Why do aminoglycosides have poor activity against anaerobic bacteria?

Due to their reliance on oxygen dependent transport into the bacterial cell

• Need O2 to form porins which are channels that allow the aminoglycoside into the cell to access the 30S subunit

Tetracyclines MOA

Inhibit bacterial protein synthesis by binding to the 30S subunit of the bacterial ribosome

• Prevents the bidning of aminoacyl-tRNA to the ribosome, leading to the inhibition of protein synthesis

Macrolides MOA

e.g. erythromycin and azithromycin

Interfere with bacterial protein synthesis by bindings to the 50S subunit of the bacterial ribosome, specifically the 23S rRNA

• Prevents the movement of the ribosome along the mRNA molecule during protein synthesis

• Inhibits the translocation step and prevents the elongation of the protein chain, thereby inhibiting the synthesis of bacterial proteins

Can also interfere with other steps of protein synthesis

• Can prevent initiation of protein synthesis by binding to the 50S subunit and blocking the association of the ribosome from the mRNA

• Have been shown to have immunomodulatory effects that contribute to their antibacterial activity

  • Can inhibit the production of pro-inflammatory molecules and modulate the immune response, which can help in the treatment of certain infections

Metronidazole Class

Nitroimidazole

Is metronidazole time or concentration dependent?

Time dependent

Metronidazole Spectrum

Anaerobic, no aerobic coverage

Lincosamides MOA

e.g. clindamycin

Bind to the 50S ribosomal subunit of bacterial ribosomes, interfering with protein synthesis

Once it enters the bacterial cell, it binds to the 23S rRNA within the 50S ribosomal subunit, specifically at a site adjacent to the peptidyl transferase center

• Prevents formation of peptide bonds between amino acids, inhibiting the elongation of the growing peptide chain during protein synthesis

Has been found to inhibit the early stages of bacterial protein synthesis by interfering with the initiation complex formation, preventing the association of the 50S and 30S ribosomal subunits, further hampering protein synthesis

Chloramphenicol MOA

Specifically targets the 50S subunit of the bacterial ribosome called the peptidyl transferase center

• Prevents formation of peptide bonds between amino acids, inhibiting the elongation of the growing peptide chain during protein synthesis

• Disruption of protein synthesis impairs bacterial growth, replication, and the production of essential proteins necessary for bacterial survival

Fluoroquinolones MOA

e.g. enrofloxacin, marbofloxacin, pradofloxacin

Target bacterial DNA replication and repair enzymes, specifically DNA gyrase and topoisomerase IV

• Bind ot the A subunit of DNA gyrase, hindering the process of negative supercoiling and the ParC subunit of topoisomerase IV, resulting in the buildup of positive supercoils

• Disruption of DNA processes leads to abnormal DNA structures, DNA strand breaks, and ultimately bacterial cell death

Sulfonamides and Trimethoprim MOA

Inhibit bacteria folate synthesis

Sulfonamides competitively inhibit the enzyme dihydropteroate synthase

Trimethoprim inhibits the enzyme dihydrofolate reductase

Combination of sulfonamides and trimethoprim creates a synergistic effect

Why don’t you want to give two 50S subunit antibiotics together?

They will compete with each other and cause competitive inhibition

Intrinsic Antimicrobial Resistance

The inherent ability of certain bacterial species to resist the effects of specific antibiotics due to natural characteristics

Acquired Antimicrobial Resistance

Occurs when bacterial acquire resistance traits through genetic changes or horizontal gene transfer

Antimicrobial Resistance through Mutation

Spontaneous genetic mutations can alter bacterial targets, such as enzymes or receptors, making them less susceptible to the action of antibiotics

Antimicrobial Resistance through Horizontal Gene Transfer

Bacteria can acquire resistance genes from other bacteria through processes like conjugation, transformation, or transduction

Antimicrobial Resistance through Conjugation

Transfer of resistance plasmids

Antimicrobial Resistance through Transformation

Allows bacteria to take up free DNA from the environment, enabling the acquisition of resistance genes

Antimicrobial Resistance through Transduction

Involves the transfer of genes via bacteriophages (viruses that infect bacteria)

What are the main mechanisms of antimicrobial resistance?

Target modificiation or bypass

Enzymatic inactivation of the antibiotic

Efflux pumps

Reduced permeability

Antibiotic modification

Antibiotic Resistance through Target Modification or Bypass

Bacteria can modify the target site of an antibiotic, reducing its binding affinity and rendering it ineffective

• Achieved by:

  • Mutation within the target site (seen with fluoroquinolone resistance and rifampin)

  • Enzymatic alteration of the binding site

    • e.g. tetracycline resistance

      • TetO and TetM interact with the ribosome and dislodge the tetracycline from its binding site

      • Tet M directly dislodges and releases tetracycline from the ribosome by an interaction between the domain IV of the 16S rRNA and the tetracycline binding site

      • This interaction alters the ribosomal conformation, preventing rebinding of the antibiotic

  • Complete replacement or bypass of the target site

    • e.g. alteration of penicillin-binding proteins (PBPs) in methicillin-resistance Sstaphylococcus pseudointermedius (MRSP)

      • MRSP has acquired the mecA gene which encodes for PBP2a, which has a significantly reduced affinity for beta-lactam antibiotics

        • When methicillin or other beta-lactam antibiotics are used, PBP2 a takes over the cell wall synthesis process, bypassing the inhibitory effects of the drug

Antibiotic Resistance through Enzymatic Inactivation of the Antibiotic

Bacteria can produce enzymes that chemically modify antibiotics, rendering them inactive

e.g. Production of beta-lactamases by various bacteria

• Beta-lactamases can hydrolyze and inactivate beta-lactam antibiotics

• To overcome this resistance mechanism, beta-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam, are used in combination with penicillin or aminopenicillins

  • These inhibitors have a structure similar to the beta-lactam ring of penicillin and they irreversibly bind to and inactivate the beta-lactamase enzymes

• Extended spectrum beta-lactamase (ESBL) are enzymes which hydrolyze extended spectrum cephalosporin such as 3rd generation cephalosporins and oxyiminomonobactam

Antibiotic Resistance through Efflux Pumps

Efflux pumps found in bacteria actively remove antibiotics from within the bacterial cell, preventing their build-up and maintaining low concentrations of drugs inside the cell

Five major families of efflux pumps

• Major facilitator superfamily (MFS)

• Small multidrug resistance family (SMR)

• Resistance-nodulation-cell-division family (RNS)

• ATP-binding cassette family (ABC)

• Multidrug and toxic compound extrusion family (MATE)

e.g. tetracycline resistance

• Tet efflux pumps, belonging to the MFS family, extrude tetracyclines using proton exchange as the energy source

e.g resistance to macrolides

• mef genes (mefA and mefE) encode the efflux pumps that expel macrolide antibiotics

• Mef pumps primarily found in S. pyogenes and S. pneumoniae, as well as other streptococci and gram-positive organisms

Antibiotic Resistance through Reduced Permeability

Bacterial decrease the permeability of their outer membrane

• Limits the entry of antibiotics within the bacterial cell and their ability to reach the intracellular target site

e.g. Gram negative bacteria such as Klebsiella pneumonia and Pseudomonas aeruginosa

Reduced permeability primarily affects hydrophilic molecules like B-lactams, tetracyclines, and certain fluoroquinolones, which relies on porins to traverse the outer membrane

• E. coli produces three major porins (OmpF, OmpC, and PhoE)

• P. aeruginoa produces OprD (protein D2)

• Changes in porin characteristics can occur through three general processes

  • A shift in the types of porins expressed

  • Alterations in the level of porin expression

  • Impairment of porin function

Changes in permeability often lead to low-level resistance and are frequently associated with other mechanisms of resistance

Antibiotic Resistance through Antibiotic Modification

Bacteria possess the ability to modify antibiotics through enzymatic reactions, leading to structural changes that render the drugs ineffective

Antibiotics most susceptible to enzymatic modifications often exert their mechanism of action by inhibiting protein synthesis at the ribosome level

Common biochemical reactions include

• Acetylation (aminoglycosides, chloramphenicol, streptogramins)

• Phosphorylation (aminoglycosides, chloramphenicol)

• Adenylation (aminoglycosides, lincosamides)

Resulting effect often involves steric hindrance, which reduces the drug’s affinity for its target

• Leads to higher MICs

e.g. presence of aminoglycoside modifying enzymes (AMEs) that covalently modify hydroxyl or amino groups within the aminoglycoside molecules

MIC

Minimum concentration to inhibit bacterial growth in a petri dish determine during susceptibility testing on a clinical sample

Breakpoint

Lowest concentration we can achieve for a given population of bacteria that will make it susceptible

Multidrug-Resistant (MDR) Bacteria

Bacteria that are resistant to at least one agent in three or more different classes of antibiotics

Extensively drug-resistant (XDR) Bacteria

Strains that are resistant to at least one agent in all but two or fewer antibiotic classes

Pandrug-Resistant (PDR) Bacteria

Resistant to all available antibiotics, including those considered as last-line treatments

Recommendations for Antibiotics in 2013 Surviving Sepsis Campaign Guidelines

Administration of IV antibiotics within the first hour of recognition of septic shock and severe septics

• Kumar A et al found administering appropriate antimicrobials within the first hour of hypotension caused by sepsis improved patient outcome with a survival rate of 79.9%

  • Survival decreased by 7.6% for every hour delay in administering appropriate antimicrobials until >36 hours had a 5% survival rate

Penicillins MOA

Inhibit cell wall synthesis, binds to penicillin binding proteins, cell lysis

Are penicillins static or cidal?

Cidal

Are penicillins time or concentration dependent?

Time

What are penicillins effective against?

Gram +, gram - cocci

Cephalosporins MOA

Inhibit cell wall syntehsis, binds to penicillin binding proteins, cell lysis

Are cephalosporins static or cidal?

Cidal

Are cephalosporins time or concentration dependent?

Concentration

What are cephalosporins effective against?

G+ and G-, anaerobes

Does not get enterococcus, pseudomonas

Trimethoprim-sulfonamides MOA

Inhibit folic acid synthesis

Are trimethoprim sulfonamides static or cidal?

Cidal

Are trimethoprim sulfonamides concentration or time dependent?

Concentration

What are trimethoprim sulfonamides effective against?

Gram + and gram -

Tetracyclines MOA

Inhibit protein synthesis: reversibly bind to 30S ribosomal subunit

Are tetracyclines static or cidal?

Static

Are tetracyclines concentration or time dependent?

Time

What are tetracyclines effective against?

G+/G-, anaerobes

Chloramphenicol MOA

Inhibit protein synthesis: reversibly bind to 50 S ribosomal subunit

Is chloramphenicol static or cidal?

Static

Is chloramphenicol concentration or time dependent?

Time

What is chloramphenicol effective against?

Gram + and gram -, anaerobes, intracellular organisms

Macrolides MOA

Inhibit protein synthesis: bind to 50S ribosomal subunit

Are macrolides static or cidal?

Static

Are macrolides time or concentration dependent?

Time

What are macrolides effective against?

G+, in general not G-

Aminoglycosides MOA

Inhibit protein synthesis: bind to 30S ribosomal subunit

Are aminoglycosides cidal or static?

cidal

Are aminoglycosides time or concentration dependent?

Concentration

What are aminoglycosides effective against?

Gram -, not good for G+ and anaerobes

Fluoroquinolones MOA

Inhibit bacterial DNA gyrase

Are fluoroquinolones cidal or static?

Cidal

Are fluoroquinolones time or concentration dependent?

Concentration

What are fluoroquinolones effective against?

Gram -, pseudomonas

Does not get anaerobes or Strep

Metronidazole MOA

Disrupts bacterial DNA through free radicals

Is metronidazole cidal or static?

Cidal

Is metronidazole time or concentration dependent?

Time

What is metronidazole effective against?

Anaerobes