Antibiotic Resistance

Alexander Fleming’s Other Experiment In 1945, a few years after Penicillin was In 1945, a few years after Penicillin was

introduced to the world, Fleming created a introduced to the world, Fleming created a strain of strain of Staphylococcus aureus Staphylococcus aureus that was that was resistant to penicillin. This was accomplished resistant to penicillin. This was accomplished by exposing by exposing s. aureuss. aureus to sub-optimal doses of to sub-optimal doses of Penicillin. Fleming warned the world about Penicillin. Fleming warned the world about antibiotic resistance during an interview with antibiotic resistance during an interview with the New York Times.the New York Times.

But the world did not listen…

Between 1945 and 1955, Penicillin was available Between 1945 and 1955, Penicillin was available to the public over the counter, without a to the public over the counter, without a prescription.prescription.

During those ten years the public did in vivo what During those ten years the public did in vivo what Fleming did in vitro:Fleming did in vitro:

People stopped taking Penicillin as soon as they People stopped taking Penicillin as soon as they began to feel better, they took Penicillin for viral began to feel better, they took Penicillin for viral infections, some reports even say Penicillin was infections, some reports even say Penicillin was used to treat male pattern baldness…used to treat male pattern baldness…

Resistance By the end of the 1950’s 50% or more of all By the end of the 1950’s 50% or more of all

Staphylococcus aureus Staphylococcus aureus strains were resistant to strains were resistant to penicillin.penicillin.

1975 MRSA (methicillin resistant 1975 MRSA (methicillin resistant Staphylococcus Staphylococcus aureusaureus).).

1988 VRE (Vancomycin resistant 1988 VRE (Vancomycin resistant enterococci) enterococci) .. 2002 VRSA (Vancomycin resistant 2002 VRSA (Vancomycin resistant Staphylococcus Staphylococcus

aureusaureus).). Today, 70% of all health care facility infections are Today, 70% of all health care facility infections are

resistant to one or more antibiotics.resistant to one or more antibiotics.

Antibiotic Diversity There is great diversity among antibiotics and among their There is great diversity among antibiotics and among their

molecular targets, here are a few examples:molecular targets, here are a few examples:

1. Penicillins, Cephalosporins, and 1. Penicillins, Cephalosporins, and Vancomycin- Target the bacterial cell Vancomycin- Target the bacterial cell wall.wall.

2. Sulfa drugs: growth factor analogues. 2. Sulfa drugs: growth factor analogues. 3. Quinolones: effect DNA girase. 3. Quinolones: effect DNA girase. 4. Tetracycline: effects the bacterial ribosome. 4. Tetracycline: effects the bacterial ribosome. 5. Rifampin: targets nucleic acid metabolism.5. Rifampin: targets nucleic acid metabolism.

All target processes specific to the bacteria without harming the host All target processes specific to the bacteria without harming the host

Eukaryote vs Prokaryote

Schematic Diagrams Of The Two Bacteria Cell Wall TypesSchematic Diagrams Of The Two Bacteria Cell Wall Types A Gram Positive Bacteria Cell and a Gram Negative Bacteria CellA Gram Positive Bacteria Cell and a Gram Negative Bacteria CellA: Peptidoglycan layerA: Peptidoglycan layer Polymer of sugars and amino acids for structure Polymer of sugars and amino acids for structure

and support. Note the difference in thickness between the two cells. and support. Note the difference in thickness between the two cells. This difference is what allows gram-positive and gram-negative This difference is what allows gram-positive and gram-negative bacteria to stain in separate colors.bacteria to stain in separate colors.

B: Cytoplasmic MembraneB: Cytoplasmic Membrane Encases the cell’s cytoplasm. Encases the cell’s cytoplasm. C: CytoplasmC: Cytoplasm Living cell substance holding all of the cell’s “organs.” Living cell substance holding all of the cell’s “organs.” D: Outer membraneD: Outer membrane Found only in gram-negative bacteria. It holds Found only in gram-negative bacteria. It holds

special chemicals toxic to animals. This membrane is highly resistant special chemicals toxic to animals. This membrane is highly resistant to many antibacterial chemicals.to many antibacterial chemicals.

Contents of Cell Wall May Protect Bacterial from Antibiotic

Crosslinking of Petidoglycans performed by transeptidases aka penicillin binding proteins (PBP)

Amino acids Amino acids

and amino sugarsand amino sugars

Penicillin binds PBP’s to prevent them from functioning in crosslinking

Mechanism of Penicillin

The Beta Lactam (4 membered ring with The Beta Lactam (4 membered ring with carbonyl) binds to the active carbonyl) binds to the active site of the site of the

transpeptidase transpeptidase enzyme enzyme

Once inside the enzyme’s active site, penicillin Once inside the enzyme’s active site, penicillin doesn’t leave, so it acts as a ‘suicide substrate.’ doesn’t leave, so it acts as a ‘suicide substrate.’

Beta Lactamases (from bacteria) have evolved to break beta lactam ring in penicillins for resistance

Vancomycin has no beta- lactam ring

Mechanism of Vancomycin

Forms multiple hydrogen bonds to the D-Forms multiple hydrogen bonds to the D-alanyl-D-alanine amino acids of the N-alanyl-D-alanine amino acids of the N-acetyl muramic acid, and N-acetyl acetyl muramic acid, and N-acetyl glucosamine monomers that are the ‘bricks’ glucosamine monomers that are the ‘bricks’ of the cell wall. This prevents the of the cell wall. This prevents the formation of the crosslinking in the walls of formation of the crosslinking in the walls of the bacteria. They are then unable to the bacteria. They are then unable to produce a wall and they will die.produce a wall and they will die.

Cell Wall Synthesis

3 Stages of cell wall synthesis3 Stages of cell wall synthesis 1. Synthesis of N-acetylmuramic acid 1. Synthesis of N-acetylmuramic acid

peptide synthesis.peptide synthesis. 2. Synthesis of N-acetylmuramic acid-N-2. Synthesis of N-acetylmuramic acid-N-

acetylglucosamine repeating chainsacetylglucosamine repeating chains 3. Cross linking of the MurNAc-GlcNac 3. Cross linking of the MurNAc-GlcNac

chains by transpeptidase.chains by transpeptidase.

Vancomycin Resistance

Vancomycin Resistance is conferred by a Vancomycin Resistance is conferred by a change from D-alanyl D-alanine to D-change from D-alanyl D-alanine to D-alanyl D-lactate or D-alanyl D-serine.alanyl D-lactate or D-alanyl D-serine.

This decreases the number of hydrogen This decreases the number of hydrogen bonds vancomycin can form with the N-bonds vancomycin can form with the N-acetylmuramic acid and N-acetyl acetylmuramic acid and N-acetyl glucosamine. In effect, increasing the Kglucosamine. In effect, increasing the Kdd..

Types of Antibiotic Resistance

1. Chemical Warfare, aka the beta 1. Chemical Warfare, aka the beta lactamase.lactamase.

2. Change in target protein structure.2. Change in target protein structure. 3. Antibiotic Efflux mechanism3. Antibiotic Efflux mechanism 4. Inaccessibility.4. Inaccessibility.

Efflux Mechanisms

Pump antibiotics out of the cell, decreasing Pump antibiotics out of the cell, decreasing the intercellular concentration of drug, and the intercellular concentration of drug, and increases the changes that the few increases the changes that the few molecules of the drug that remain inside the molecules of the drug that remain inside the cell can be degrade by enzymes (especially cell can be degrade by enzymes (especially beta lactamses).beta lactamses).

Inaccessibility

If a drug can’t access it’s target, it can’t work.If a drug can’t access it’s target, it can’t work. Example: Gentamicin effects the bacterial Example: Gentamicin effects the bacterial

ribosome, and is actively transported across the ribosome, and is actively transported across the gram negative cell membrane through porin gram negative cell membrane through porin channels. A mutation to one of the proteins channels. A mutation to one of the proteins involved in this pathway slows entrance of involved in this pathway slows entrance of gentamicin into the cell, therefore resulting in gentamicin into the cell, therefore resulting in resistance.resistance.

Combating Antibiotic Resistance

CDC’s approach to combat antibiotic resistance:CDC’s approach to combat antibiotic resistance: 1. Vaccination1. Vaccination 2. More appropriate use of and attention to indwelling 2. More appropriate use of and attention to indwelling

catheters.catheters. 3. Early involvement of Infectious Disease Specialists3. Early involvement of Infectious Disease Specialists 4. Choosing more appropriate/specialized antibiotics 4. Choosing more appropriate/specialized antibiotics

(using ‘broad spectrum drugs as little as possible’)(using ‘broad spectrum drugs as little as possible’) 5. Appropriate use of prophylactic antibiotics for surgery. 5. Appropriate use of prophylactic antibiotics for surgery. 6. Stricter infection control, especially handwashing!6. Stricter infection control, especially handwashing!

What you can do:

Don’tDon’t ask your doctor for antibiotics. ask your doctor for antibiotics. If prescribed antibiotics take them for the If prescribed antibiotics take them for the

full course of treatment, and at equally full course of treatment, and at equally spaced time intervals as prescribed by your spaced time intervals as prescribed by your doctor.doctor.

WASH YOUR HANDS!!!!WASH YOUR HANDS!!!!

Questions?

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References: Medical BiochemistryMedical Biochemistry, N.V. Bhagavan. Jones and Bartlett , N.V. Bhagavan. Jones and Bartlett

Publishing, 1992. Pages 345-350.Publishing, 1992. Pages 345-350. Goodman and Gilman’s The Pharmacological Basis of Therapeutics Goodman and Gilman’s The Pharmacological Basis of Therapeutics

1111thth edition. edition. L. Brunton, J. Lazo, and K. Parker. McGraw-Hill L. Brunton, J. Lazo, and K. Parker. McGraw-Hill Medical Publishing Division, 2006. Chapters 42, 43, 44.Medical Publishing Division, 2006. Chapters 42, 43, 44.

Cdc.govCdc.gov Textbookofbacteriology.netTextbookofbacteriology.net Also, special thanks to Debra Dunaway-Mariano, PhD professor of Also, special thanks to Debra Dunaway-Mariano, PhD professor of

chemistry at UNM. (if you’re interested in this topic you might want chemistry at UNM. (if you’re interested in this topic you might want to take her ‘boilogical chemistry’ course, it was a big help in preparing to take her ‘boilogical chemistry’ course, it was a big help in preparing for this presentation, as well as being a really cool class.)for this presentation, as well as being a really cool class.)

Additional SlidesAdditional Slides

Beta Lactamase

Hydrolyzes the Carbonyl portion of the beta lactam ring. Hydrolyzes the Carbonyl portion of the beta lactam ring. The ring breaks because of strain, and the business end The ring breaks because of strain, and the business end of penicillin is no longer able to bind the transpeptidase of penicillin is no longer able to bind the transpeptidase enzyme. Kenzyme. Kdd actually approaches infinity. actually approaches infinity.

Examples: Examples: Haemophylis influenzaeHaemophylis influenzae secretes a beta secretes a beta lactamase, making these strains resistant to all but lactamase, making these strains resistant to all but methicillin.methicillin.

However, it has been discovered that clavulanic acid acts However, it has been discovered that clavulanic acid acts as a beta-lactamase inhibitor. The antibitoic Augmentin as a beta-lactamase inhibitor. The antibitoic Augmentin is actually amoxicillin combined with clavulanic acid.is actually amoxicillin combined with clavulanic acid.

Clavulanic acidBeta-lactamase inhibitor.