Station 5 - Antibiotics Resistance

Question 1

Describe the concepts of selective toxicity in relation to antibiotics

Answer 1

Principles of Antibiotics as Therapeutic Agents:

1) Selective Toxicity:

• Refers to the ability of a drug to selectively kill or inhibit the growth of microbial targets while causing minimal or no harm to the host
• Due to the differences in structure and metabolic pathways between host and pathogen
• Harm microorganisms, not the host
• Target in microbe, not host (if possible)
• Difficult for viruses (intracellular), fungi and parasites
• Variation between microbes
• Effect on commensals

2) Therapeutic Margin: Active dose (MIC) vs Toxic effect

Selective toxicity examples:

1) Beta-Lactam Antibiotics (Penicillins):

• They target the bacterial cell wall, a structure that human cells lack
• Inhibit enzymes (penicillin-binding proteins) involved in peptidoglycan synthesis, a key component of the bacterial cell wall; osmotic lysis

2) Glycopeptide Antibiotics (Vancomycin):

• Glycopeptides inhibit cell wall synthesis via binding directly to peptidoglycan precursors
  -Preventing their incorporation into the growing cell wall

3) Macrolide Antibiotics (Erthromycin):

• Target the bacterial ribosome, specifically the 50S subunit, and inhibit protein synthesis
• Bacterial ribosomes are 70S (50S + 30S subunit) whereas human ribosomes are 80S (60S +40S subunit)
• The difference in ribosome structure allows macrolides to selectively inhibit bacterial protein synthesis

4) Aminoglycosides (Gentamicin, Streptomycin):

• Target 30S subunit of the bacterial ribosome, interfering with protein synthesis

Question 2

Define the biological and pharmaceutical origins of antibiotics

Answer 2

Biological Origins:

• compounds produced by microorganisms; advantage to the producing organism by inhibiting the growth of competitors
• Bacterium-derived antibiotics: Streptomyces (actinomycetes genus)
• Fungus-derived antibiotics: Penicillin

Pharmaceutical Origins:

• Chemically modify naturally occurring antibiotics to improve its efficacy, safety, or spectrum of activity
• Semi-synthetic antibiotics: methicillin, ampicillin, and amoxicillin are chemically modified semi-synthetic penicillin that are more effective against a broader range of bacteria or more resistant to bacterial mechanisms of resistance
• Fully synthetic antibiotics: fluoroquinolones (ciprofloxacin, levofloxacin), are fully synthetic, designed and created entirely in the lab. Developed based on the knowledge of bacterial targets such as DNA gyrase and topoisomerase IV

Question 3

Define the terms: bactericidal, bacteriostatic, broad and narrow spectrum, MIC

Answer 3

1) Bactericidal:

• Directly kill bacteria by interfering with bacterial cell wall synthesis, DNA replication, or protein production, e.g. penicillin and ciprofloxacin

2) Bacteriostatic:

-Inhibit the growth or multiplication of bacteria by interfering with bacterial protein production, DNA replication, and bacterial cellular metabolism

3) Broad Spectrum:

• Effective against a wide range of both Gram-positive and Gram-negative bacteria
• Useful when it is not possible to determine the specific type of bacterium causing an infection
  also used in treating mixed infections and preventing infections in patients with weakened immune systems. E.g. amoxicillin and ciprofloxacin

4) Narrow Spectrum:

• Effective against a select group of bacterial types, only gram +ve/-ve or only against a specific genus of bacteria
• Cause less disruption to the normal microbiota. E.g. dicloxacillin and azithromycin

MIC (Minimum Inhibitory Concentration):

• lowest concentration of an antimicrobial agent that prevents the visible growth of a microorganism in vitro (in a test tube or petri dish)
• The MIC is used in diagnostic laboratories to determine the susceptibility of microorganisms to different antibiotics
• Antibiotics with low MICs are more effective than those with higher MICs
• This helps clinicians choose the most effective antibiotic to treat a particular infection

Question 4

Describe the categories of antibiotics, their modes of action, and targets, giving a named example of an antibiotic for each category

Answer 4

1) Cell Wall Synthesis Inhibitors:

• preventing the formation of the bacterial cell wall; osmotic lysis
  E.g. Penicillin - inhibit the final cross-linking of peptidoglycan chains

2) Protein synthesis inhibitors:

• Inhibit bacterial protein synthesis, affecting the bacterial ribosomes without harming human ribosomes due to structural differences
  E.g. Erythromycin - bind to the 50S subunit, preventing elongation

3) Folic Acid Synthesis Inhibitors:

• These antibiotics interfere with the synthesis of folic acid, a critical nutrient for bacteria; inhibiting DNA, RNA, and protein synthesis
  E.g. Sulfonamides: structural analogues of para-aminobenzoic acid (PABA), a precursor for folic acid synthesis

4) Metabolic Pathway Inhibitors:

• Hinder certain metabolic pathways vital to the bacteria’s survival
  E.g. Quinolones (e.g., ciprofloxacin): inhibit bacterial DNA gyrase and topoisomerase IV, which are crucial enzymes for bacterial DNA replication, transcription, repair, and recombination

5) Cell Membrane Disruptors:

-Disrupting the bacterial cell membrane, causing cell contents to leak out, leading to cell death
E.g. Polymyxins: bind to the bacterial cell membrane, altering its structure and leading to increased permeability, leakage of cellular contents, and cell death, particularly Gram-negative

Question 5

Explain in broad principles when antibiotics are used and how they are delivered

Answer 5

When Antibiotics are used:

1) Treatment of bacterial infections:

• Antibiotics are typically prescribed when a patient has a confirmed bacterial infection, such as pneumonia, UTI, skin infections
• Antibiotic selected is usually based on the type of bacteria causing the infection, as determined by microbiological testing, along with the patient’s overall health status

2) Prophylactic use:

• Antibiotics can also be used to prevent infections, particularly in certain at-risk populations
• This includes patients undergoing surgery (to prevent post-operative infections), patients with certain types of heart disease before dental procedures (to prevent bacterial endocarditis), and people exposed to a person with a bacterial infection that can easily spread, such as meningitis

3) Empiric Therapy:

• When the exact cause of an infection is unknown, but a bacterial cause is suspected, empiric therapy may be started
• Based on the types of bacteria that are most likely causing the infection and local resistance patterns

How Antibiotics are Delivered:

1) Orally:

• In the form of tablets, capsules, or liquid suspensions. The medication is then absorbed through the digestive system

2) Intravenous (IV):

• For severe infections or in cases where the patient cannot take oral medications
• This allows for rapid and complete absorption of the drug into the bloodstream

3) Topical:

• For skin and eye infections, antibiotics may be applied directly to the affected area in the form of creams, ointments, or eye drops

4) Intramuscular:

• Some antibiotics, such as penicillin, may be given as an intramuscular injection. This is less common than oral or intravenous routes

5) Inhalation:

• Certain antibiotics can be given by inhalation, especially for respiratory infections in cystic fibrosis patients

Question 6

Global Treat

Answer 6

1) Increased Mortality and Morbidity:

• first-line antibiotics, which are typically safer and more affordable, might no longer be effective
• Patients might have to resort to second and third-line antibiotics, which could be more toxic, less effective, and more expensive
• E.g. the rise in methicillin-resistant Staphylococcus aureus (MRSA) infections, which are more difficult to treat and can be fatal

2) Treat to Modern Medicine:

• Surgeries and procedures such as organ transplants, joint replacements, chemotherapy and premature infants rely on antibiotics to prevent and control the risk of bacterial infections
• These procedures could become high-risk treatments due to fear of untreatable post-operative infections

3) Economic Burden:

• Increased healthcare costs due to prolonged illness, extended hospital stays, the necessity for more expensive drugs, and additional tests and procedures

4) Increased Spread of Disease:

• Resistance can cause bacterial infections to be more difficult to control, leading to faster and wider spread of the disease
• I.e. multi-drug resistant tuberculosis (MDR-TB) is much harder to treat; resistant to the two most potent TB drugs
• Requiring longer treatment times and more expensive and toxic drugs

5) Emergence of ‘Superbugs’:

• ‘Superbugs’ are bacteria that have become resistant to several types of antibiotics
  strains that are nearly impossible to treat
• E.g. Carbapenem-resistant Enterobacteriaceae (CRE) are resistant to all or nearly all antibiotics, making them difficult to treat

6) Overuse in Agriculture:

• The use of antibiotics in livestock to promote growth and prevent diseases in crowded or unsanitary conditions contributes significantly to the emergence of antibiotic resistance
• These antibiotics can enter the human body through the food chain, promoting the development of antibiotic resistance

Question 7

Describe the main mechanisms of antibiotic resistance

Answer 7

1) Drug Inactivation or Modification:

• The bacteria produce enzymes that can chemically modify the antibiotic, rendering it ineffective
• E.g. production of beta-lactamase enzymes by Staphylococcus aureus and E. coli
• These enzymes cleave the beta-lactam ring, a common structure in penicillins and cephalosporins, rendering the drugs ineffective, further divided into two types:
  Adenylating enzymes: They add an AMP group to the antibiotic, changing its structure and function
  Phosphorylating enzymes: They add a phosphate group to the antibiotic, inactivating it

2) Alteration of Target Site:

• The target of an antibiotic can undergo changes, which prevents the antibiotic from binding efficiently
  These changes can occur due to random mutations or horizontal gene transfer
• E.g. MRSA alters its penicillin-binding protein (PBP) target to a variant with lower affinity for beta-lactam antibiotics, thus evading the action of methicillin and other similar drugs

3) Efflux Pumps:

• Bacteria can possess transmembrane proteins known as efflux pumps, which actively transport antibiotics and other toxins out of the cell
• These pumps can have broad specificity, meaning they can pump out a range of different antibiotics, contributing to multi-drug resistance
• E.g. the AcrAB-TolC efflux pump in E.coli can expel a broad range of antibiotics

4) Bypassing the Antibiotic Effect:

• Bacteria can develop alternative metabolic pathways that circumvent the inhibitory effect of the antibiotic
• E.g. sulfonamide-resistant bacteria can use alternative sources of folate, bypassing the need for the enzyme that is targeted by the antibiotic

5) Impermeability or Decreased uptake:

• Bacteria can alter the permeability of their outer membranes, making it more difficult for antibiotics to penetrate the cell
• This is often seen in Gram-negative bacteria, which have an additional outer membrane that can be modified to prevent the entry of antibiotics
• E.g. Pseudomonas aeruginosa is resistant to many antibiotics because of the low permeability of its outer membrane and the action of efflux pumps

Question 8

Explain the different mechanisms for penicillin resistance

Answer 8

1) Beta-Lactamase Production:

• Beta-lactamase enzymes break the beta-lactam ring, which is the core chemical structure of penicillins and other beta-lactam antibiotics
• When this ring is intact, it interferes with bacterial cell wall synthesis by binding to penicillin-binding proteins (PBPs)
• Once this ring is cleaved by the enzyme, the antibiotic can no longer inhibit these proteins and loses its antimicrobial activity
• Beta-lactamase enzymes are often plasmid-encoded, which means they can be easily transferred between bacteria through horizontal gene transfer

2) Alteration of Penicillin-Binding Proteins (PBPs):

• PBPs are integral membrane proteins that participate in the final stages of assembling the bacterial cell wall, and are the primary targets of beta-lactam antibiotics
• Some bacteria, however, have developed modified PBPs with lower affinity for beta-lactam antibiotics
• This means the antibiotic cannot bind effectively to these altered PBPs and therefore cannot inhibit cell wall synthesis
• E.g. MRSA acquires mecA gene that codes for PBP2a, an altered PBP with reduced affinity for beta-lactams

3) Efflux Pumps:

• Some bacteria possess efflux pumps in their cellular membranes that actively expel certain antibiotics out of the cell
• These pumps can significantly reduce the intracellular concentration of antibiotics, decreasing their ability to reach effective levels seen in Gram-negative bacteria due to the presence of an outer membrane, and it often contributes to multidrug resistance

4) Reduced Permeability:

• Gram-negative bacteria possess an outer membrane that acts as a barrier to many toxic substances, including antibiotics
• This membrane is selectively permeable due to the presence of porins, protein channels that allow small, hydrophilic molecules to cross
• By altering the structure or quantity of these porins, bacteria can decrease the permeability of the membrane to certain antibiotics, including penicillins, reducing their concentration inside the cell

Question 9

Differentiate acquired from intrinsic antibiotic resistance

Answer 9

Intrinsic Resistance:

• Naturally occurring, without previous exposure to an antibiotic, genetically encoded and is a typical characteristic of a certain bacterial species

Mechanisms for intrinsic resistance include:

1) Lack of uptake or decreased permeability:

• Some antibiotics are unable to penetrate the cell wall or outer membrane of certain bacteria, rendering them ineffective

2) Presence of efflux pumps:

• These are proteins that actively pump out antibiotics from the bacterial cell, reducing the intracellular concentration of the drug

3) Antibiotic modification or degradation:

• Some bacteria produce enzymes that modify or degrade antibiotics, rendering them ineffective

4) Lack of target site:

• The antibiotic’s target site may not exist in the bacterial cell, rendering the antibiotic useless

Acquired Resistance:

• when a bacterium that was initially sensitive to an antibiotic becomes resistant, usually as a result of genetic mutation or the acquisition of resistance genes from another bacterium.

1) Mutations:

• Spontaneous mutations in the bacterial genome can lead to antibiotic resistance. For example, a mutation could alter the target site of the antibiotic, decreasing its effectiveness
• Alternatively, a mutation could lead to the overproduction of a protein that pumps the antibiotic out of the cell, decreasing the intracellular concentration of the antibiotic
• Mutations can also cause the overproduction of the antibiotic’s target, effectively diluting the drug

2) Horizontal gene transfer:

• Bacteria can acquire resistance genes from other bacteria through mechanisms like transformation (uptake of DNA from the environment) transduction (transfer of DNA from one bacterium to another by a bacteriophage)
  or conjugation (direct transfer of DNA from one bacterium to another via a plasmid)
• E.g. MRSA has acquired resistance to many beta-lactam antibiotics through the acquisition of a gene (mecA gene) that encodes a modified penicillin-binding protein (PBP2a) and genes that encode β-lactamase enzymes, the gene is carried on plasmid; mobile, spread rapidly

Question 10

Describe the microbiological methods for testing antibiotic susceptibility

Answer 10

1) Disk Diffusion Method (Kirby-Bauer Test):

• bacteria are spread across a plate of agar medium
• Small disks impregnated with known concentrations of antibiotics are placed on the plate
• The plate is incubated, and the bacteria are allowed to grow
• If the bacteria are susceptible to the antibiotic, a clear ring, or zone of inhibition, will appear around the disk where bacterial growth has been prevented
• The diameter of these zones is measured and compared to standard tables to determine whether the microorganism is sensitive, resistant, or intermediately resistant to the antibiotic

2) Minimum Inhibitory Concentration (MIC) Testing:

• This method determines the lowest concentration of an antibiotic that inhibits the growth of a bacterium
  involves preparing a series of dilutions of the antibiotic in a liquid growth medium, inoculating each with the test organism, and incubating them.
• The MIC is the lowest concentration of the antibiotic that prevents visible growth of the bacteria
• MIC testing can be performed using broth dilution (in tubes or in wells of a microtiter plate) or by the Etest, a commercially available system that combines the principles of both disk diffusion and MIC

3) Broth Dilution Test:

• This method is similar to the MIC test but done in a broth
• Different concentrations of antibiotics are added to tubes or wells of broth containing the bacteria, and these are then observed for bacterial growth

4) Gradient Diffusion Method (E-test):

• E-test is another method used to determine the MIC
  It uses a plastic strip that has a gradient of antibiotic concentrations
• One end of the strip contains a high concentration of the antibiotic, and the other end contains a low concentration
• The strip is placed on an agar plate that has been inoculated with the bacteria
• After incubation, an elliptical zone of inhibition is produced
• The intersection of the ellipse with the strip indicates the MIC

Question 11

Give specific examples of antibiotics, their targets, and the basis of resistance for that antibiotic

Answer 11

1) Penicillins (Amoxicillin):

• Penicillins target bacterial cell wall synthesis by binding to proteins called penicillin-binding proteins (PBPs), which are needed for the cross-linking of the peptidoglycan layer
• Resistance often arises through the acquisition of beta-lactamase enzymes, which can hydrolyse the beta-lactam ring of penicillins and render them ineffective
• Some bacteria also alter the structure of their PBPs to reduce penicillin’s affinity, making them less susceptible to the drug

2) Fluoroquinolones (Ciprofloxacin):

• Target bacterial DNA gyrase and topoisomerase IV, enzymes that are necessary for DNA replication
• Resistance can occur through mutations in the genes encoding these enzymes, reducing the binding of the antibiotic
• Bacteria can also develop resistance through efflux pumps that remove the antibiotic from the bacterial cell

3) Tetracyclines (Doxycycline):

• Inhibit protein synthesis by binding to the 30S subunit of the bacterial ribosome and preventing the attachment of tRNA
• Resistance is often due to the acquisition of efflux pumps or ribosomal protection proteins, which prevent the drug from reaching its target

4) Aminoglycosides (Gentamicin):

• Aminoglycosides also target the 30S ribosomal subunit, causing misreading of the mRNA and inhibiting protein synthesis
• Bacteria can develop resistance through the production of aminoglycoside-modifying enzymes, which chemically modify the drug and prevent it from binding to its target

5) Macrolides (Erythromycin):

• Bind to the 50S subunit of the bacterial ribosome, inhibiting protein synthesis
• Resistance can occur through the methylation of the ribosomal target, blocking the binding of the drug, or through efflux pumps

6) Sulfonamides and Trimethoprim (Co-trimoxazole):

• These drugs inhibit folic acid synthesis, an essential component for bacterial DNA replication
• Sulfonamides inhibit the enzyme dihydropteroate synthase, and trimethoprim inhibits dihydrofolate reductase
• Resistance often involves the acquisition of alternative enzymes that are not inhibited by these drugs

7) Beta-Lactam/Beta-Lactamase Inhibitor combinations (Amoxicillin-Clavulanate):

• In this combination, the beta-lactamase inhibitor protects the penicillin from destruction, enhancing its effectiveness
• Resistance can still occur through the production of beta-lactamases that are not inhibited by the beta-lactamase inhibitor, or by alterations in PBPs

Question 12

Discuss the role of antibiotic stewardship in the control and prevention of antibiotic resistance

Answer 12

Antibiotic stewardship is a coordinated program that promotes the appropriate use of antibiotics to improve patient outcomes, reduce microbial resistance, and decrease the spread of infections caused by multidrug-resistant organisms

They are typically multidisciplinary, involving infectious disease physicians, pharmacists, clinical microbiologists, and infection control professionals.

1) Prospective Audit and Feedback:

• This involves the review of antimicrobial prescriptions after the therapy has started
  Feedback is then provided to the prescriber about the appropriateness of the antibiotic choice, dose, duration, and de-escalation opportunities

2) Antimicrobial Formulary Restrictions and Pre-authorisation Requirements:

• Some antibiotics known to drive resistance may be restricted and require pre-approval before they can be prescribed

3) Education and Guidelines:

• Providing clinicians with information about antibiotic resistance trends, optimal prescribing strategies, and the latest treatment guidelines is a crucial part of stewardship
• Regular seminars, workshops, or online modules may be part of this educational effort

4) Antimicrobial Cycling and Rotation:

• This strategy involves regularly changing the class of antibiotics used within a healthcare setting to reduce the selection pressure on bacteria

5) Dose Optimisation:

• This involves adjusting the dose of antibiotics based on individual patient characteristics (like kidney function) and the characteristics of the antibiotic

6) De-escalation and Duration of Therapy:

• Once culture results are available, antibiotics can often be narrowed to target the specific pathogens identified (“de-escalation”)
• Also, limiting the duration of therapy to the shortest effective period can help prevent resistance

7) Rapid Diagnostic Testing:

• Newer molecular diagnostic techniques can identify bacteria and their resistance genes more rapidly than traditional culture methods, allowing for more targeted antibiotic use