IAS59 Antimicrobials and Immunization

An antimicrobial is a substance that kills or inhibits the growth of an infectious microorganism.

The categories of antimicrobial agents include:

1. Antibiotics/Anti-bacterial agents - against bacteria
2. Anti-viral agents - against viruses
3. Anti-fungal agents - against fungi
4. Anti-parasitic agents - against parasites

The 'discovery void' refers to the lack of new classes of antibiotics licensed for use since the 1980s, indicating a stagnation in antibiotic discovery despite the ongoing need for new treatments due to antibiotic resistance.

The following new antibiotics were approved by the FDA:

1. Ceftaroline (Nov-2010)
2. Bedaquiline (Dec-2012)
3. Ceftolozane-tazobactam (Dec-2014)
4. Ceftazidime-avibactam (Feb-2015)

The lack of new antibiotic classes since the 1980s raises concerns about the effectiveness of current treatments against resistant bacteria, potentially leading to increased morbidity and mortality from infections that were previously treatable.

1. By chemical structure
2. By activity: bacteriostatic or bactericidal
3. By target site

The main members of the beta-lactam family include:

The fluorine atom at the C6 position of fluoroquinolones increases:

• Potency
• Spectrum
• Tissue penetration

Examples of fluoroquinolones include Ciprofloxacin, Levofloxacin, and Moxifloxacin.

Quinolones inhibit:

• DNA gyrase
• Topoisomerase IV

This action blocks DNA replication and provides broad-spectrum antibacterial activity.

Vancomycin is a large glycopeptide molecule. It is known for its effectiveness against certain types of bacteria, particularly Gram-positive bacteria.

Doxycycline has four cyclical structures and different sidechains, including three sugars (glycoside).

Aminoglycosides differ in terms of their amino group, hydroxy group, and their position on the structure.

Tetracyclines are a class of antibiotics characterized by their four-ring structure and are used to treat a variety of infections.

Macrolides antibiotics have a large cyclical ring structure, typically with 14 members, and differ in terms of their side chains from erythromycin.

Telithromycin was approved by the US FDA in 2004 and was developed through 12 steps from erythromycin.

Cethromycin is a clinical candidate developed through 9 steps from erythromycin, while Solithromycin was developed through 16 steps from erythromycin.

• Immunological reactivity: Potential for drug allergy due to cross-reaction.
• Pharmacological properties: Includes absorption, tissue distribution, contraindications (e.g., in children, pregnancy), and toxicities.
• Mechanism of action: Often share the same binding site(s) in bacteria.
• Bacteria resistance mechanisms: Similarities in how bacteria may resist these agents.

Gram-positive bacteria have a thick layer of peptidoglycan, while Gram-negative bacteria have a thinner layer of peptidoglycan located between an outer membrane and a cytoplasmic membrane.

The two key enzymatic reactions in peptidoglycan synthesis are:

1. Transglycosylation - links NAG-NAM backbone.
2. Transpeptidation - forms cross-links between NAM units.

The backbone of the peptidoglycan structure consists of alternating units of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM).

Cross-links, formed by short peptide chains connecting NAM units, provide rigidity to the peptidoglycan structure, enhancing the strength of the bacterial cell wall.

Transglycosylase catalyzes the formation of glycan chains in the peptidoglycan layer, linking N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM).

Peptide bridges cross-link the glycan chains (NAG-NAM) in the peptidoglycan layer, providing structural integrity and varying among different bacterial species.

Transpeptidase catalyzes the cross-linking of peptide bridges between glycan chains, which is essential for maintaining the strength and rigidity of the bacterial cell wall.

The structures of peptide bridges vary among bacterial species, which can affect the susceptibility of bacteria to certain antibiotics targeting cell wall synthesis.

The β-lactam ring binds to the transpeptidase enzyme at its serine active site, opening the ring and covalently attaching to the enzyme, which blocks its activity. This inhibits peptide cross-linking in peptidoglycan, weakening the cell wall and leading to bacterial lysis.

PBPs are classified into two groups:

1. Class A PBPs: Enzymes with both transpeptidase (TPase) and transglycosylase (TGase) activity.
2. Class B PBPs: Enzymes with only transpeptidase (TPase) activity.

Vancomycin binds to the terminal D-Ala-D-Ala portion of peptidoglycan peptide chains. This binding blocks cross-linking and the formation of the NAG-NAM backbone, preventing proper assembly of the cell wall, which is particularly effective against Gram-positive bacteria due to their inability to penetrate the outer membrane of Gram-negatives.

The large molecular size of vancomycin prevents proper assembly of the bacterial cell wall, making it effective in blocking the synthesis of the cell wall, particularly in Gram-positive bacteria.

The learning objective is to describe how to assess the in vitro activity of antibacterial agents in the laboratory.

The term 'bactericidal' indicates that the action of the agent will kill the targeted microbe, defined as a >99.9% decrease in the initial inoculum.

'Bacteriostatic' indicates that the action of the agent will inhibit the growth of the targeted microbe but will not kill it, defined as preventing visible growth.

The effectiveness can be assessed by comparing growth curves, where bactericidal agents show a dramatic decline in the number of organisms, while bacteriostatic agents maintain a constant level of organisms without growth.

MIC stands for Minimal Inhibitory Concentration, and MBC stands for Minimal Bactericidal Concentration. They are measured quantitatively to assess the effectiveness of antibacterial agents.

The growth curve illustrates the number of organisms over time under different conditions, showing that bactericidal agents kill bacteria while bacteriostatic agents prevent growth without killing.

The MIC is the lowest concentration of an antibacterial agent that inhibits visible growth of bacteria after overnight incubation (16-24 hours).

The MBC is the lowest concentration of an antibacterial agent that kills the initial bacterial inoculum by ≥99.9%.

The MIC and MBC of an antibiotic are usually within 1 to 2 dilution differences from each other.

Examples of bacteriostatic agents include:

• Macrolides
• Clindamycin
• Tetracyclines
• Chloramphenicol
• Sulphonamides
• Trimethoprim

Examples of bactericidal agents include:

• Beta-lactams
• Vancomycin
• Aminoglycosides
• Fluoroquinolones

The designation is arbitrary; an agent can be bacteriostatic against one organism but bactericidal against another. Additionally, an agent may be bacteriostatic at low concentrations but bactericidal at higher concentrations, and this is also affected by the bacterial inoculum.

For lungs, skin, soft tissue, and intra-abdominal infections, there is no major difference if bacteria are susceptible.

Bactericidal agents are preferred for meningitis, endocarditis, and neutropenic infections for faster pathogen clearance.

The main outcome measures were clinical cure rates and overall mortality.

Neither clinical cure rates nor mortality rates were significantly different between patients treated with bactericidal drugs and those treated with bacteriostatic drugs.

Linezolid showed a benefit in clinical cure rates, while tigecycline was associated with increased mortality.

The categorization of antibiotics into bacteriostatic and bactericidal is unlikely to be relevant in clinical practice for abdominal infections, skin and soft tissue infections, and pneumonia.

The different types of vaccines include:

Passive immunization involves the direct transfer of antibodies from one individual to another. This can occur naturally, such as through maternal antibodies passed to a fetus, or artificially, through antibody-containing blood products. It provides immediate, but temporary, protection against pathogens.

Active immunization involves the stimulation of the immune system to produce its own antibodies against a pathogen. This can be achieved through vaccination, where a harmless form of the pathogen is introduced to the body, prompting an immune response that provides long-lasting protection.

The process of artificially inducing immunity or providing protection from disease.

A vaccine is an antigenic preparation used to induce immunity.

The purpose of active immunization is to induce 'acquired' immunity by priming the immune system with an antigen, which leads to preexisting immunity that can prevent infection and elicit a secondary response upon exposure, resulting in mild or subclinical infection with little or no damage.

Active immunization involves the production of specific antibodies and/or T lymphocytes in response to an antigen.

The secondary response in active immunization is characterized by a rapid increase in antibody levels upon re-exposure to the antigen, often resulting in a higher antibody response compared to the primary response, which has a longer lag time.

• Mimic natural infection
• Stimulate T-lymphocytes 'naturally'
• Induce mucosal immunity
• Protect the unvaccinated through:
  • Infectious vaccine strain
  • Herd immunity

• Vaccine strain could cause disease:
  • In healthy recipients (rare risk of reversion to virulence)
  • In immunocompromised recipients:
    • Immunized before diagnosis of condition
    • Person-to-person transmission of vaccine strain

Inactivated vaccines should be used when it is impossible to attenuate the pathogen, when the risk of using a live attenuated vaccine is too high, or when there is unknown stability with a risk of reversion.

Vaccines can be inactivated using the following methods:

1. Heat

   • Example: Vibrio cholera

2. Chemical

   • Formaldehyde
     • Examples: Hepatitis A, Diphtheria toxin, Tetanus toxin
   • Beta-propiolactone
     • Example: Influenza virus

• Less immunogenic
• Poorer stimulation of memory cells
• Shorter duration of protection

Adjuvants are substances used to boost the immune response. They are non-specific immunostimulants, with aluminum salts being the most widely used. They are effective at inducing antibody responses but less active in inducing cell-mediated immunity.

The potency of Prevnar 13 is determined by:

1. Quantification of each of the saccharide antigens
2. Saccharide to protein ratios in the individual glycoconjugates

Each 0.5 mL dose contains approximately:

• 2.2 µg of each of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 23F saccharides
• 4.4 µg of 6B saccharides
• 34 µg CRM197 carrier protein
• 100 µg polysorbate 80
• 295 µg succinate buffer
• 125 µg aluminum as aluminum phosphate adjuvant

Protein conjugation improves the immune response to polysaccharide antigens by allowing T cells to participate, which is crucial since many polysaccharide antigens fail to stimulate T cells effectively, especially in children under 2 years old.

Prevnar 13 is a pneumococcal 13-valent conjugate vaccine that contains saccharides of the capsular antigens of Streptococcus pneumoniae serotypes linked to the non-toxic diphtheria CRM197 protein.

Polysaccharides alone stimulate B cells through cross-linking the B-cell receptor, leading to antibody production but depleting the memory B-cell pool. In contrast, polysaccharides conjugated to a protein are processed by B cells, allowing T-cell help, which promotes the production of both plasma cells and memory B cells, resulting in a more robust immune response.

The lack of T-cell involvement leads to a non-immunogenic response in children under 2 years, resulting in decreased production of memory B cells and diminished subsequent immune responses.

By age 1, children in Hong Kong are recommended to receive a total of 12 injections including:

• BCG (Single dose only)
• HBV (Hepatitis B vaccine #1, #2, #3)
• DTaP-IPV (Diphtheria, Tetanus, acellular Pertusis & inactivated poliovirus vaccine #1, #2, #3, Booster)
• PCV13 (13-valent pneumococcal conjugate vaccine #1, #2, Booster (12-15m))
• MMR (Measles, Mumps & Rubella vaccine #1, #2)
• Varicella (Vaccine #1)
• Influenza (annually for ages 6-23 months)

By age 1, children in the United States are recommended to receive up to 21 injections including:

• Hepatitis B
• Rotavirus
• Diphtheria
• Tetanus
• Pneumococcal
• Influenza
• Measles/Mumps/Rubella (MMR)
• Varicella
• Hepatitis A
• Human Papillomavirus
• Meningococcal

The key differences include:

1. Vaccine availability: Different access to vaccines in each region.
2. Efficacy: Variations in how effective the vaccines are perceived to be.
3. Safety: Differences in safety profiles and monitoring.
4. Disease burden: The prevalence and impact of diseases in each region.
5. Availability of treatment: Access to treatments for diseases.
6. Affordability or cost-effectiveness: Economic factors influencing vaccine distribution and uptake.

Passive immunization is a method that:

• Does not involve the immune system: It provides temporary protection without stimulating the body's immune response.
• Involves administration of exogenous antibodies or memory T cells: These are produced outside the body and given to the individual.
• Can be used as prophylaxis or replacement therapy: It serves to prevent disease or replace missing antibodies.
• Includes trans-placental transfer of antibodies: This is a natural form of passive immunization where antibodies are transferred from mother to fetus.

A sterile solution for parenteral administration containing antibody from unselected pools of donor blood, typically from over 1000 donors, providing a more diverse pool of antibodies.

Specific preparations obtained from donor pools pre-selected for a high antibody content against a specific disease, such as Hepatitis B Ig, Varicella-zoster Ig, and Tetanus Ig, mostly given as secondary prophylaxis.

Specific equine IgG is used for the treatment of wound and food borne forms of botulism, while infant botulism is treated with human botulism immune globulin (BabyBIG).

Hyper-immune IVIG is used for prophylaxis, most often in kidney transplant patients to prevent CMV infection.

Pooled human Ig is used for the prevention of Hepatitis A and measles infection, as well as for the treatment of congenital or acquired immunodeficiency.

Hepatitis B Ig is used for post-exposure prophylaxis and prevention in high-risk infants, and is administered alongside the Hepatitis B vaccine.

Rabies Ig is administered for post-exposure prophylaxis in conjunction with the rabies vaccine to prevent the onset of rabies after exposure.

Tetanus Ig is used for the treatment of tetanus infection, providing immediate passive immunity.

Vaccinia Ig is used for the treatment of progressive vaccinia infection, including eczema and ocular forms, usually resulting from smallpox vaccination in immunocompromised individuals.

Varicella-zoster Ig is used for post-exposure prophylaxis in high-risk individuals to prevent chicken-pox infection after exposure.

• Immediate protection is provided.
• Useful for individuals unable to respond immunologically.

1. Protection is relatively short-lasting (IgG has a half-life of approximately 21 days).
2. Use of antiserum raised in animals can cause serum sickness.
3. There is a residual risk of blood-borne infections (e.g., HIV, HBV, HCV).