Antimicrobial Mechanisms and Drug Action

• MIC1204 covers an introduction to microbiology and immunology
• The course also examines antibiotics, resistance, and superbugs

Antimicrobial Terms

• Biocide is a chemical or physical agent, usually broad spectrum, that inactivates microorganisms
• Bacteriostatic describes a biocide's ability to inhibit bacterial multiplication, which is reversible
• Bactericidal refers to a biocide's property of killing bacteria
• Septic indicates the presence of pathogenic microbes in living tissues or associated fluids
• Antiseptic is a biocide or product that destroys or inhibits microorganism growth on living tissue or biologic fluids
• Aseptic describes being free of microorganisms, or using methods to keep free of them
• Preservation refers to preventing microorganism multiplication in formulated products, including pharmaceuticals and foods
• Antibiotics are naturally occurring and synthetically derived compounds that inhibit or destroy selective bacteria at low concentrations

Disinfection and Topical Antimicrobial Agents

• Disinfectants are used in the inanimate environment
• For tabletops and instruments, Lysol or other phenolic compounds, formaldehyde, aqueous glutaraldehyde, or quaternary ammonium compounds are used
• For excreta, bandages, and bedpans, sodium hypochlorite or Lysol, or other phenolic compounds are used
• To disinfect the air, propylene glycol mist or aerosol or formaldehyde vapor can be used
• Ethylene oxide gas is for heat-sensitive instruments; the residual gas must be removed by aeration
• For skin or wound disinfection, soap and water, or soaps and detergents containing hexachlorophene, trichlorocarbanilide, or chlorhexidine are used
• Other disinfectants for skin and wounds are tincture of iodine, ethyl alcohol, isopropyl alcohol, povidone-iodine, peracids or nitrofurazone jelly or solution
• Topical drugs for skin or mucous membrane infections include:
• Nystatin cream, candicidin ointment, or miconazole creams for candidiasis
• Mafenide acetate cream or silver sulfadiazine for burns
• Undecylenic acid powder or cream, tolnaftate cream, or azole cream for dermatophytosis
• Bacitracin-neomycin-polymyxin ointment or potassium permanganate for pyoderma
• Malathion or permethrin lotion for pediculosis
• Mupirocin for nasal decolonization
• Topical applications of drugs for the eyes include:
• Erythromycin or tetracycline ointment for gonorrhea prophylaxis
• Sulfacetamide ointment, gentamicin, or tobramycin ointment, ciprofloxacin ointment, moxifloxacin ophthalmic solution, gatifloxacin solution, or levofloxacin solution for bacterial conjunctivitis

Modes of Antimicrobial Action

• Microbes adapt to living in particular environments, so they can be targeted in situ or in vivo
• Actions used to target microbes include:
  • DNA damage
  • Denaturation of proteins
  • Disruption of the cell membrane/wall
  • Disruption of free sulfhydryl groups
  • Chemical antagonism

DNA Damage

• Physical and chemical agents damage DNA, including ionizing/UV radiations and DNA-reactive chemicals
• Alkylating agents react covalently with purine and pyrimidine bases to form DNA adducts or interstrand cross-links
• Radiation damages DNA in several ways
• UV light induces cross-linking between adjacent pyrimidines on polynucleotide strands, forming pyrimidine dimers
• Ionizing radiation produces breaks in single and double strands
• Radiation-induced and chemically-induced DNA lesions kill the cell mainly by interfering with DNA replication

Denaturation of Proteins

• Proteins exist in a folded, three-dimensional state, determined by intramolecular noncovalent interactions like ionic, hydrophobic, hydrogen bonds, or covalent disulfide linkages
• The tertiary structure of a protein is readily disrupted by physical or chemical agents causing the protein to become nonfunctional
• Disruption of the tertiary structure of a protein is called protein denaturation

Disruption of Cell Membrane or Wall

• The cell membrane is a selective barrier that allows some solutes vs. excluding others
• Many compounds are actively transported through the membrane, concentrating within the cell
• Enzymes are involved in the biosynthesis of cell envelope components
• Substances may concentrate at the cell surface, altering the physical and chemical membrane properties, preventing its normal functions
• The cell wall protects the cell against osmotic lysis as a corseting structure
• Agents that destroy the wall, such as lysozyme, which cleaves sugar linkages, or prevent its synthesis may bring about cell lysis
• β-lactam known as carbapenem can block enzymes that construct the cell wall

Disruption of Free Sulfhydryl Groups

• Enzymes containing cysteine have side chains terminating in sulfhydryl groups
• Coenzymes like coenzyme A and dihydrolipoate also contain free sulfhydryl groups
• The enzyme can only function if the sulfhydryl groups remain free and reduced
• Oxidizing agents interfere with metabolism by forming disulfide linkages between neighboring sulfhydryl groups: R-SH + HS-R —> R—S—S-R (+2H)
• Metals like mercuric ions interfere by combining with sulfhydryls of sulfhydryl-containing enzymes

Chemical Antagonism

• Enzymes perform critical functions enabling cellular activities for an organism's survival
• A chemical agent interfering with the normal reaction between an enzyme and its substrate is chemical antagonism
• The antagonist has chemical affinity for combining with some part of the enzyme, which prevents attachment of the normal substrate
• Compounds structurally resembling a substrate can also have an affinity for the enzyme
• The analog will displace the normal substrate and prevent the proper reaction from taking place

Antibiotics

• Antibiotics are naturally occurring and synthetically derived organic compounds that inhibit or destroy selective bacteria at low concentrations
• The selection of antimicrobial drugs depends on diagnosis and susceptibility, two specific considerations

Diagnosis Considerations

• Specific etiologic diagnosis must be formulated, often based on clinical impressions
• Relation between clinical picture and causative agent sufficiently constant to permit antibiotic choice based on clinical impression alone
• Obtain a representative specimen for bacteriologic study before giving antimicrobial drugs as a safeguard against diagnostic error
• In most infections, the causative agent and clinical picture relationship isn't constant, making it crucial to obtain specimens for bacteriologic identification
• Chemotherapy can be started based on the "best guess" once specimens are secured
• Then, the initial regimen can be modified after lab procedures confirm the causative agent

Diagnosis Continued

• Factors for the "best guess":
  • Site of infection (e.g., pneumonia, urinary tract infection)
  • Age of the patient (e.g., meningitis: neonatal, young child, adult)
  • Infection acquisition location (hospital vs. community)
  • Mechanical predisposing factors (indwelling vascular catheter, urinary catheter, ventilator, exposure to vector)
  • Predisposing host factors (immunodeficiency, corticosteroids, transplant, cancer chemotherapy)
• The drug of choice can often be selected on the basis of current clinical experience if the causative agent is known
• Lab tests for antibiotic susceptibility are necessary to determine the drug when that is not the case

Susceptibility Considerations

• Lab tests for antibiotic susceptibility are indicated when:
  • The microorganism is a type that is often resistant to antimicrobial drugs
  • An infectious process is likely to be fatal unless treated (e.g., meningitis, septicemia)
  • An infection needs drugs that are rapidly bactericidal, not merely bacteriostatic (e.g., infective endocarditis)

Dangers Of Indiscriminate Use

• Indications for antibiotic administration:
  • Widespread sensitization of the population with hypersensitivity, anaphylaxis, rashes, fever, blood disorders, cholestatic hepatitis, and collagen-vascular diseases
  • Changes in the normal microbiota of the body with disease from “superinfection” caused by overgrowth of drug-resistant organisms
  • Masking serious infection without eradicating it, but clinical manifestations of an abscess may be suppressed while the infectious process continues
  • Direct drug toxicity, granulocytopenia, or thrombocytopenia with cephalosporins and penicillins, and renal or auditory nerve damage from aminoglycosides is also a concern
  • Drug resistance development in microbial populations from elimination of sensitive microorganisms and their replacement by resistant microorganisms

Major Antibiotic Classes

• Penicillins
• Cephalosporins
• Other B-Lactam Drugs
• Tetracyclines
• Glycylcyclines
• Chloramphenicol
• Erythromycins
• Clindamycin and Lincomycin
• Glycopeptides and Lipopeptides

Penicillin

• Penicillin's discovery is a prime example of scientific serendipity
• Scottish bacteriologist Alexander Fleming noticed mold growing on a petri dish containing Staphylococcus aureus bacteria in 1928
• The mold was later identified as Penicillium notatum, which inhibited the bacteria's growth
• This led to the isolation and characterization of the first antibiotic

Antibiotic Resistance

• Review of common antimicrobial actions of antibiotics: DNA Damage, Denaturation of Proteins, Disruption of cell membrane or cell wall, Disruption of free sulfhydryl groups, and Chemical antagonism
• These elimination methods target a microbe's physical characteristic
• Resistance to antibiotic compounds can occur in several ways:
  • Non-genetic origin of resistance
  • Chromosomal resistance
  • Extrachromosomal resistance
• Most drug-resistant microbes emerge as a result of genetic change and subsequent selection by antimicrobial drugs

Nongenetic Origin of Drug Resistance

• Active bacteria replication is required for most antibacterial drug actions
• Metabolically inactive microorganisms may be phenotypically resistant to drugs, although their offspring are still be susceptible
• Mycobacteria can survive in tissues for years while restrained by the host's defenses and do not multiply
• Persisting organisms are resistant to treatment
• These organisms cannot be eradicated by drugs. However, they become susceptible when they start to multiply after suppression of cellular immunity in the patient
• Microorganisms may lose a drug's specific target structure, like when penicillin-susceptible organisms change to cell wall-deficient L forms lacking cell walls during penicillin administration
• These L forms are resistant to cell wall-inhibitor drugs and remain so for generations
• Microorganisms may infect sites where antimicrobials are excluded or inactive; for example, aminoglycosides like gentamicin are not effective in treating Salmonella enteric fevers since the salmonellae are intracellular and the aminoglycosides cannot enter the cells

Chromosomal Resistance

• Chromosomal resistance is a result of spontaneous mutation in a locus controlling susceptibility to an antimicrobial drug
• The antimicrobial drug presence selects to suppress susceptible organisms and favor growth of resistant mutants
• Spontaneous mutation occurs with a frequency of ~10–12—10-7 & is an infrequent cause of clinical drug resistance emergence
• However, chromosomal mutants resistant to rifampin occur with high frequency (~10−7−105) and treatment fails because of this
• Chromosomal mutants are commonly resistant by virtue of a structural receptor change for a drug
• P 12 protein on the bacterial ribosome's 30S subunit serves as a receptor for streptomycin attachment, while mutation in the gene controlling this protein causes streptomycin resistance
• Mutation can also result in the loss of Penicillin Binding Proteins (PBP), making mutants resistant to B-lactam drugs

Extrachromosomal Resistance

• Bacteria often contain extrachromosomal genetic elements called plasmids
• Some plasmids carry genes for resistance to one, and often several- antimicrobial drugs
• Plasmid genes control forming enzymes capable of destroying the antimicrobial drugs
• This leads to plasmids determining resistance to penicillins and cephalosporins by carrying genes for forming B-lactamases
• Plasmids code for enzymes that acetylate, adenylate or phosphorylate aminoglycosides, for enzymes determining active transport of tetracyclines across the cell membrane, and for others
• Genetic material and plasmids can be transferred by transduction, transformation, and conjugation

Resistance Mechanisms

• Antimicrobial resistance from these origins can be effected by multiple mechanisms:
  • Inactivation of B-lactam molecules by microbial production of B-lactamases
  • Decreased target site penetration (e.g., resistance of Pseudomonas aeruginosa)
  • Alteration of target site PBPs (e.g., penicillin resistance in pneumococci)
  • Efflux from the periplasmic space through specific pumping mechanisms

Notable Examples of Superbugs

• Methicillin-resistant Staphylococcus Aureus (MRSA)
• Carbapenem-resistant Enterobacteriaceae (CRE)
• Extensively drug-resistant tuberculosis (XDR-TB)
• Antibiotic-resistant Klebsiella (Nakry)

Methicillin-Resistant Staphylococcus Aureus (MRSA)

• Staphylococcus Aureus (SA) in 1944 was susceptible to penicillin G
• Penicillin use lead to population resistance, accounting for 65-85% of collected samples
• B-lactamase was the chief method of resistance
• Lactamase-resistant penicillins like nafcillin, methicillin, and oxacillin provided respite
• Strains then eventually mutated into resistance
• Vancomycin remains the chief option, though evidence of genetic mutation conferring resistance has been observed resulting in vancomycin-resistant staphylococcus aureus (VRSA_

Limitation Of Drug Resistance

• The healthcare setting should take the following into consideration for the high frequency of drug resistance emergence:
  • Maintain sufficiently high drug levels in tissues to inhibit the original and first-step mutants
  • Simultaneously administer two drugs that do not give cross-resistance, and delay the emergence of mutants like rifampin and isoniazid [INH] in treating tuberculosis
  • Avoid microorganism exposure to a particularly valuable drug by limiting its use, especially in hospitals

Explore antimicrobial mechanisms, including cell lysis by penicillin and disruption of cellular metabolism by oxidizing agents. Learn about enzyme inhibition, genetic material interference, and protein denaturation. Understand antimicrobial drug selection based on mechanisms.