article : In Vitro Antibiotic Susceptibility Testing

Performed to determine the susceptibility of the organism isolated from the diseased host; Generate antibiogram(s)

Broth Dilution MIC test

Agar Dilution MIC test

Broth/Agar MBC test

Agar Diffusion (Kirby-Bauer Disk) Test: Measure diameter of zone of inhibition; Read as susceptible, intermediate, or resistant

Factors influencing zones of inhibition on agar

Concentration of bacteria spread onto agar plate
Pathogen susceptibility
Antibiotic diffusion effects
Agar depth
Growth rate
Temperature
Nutrient availability
Drug antagonists

Factors influencing diffusion of antibiotic

Concentration of antibiotic: Kirby-Bauer disks, E-tests have standardized concentrations
Molecular weight of antibiotic
Water solubility of antibiotic
pH and ionization
Binding to agar

Modern Commercial Kits (e.g., E-test strips (up to six) on an agar plate and can be read out as MIC)

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article : Physiological and Biochemical Mechanisms of Drug Resistance

Bacteria may Demonstrate any of Five General Mechanisms of Antibiotic Resistance:

1. Lack of entry; Decreased cell permeability
2. Greater exit; Active efflux
3. Enzymatic inactivation of the antibiotic
4. Altered target; Modification of drug receptor site
5. Synthesis of resistant metabolic pathway

These Mechanisms can be Grouped into Three Broad Categories:

Permeability Mechanisms

Lack of entry; Decreased cell permeability

Greater exit; Active efflux

Enzymatic Inactivation of the Antibiotic Altered Target or Pathway

Altered target; Modification of drug receptor site

Synthesis of resistant metabolic pathway

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article : Antibiotic Resistance

Bacterial Resistance to Antibiotics is either:

1. Intrinsic (inherent) or phenotypic
2. Acquired via acquisition of foreign resistance genes
3. Acquired via mutational events in the native genome

Intrinsic Resistance: organism is inherently not susceptible to the antibiotic

Phenotypic Resistance (non-genetic) (e.g., non-growing cells; gram-negative, outer cell membrane)

Genotypic Resistance: Exchange of r-Determinants (Genes that confer resistance to specific antimicrobial agents); Transfer and recombination of resistant mutant genes is possible through normal bacterial genetic exchange mechanisms (conjugation; transduction; transformation)

Plasmids: Multidrug (multiple) resistance is possible; Can cross species barrier and closely related strains may acquire r-determinants

Plasmid: covalently closed circular extrachromosomal DNA

Dispensable
May carry genes for drug resistance; metabolic enzymes; virulence factors (e.g., exotoxins)
Restricted or broad host range
Small size (~5 Md) are non-conjugal; Large (20-200 Md) can be conjugal

Plasmid transfer between cells

Bacterial conjugation ("sex"):Replication and transfer of the conjugal plasmid via cell-to-cell contact through an F-pilus encoded by tra (transfer) genes
Transduction: Transferred by phage
Transformation possible

Transposons (Tn) (plasmid or chromosomal): Genes transferable within a replicon via self-excision; Multidrug resistance is possible; Can cross species barrier

Integrons found on transposons or plasmids; Contains the gene and the site for incorporating resistance genes as cassettes allowing expression of the genes; Multidrug resistance is possible

Selective chromosomal mutations (single drug resistance)

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article : Mechanism of Action of Beta-Lactam Antibiotics

Structures of Beta-Lactam Antibiotics:

Beta-lactam Ring Structure
Structure of Penicillins
Structure of Cephalosporins

Bactericidal Against Actively Growing Cells

Drug Covalently Links to Cytoplasmic Membrane Regulatory Enzymes (a.k.a., Penicillin binding proteins (PBP))

PBPs function in cell to catalyze crosslinking of peptidoglycan chains

PBPs are transpeptidases

Beta-Lactam Antibiotics at Concentrations >MIC:

Bind to PBPs

Disrupt synthesis of peptidoglycan

Resultant release of autolysins (autolytic enzymes)

Autolysins enzymatically degrade cell wall forming spheroplast (osmotically-sensitive cell lacking rigidity of cell wall)

Cells lyse; Bacterial cell killed

For E. coli, at Concentrations <MIC:

Septum formation is interrupted

Filamentous, multinucleated cells are observed with cells continuing to divide but septa (new cell walls that separate daughter cells) do not form

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