Antibiotic Susceptibility & Resistance

Antibiotic Susceptibility Test and Resistance

• Determines the effectiveness of antibiotics against specific bacteria.

Antibiotic

• A substance produced by microorganisms that inhibits the growth of other microorganisms.

Selective Toxicity of Antimicrobials

• Antimicrobials should target pathogens while leaving host cells unharmed.

Spectrum of Action

• Antimicrobials can have narrow or broad spectra, affecting different ranges of microorganisms.

Cellular Targets of Antibiotics

• Antibiotics disrupt cell wall synthesis.
• Antibiotics disrupt protein synthesis.
• Antibiotics prevent nucleic acid replication, and transcription.
• Antibiotics cause injury to the plasma membrane.
• Antibiotics inhibit the synthesis of essential metabolites.

Bactericidal vs. Bacteriostatic Antibiotics

• Bactericidal antibiotics kill target bacterial cells via various mechanisms.
• Beta-lactam drugs, such as penicillin, and bacitracin/vancomycin, kill bacterial cells by interfering with cell wall synthesis.
• Bacteriostatic antibiotics inhibit the growth of target microbes, allowing elimination by the host's immune system.
• Tetracycline is a bacteriostatic antibiotic that interferes with protein synthesis.
• Sulfadrugs are bacteriostatic and inhibit folic acid synthesis.

Mechanisms of Resistance Against Antibiotics

• Microbes resist antibiotics by blocking entry of the drug.
• Microbes resist antibiotics by inactivating the drug with enzymes.
• Microbes resist antibiotics by altering the drug's target sites.
• Microbes resist antibiotics by efflux of the drug from the cell.

Antibiotic Resistance

• Resistance to antibiotics can be acquired through mutations or genetic exchange like R plasmids.
• Examples of emerging resistance include MRSA, VRSA, VRE, MDR-TB, and ESBLs.

Antimicrobial Susceptibility Tests

• Tests such as the Kirby-Bauer method, E-test, and broth dilution method, help physicians determine the most effective antibiotic for a pathogen.

Kirby-Bauer Test

• A disc-diffusion method comparing the effectiveness of different antibiotics by measuring zones of inhibition,
• Organisms are classified as susceptible, intermediate, or resistant, according to the inhibition zone size.

E-Test

• A diffusion method that determines the minimum inhibitory concentration (MIC) by using a plastic strip with an antibiotic concentration gradient.
• The MIC is read from the scale on the strip.
• The minimum bactericidal concentration (MBC) can be calculated via broth dilution.

Calculating MIC

• The minimum inhibitory concentration is the lowest concentration that halts the growth in vitro.

Kirby Bauer Method Materials

• General lab materials include Bunsen burner, sterile swab Mueller-Hinton plates or TSA plates, McFarland standard, sterile saline solution, sterile paper disks, tweezers/ethyl alcohol, and incubator.
• Common Antibiotic disks include Penicillin, Gentamicin, Tetracycline, and Chloramphenicol.
• Common bacteria species include E.coli, K. aerogenes, B. subtilis, and S. epidermidis.
• The Kirby-Bauer method assesses data through inhibition zones and reference tables.
• The Kirby-Bauer method typically uses the 0.5 McFarland turbidity standard.

Kirby Bauer Method Steps

• Prepare a bacterial lawn on the Mueller Hinton plate then place the antibiotic discs on the seeded plate.
• The plates are incubated in an inverted position for 18-24 hours at 35C
• Check for growth of the control, record presence or absence of the inhibition zones, then measure the diameter of the inhibition zones in mm using the ruler (metric system)
• Finally assess the data using the reference tables

Kirby Bauer Plates

• Each student prepares one plate, testing four species with the same antibiotics in consistent positions on the plate.
• The disk labeled C indicates the control disk.

Analysis of Kirby-Bauer

• Positive growth around the control disk signifies a valid test.
• No inhibition zone around a disk indicates resistance.
• Inhibition zone presence requires comparing the zone size to a reference table to determine susceptibility.

Assessing Data

• The diameter of the inhibition zone can be used to determine if the bacteria are resistant.

Antimicrobial Chemical Agents

• Disinfectants kill or inhibit microbes on inanimate objects/surfaces.
• Antiseptics kill or inhibit microbes on skin or tissue.
• "-Static" means the agent inhibits microbe growth.
• "-Cidal" means the agent kills microbes.

Factors Affecting Antimicrobial Agent Effectiveness

• Effectiveness depends on the numbers/types of microbes,accessibility to target microbes, concentration, temperature, and pH.
• Organic materials like blood, saliva, or vomit can interfere with accessibility of antimicrobial chemicals.

Common Disinfectants/Antiseptics

• Effective disinfectants/antiseptics include Bleach (0.5% and 5%), Ethanol (70% and absolute), Lysol (1% and 5%), Listerine, Betadine (10%), and Hydrogen peroxide (1.5% and 3.0%).
• Bleach has sodium or calcium hypochlorite with chlorine and halogen.
• Ethanol is alcohol for disinfecting and antisepsis.
• Lysol has benzalkonium chloride, hydrogen peroxide, ethyl alcohol, and phenol.
• Listerine is essential oils like menthol or thymol.
• Betadine is iodophor: iodine and halogen.
• Hydrogen peroxide is an oxidizing agent.

Disk-Diffusion Method Materials

• Necessary materials include a Bunsen burner, sterile swabs, TSA plates, sterile paper disks, disinfectants/antiseptics, sterile deionized water, McFarland standard, tweezers with ethyl alcohol, and an incubator set at 37C.
• Common bacterial species include Escherichia coli, Klebsiella aerogenes, Bacillus subtilis, and Staphylococcus epidermidis.

Methods for Disinfectants

• Bacterial lawn is prepared on TSA plates.
• Place paper discs impregnated with agents using sterile tweezers.
• An inverted plate is incubated for 18-24h at 35-37C.
• Record growth around control disk.
• Record presence or absence of inhibition zones around antimicrobial disks.