Antibiotic Susceptibility & Resistance

• This laboratory session covers antibiotic susceptibility testing, resistance, and antimicrobial chemicals as disinfectants/antiseptics

Antibiotic Susceptibility Test

• Determines the effectiveness of antibiotics against specific bacteria
• Involves selective toxicity, spectrum of activity, and bacterial cellular targets
• Can determine the bacteriostatic/bactericidal effect of antibiotics
• Used to identify resistance to antibiotics and study selected cases of emerging resistance
• Includes methods like the Kirby-Bauer test
• Selective Toxicity*
• A key aspect of antibiotics where antimicrobials target pathogens, not host cells
• Spectrum of Action*
• Can be narrow (effective against a limited range of bacteria) or broad (effective against a wide range of bacteria)
• Cellular Targets*
• Antibiotics can target the cell wall, protein synthesis, nucleic acid replication/transcription, the plasma membrane, or essential metabolite synthesis

Antibiotics

• Antibiotics are antimicrobial drugs used for patient therapy
• Are substances produced by certain microbes
• In small amounts, they inhibit the growth of other microorganisms
• Bactericidal Antibiotics*
• Kill target bacterial cells through various mechanisms depending on the antibiotic
• Beta-lactam drugs (e.g., penicillin) and bacitracin/vancomycin interfere with cell wall synthesis, killing bacterial cells
• Bacteriostatic Antibiotics*
• Inhibit the growth of target microbes
• Do not kill bacteria directly; the host's immune system eliminates them
• Tetracycline interferes with protein synthesis
• Sulfadrugs inhibit folic acid synthesis

Mechanisms of Resistance Against Antibiotics

• Resistance can be acquired through mutations or genetic exchange, such as R plasmids
• Emerging Resistance Examples*
• MRSA
• VRSA
• VRE
• MDR-TB
• ESBLS

Antimicrobial Susceptibility Tests

• Used to determine the most effective antibiotic against a given pathogen
• Includes the Kirby-Bauer method, E-test, and broth dilution method
• Kirby-Bauer Test*
• A disc-diffusion method for measuring of antibiotic effectiveness
• Compares the effectiveness of different antibiotics by measuring the zones of inhibition
• Organisms are classified as susceptible, intermediate, or resistant based on zone size
• E-Test*
• An advanced diffusion method used to calculate the Minimum Inhibitory Concentration (MIC)
• The MIC is the minimum amount of drug needed to inhibit the growth of the bacterial species or strain
• Plastic-coated strips contain an increasing concentration gradient of the antibiotic
• The MIC can be read from a scale printed on each strip
• The Minimum Bactericidal Concentration (MBC) of a drug, which kills the the target microbe in vitro, can be calculated using broth dilution

Kirby Bauer Method

• Involves the use of standard conditions
• Assesses data by measuring the inhibition zones and using reference tables
• Uses 0. 5 McFarland turbidity standard to determine the cell concentration of the broth culture for the test
• Materials Typically Used*
• Bunsen burner
• Penicillin
• Sterile swab
• Mueller-Hinton plates or TSA plates
• McFarland and sterile saline standards
• Sterile paper disks
• Tweezers/ethyl alcohol
• Incubator
• Procedure*
• Prepare a bacterial lawn on the Mueller Hinton plate and place antibiotic discs on the seeded plate
• Incubate plates in an inverted position (18-24h, 35°C)
• After incubation, record growth, presence/absence of inhibition zones, and measure the diameter of inhibition zones to assess the data against reference tables

Analysis of Results with the Kirby Bauer Method

• Growth on a control disk indicates positive growth around the disk with no antibiotics
• No inhibition zone around a disk indicates resistance
• A zone with inhibition may or may not mean susceptibility
• Measure the zone diameter and compare to a zone diameter on a reference chart to determine if an organism is susceptible, intermediate, or resistant

Antimicrobial Chemical Agents

• Can be disinfectants or antiseptics
• Disinfectants*
• Chemical agents that kill microbes, including pathogens, on inanimate objects or surfaces
• Antiseptics*
• Chemical agents that kill microbes, including pathogens, on skin or other tissue
• "-Static" vs. "-cidal"*
• "-Static" agents inhibit growth
• "-Cidal" agents kill the target microbe

Factors Affecting Effectiveness

• Effectiveness is affected by the numbers and types of microbes present and accessibility to the target microbes
• Other factors include the concentration of agent, the temperature, and the pH
• Organic materials (blood, saliva, vomit, or fecal matter) can interfere with the accessibility of antimicrobial chemicals in health care facilities

Common Antimicrobial Chemicals

• Bleach (0. 5% and 5%): sodium hypochlorite or calcium hypochlorite, chlorine, halogen
• Ethanol (70% and absolute): alcohol
• Lysol: contains benzalkonium chloride, hydrogen peroxide, ethyl alcohol, or phenol-based compounds
• Listerine: essential oils like menthol or thymol
• Betadine: iodophor, iodine, halogen
• Hydrogen Peroxide: oxidizing agent