Antibiotic Resistance Testing

Antibiotic Resistance Development studies in bacteria can be assessed by single- or multi-step resistance studies. Single-step resistance studies involve the formation of resistant mutants in one exposure to the antimicrobial agent, while multi-step resistance studies involve exposing the bacteria to the antimicrobial agent over many passages (subcultures), allowing for formation of resistance mutations over time. Results from resistance studies can help predict the frequency of resistant mutants that appear after exposure to an antimicrobial agent, as well as the types of resistant strains that might develop or emerge in the clinic.

Single step resistance studies:

Spontaneous Mutation Frequency: This assay can determine the frequency at which mutants emerge when exposed to inhibitory concentrations of an antimicrobial agent (see illustration below).

Mutant characterization: The stable mutants isolated from the spontaneous mutation frequency studies are characterized by an MIC broth dilution assay (following CLSI guidelines) for their antibiotic resistance to the antimicrobial agent they were selected on. This can confirm that the mutants are resistant mutants, and also can determine the level of resistance to the antimicrobial agent. Further genetic characterization of these mutants can help provide a better understanding of the resistance mechanism of the bacteria to the antimicrobial agent.

Mutant Prevention Concentration (MPC): This assay can determine the concentration of the antimicrobial agent that prevents the bacterial isolate from forming resistant colonies. The MPC is the lowest in vitro concentration of antimicrobial agent at which no resistant mutants are recovered.

Spontaneous Mutation Frequency. This figure illustrates the general workflow for conducting a single-step, spontaneous mutation frequency assay. (1) Liquid agar containing a known concentration of antibiotics (typically 4-fold or 8-fold the MIC value of the bacteria being assayed) is prepared, poured into petri dishes, and allowed to solidify. (2) A parental bacteria culture is prepared and the bacterial density in CFU/mL is determined. (3) The parental bacteria culture is spread onto the agar plate containing antibiotic and incubated for up to 3 days. (4) The number of CFUs recovered is enumerated and this value is compared to the parental bacterial density to generate a frequency of mutation.

Multi step resistance studies:

Serial Passage: This assay develops resistance in bacteria by continuously passaging bacteria grown in sub-MIC (sub-inhibitory) concentrations of antimicrobial agent.

Serial Passage. Illustration of serial passage assay. A parent culture is prepared and used to inoculate a serially diluted series of antimicrobial compound. The culture is incubated and on day 1 the MIC concentration is determined to be the lowest concentration in which no growth is observed. The sub-MIC concentration culture is used to inoculate a second serially diluted series of antimicrobial compound and incubated overnight. The next day the MIC (which may remain the same or increase) is determined and the sub-MIC concentration culture from the latest passage is used to inoculate a new series of diluted antimicrobial compound. The process continues for up to 21 days or until a predetermined end point concentration is reached.

Reference:

Antimicrobial Agents and Chemotherapy, Apr. 2006, p. 1228–1237 Vol. 50, No. 4, Mani et al, In Vitro Characterization of the Antibacterial Spectrum of Novel Bacterial Type II Topoisomerase Inhibitors of the Aminobenzimidazole Class.