Developments in the Mutant Prevention Concentration: A Novel Approach to Antimicrobial Susceptibility/Resistance Issues
The mutant prevention concentration (MPC) is defined as the lowest antimicrobial concentration required to inhibit the growth of the least susceptible bacterial cell based on an inoculum of ≥10⁹ colony forming units (CFUs). The current protocol for MPC testing is technically demanding and time-consuming which limits its implementation into clinical microbiology laboratories. In an attempt to simplify the current MPC protocol we developed a modified MPC method, the microbroth dilution method, which requires two fewer days to complete than the current or traditional method. MPC values were consistent for all organisms and strains tested using both the traditional MPC method and the modified microbroth dilution MPC method. Tigecycline is the first of a new class of compound – glycylcyclines- with potent in vitro activity against Gram-positive organisms including penicillin-resistant and multi-drug resistant Streptococcus pneumoniae (SP) and methicillin-resistant Staphylococcus aureus (MRSA). We measured minimum inhibitory concentration (MIC) and MPC values for tigecycline against 47 clinical isolates of SP and found that the MPC₉₀ values were >500 fold higher than the MIC₉₀ values. To determine if MPC testing of tigecycline against SP is impacted by blood in the medium, we developed a new medium able to sustain the growth of SP without the need for blood; solidified Todd-Hewitt broth (sTHB). The MPC₉₀ values of tigecycline against SP on sTHB were only 2 fold higher than the MIC₉₀ values. When blood was added to the sTHB, the MPC₉₀ values again became much greater than the MIC₉₀ values (> 256 fold higher). MPC results for Staphylococcus spp. against tigecycline were not impacted by blood in the medium. Benzalkonium chloride (BAK) is a cationic surface-acting agent that acts on bacterial cells by disrupting the intermolecular interaction of the lipid bilayer. To determine if the fluoroquinolones gatifloxacin (Gfx) and moxifloxacin (Mfx) are more active (lower MIC values) in the presence of BAK, we conducted MIC, MPC, and time-kill assays. MIC testing showed that in the presence of 3.125 to 50 µg/ml of BAK, the MIC of Gfx and Mfx decreased by 8- to 5000-fold against clinical isolates of methicillin-susceptible Staphylococcus aureus (MSSA), MRSA, Coagulase-negative Staphylococci(CNS), SP, Escherichia coli (EC), and Pseudomonas aeruginosa (PA). MPC testing showed that the presence of 7 to 10 µg/ml of BAK, the MPC of Gfx and Mfx decreased by 32- to 1000-fold against clinical isolates of MRSA. Conventional time-kill studies (using a bacterial load of 10⁵ CFUs) showed that the killing activity of Gfx against clinical MRSA isolates was enhanced in the presence of BAK with a log₁₀-reduction (percent kill) of 1.6 (76.08%) for Gfx alone at 180 minutes compared to a log₁₀-redecution (percent kill) of 5.4 (100%) for Gfx plus BAK at 180 minutes. Alexidine (Alx) is a bisbiguanide that has been used as an effective disinfectant in the dental industry and is potentially being developed for use as an antimicrobial agent for ocular infections. We conducted susceptibility testing of Alx using MIC testing, MPC testing, and time-kill assays against Gram-positive and Gram-negative pathogens. MIC testing showed that Alx is more active against Gram-positive pathogens than Gram-negative pathogens and showed better activity than the fluoroquinolones Gfx, Mfx, and levofloxacin (Lfx) against MRSA. The MPC values measured for MRSA and MSSA against Alx were non-reproducible using the traditional MPC method. Using the microbroth dilution MPC method, MPC₉₀ values were found to be 32 fold higher than the MIC₉₀ values. If the experimentally determined MPC values are “true” MPC values, initial MPC testing indicates that Alx may have a high likelihood for selecting for resistance, however, if the MPC values are not accurate it may be necessary to modify the MPC protocol in order to complete MPC testing of Alx against MRSA and MSSA. Conventional time-kill studies (using a bacterial load of 10⁵ CFUs) measured bactericidal activity (> 3 log₁₀-reduction) against MRSA, MSSA, SP, and PA.
Mutant Prevention Concentration, Alexidine, Benzalkonium chloride, Tigecycline, Antimicrobial Resistance
Master of Science (M.Sc.)
Microbiology and Immunology
Microbiology and Immunology