Effects of different mechanisms on antimicrobial resistance in <i>Pseudomonas aeruginosa</i> : a strategic system for evaluating antibiotics against gram-negative bacteria

Our previous studies constructed a strategic system for testing antibiotics against specific resistance mechanisms using <i>Klebsiella pneumoniae</i> and <i>Acinetobacter baumannii</i>. However, it lacked resistance mechanisms specifically expressed only in <i>Pseudomonas</i> species. In this study, we constructed this system using <i>Pseudomonas aeruginosa</i>. In-frame deletion, site-directed mutagenesis, and plasmid transformation were used to generate genetically engineered strains with various resistance mechanisms from two fully susceptible <i>P. aeruginosa</i> strains. Antimicrobial susceptibility testing was used to test the efficacy of antibiotics against these strains in vitro. A total of 31 engineered strains with various antimicrobial resistance mechanisms from <i>P. aeruginosa</i> KPA888 and ATCC 27853 were constructed, and the same antibiotic resistance mechanism showed a similar effect on the MICs of the two strains. Compared to the parental strains, the engineered strains lacking porin OprD or lacking the regulator genes of efflux pumps all showed a ≥4-fold increase on the MICs of some of the 19 antibiotics tested. Mechanisms due to GyrA/ParC mutations and β-lactamases also contributed to their corresponding resistance as previously published. The strains constructed in this study possess well-defined resistance mechanisms and can be used to screen and evaluate the effectiveness of antibiotics against specific resistance mechanisms in <i>P. aeruginosa</i>. Building upon our previous studies on <i>K. pneumoniae</i> and <i>A. baumannii</i>, this strategic system, including a <i>P. aeruginosa</i> panel, has been expanded to cover almost all the important antibiotic resistance mechanisms of gram-negative bacteria that are in urgent need of new antibiotics.IMPORTANCEIn this study, an antibiotic assessment system for <i>P. aeruginosa</i> was developed, and the system can be expanded to include other key pathogens and resistance mechanisms. This system offers several benefits: (i) compound design: aid in the development of compounds that can bypass or counteract resistance mechanisms, leading to more effective treatments against specific resistant strains; (ii) combination therapies: facilitate the exploration of combination therapies, where multiple antibiotics may work synergistically to overcome resistance and enhance treatment efficacy; and (iii) targeted treatments: enable healthcare providers to prescribe more targeted treatments, reducing unnecessary antibiotic use and helping to slow the spread of antibiotic resistance. In summary, this system could streamline the development process, reduce costs, increase the success rate of new antibiotics, and help prevent and control antimicrobial resistance.