Pooling isolates to address the diversity in antimicrobial susceptibility of Pseudomonas aeruginosa in cystic fibrosis

The intrasample diversity in antibiotic resistance of Pseudomonas aeruginosa in people with cystic fibrosis (pwCF) may contribute to the low predictive value of antibiotic susceptibility testing (AST). Since often only few isolates per AST are included, a possible solution to decrease the diversity is pooling isolates, yet a comprehensive study on this approach is lacking. This study aims to characterize the intrasample diversity in antibiotic resistance of P. aeruginosa and to assess if pooling isolates could lower diversity. In 15 pwCF, 30 P. aeruginosa isolates were collected per patient sputum sample, and the minimal inhibitory concentration (MIC) was determined for three antibiotics commonly used to treat cystic fibrosis patients to evaluate intrasample diversity. Next, the minimal number of isolates necessary to be pooled for a consistent MIC was determined. Finally, we assessed if the MIC result of pooled versus single isolates could influence clinical interpretation. Intrasample MIC diversity was observed in most samples, with the highest frequencies for aztreonam and ceftazidime. Pooling isolates decreased diversity, and for each sputum sample, a consistent MIC was obtained after pooling two to nine isolates. The minimal number of pooled isolates for consistent MIC positively correlated with MIC diversity in single isolate testing. MIC diversity and categorical disagreement were correlated, and the level of categorical disagreement decreased in most samples when pooling nine isolates. Also, pooling isolates did not decrease the frequency of resistance detection in most samples. In conclusion, intrasample MIC diversity clearly impacts AST outcome for P. aeruginosa in pwCF, and pooling isolates is a promising approach to lower diversity. IMPORTANCE People with cystic fibrosis (pwCF) often suffer from chronic lung infections with Pseudomonas aeruginosa. While antibiotics are still commonly used to treat P. aeruginosa infections, there is a high discordance between in vitro and in vivo antibiotic efficacy, which contributes to suboptimal antibiotic therapy. In the present study, we found that isolates from the same sputum sample had highly diverse antibiotic resistance profiles [based on the minimal inhibitory concentration (MIC)], which may explain the reported discrepancy between in vitro and in vivo antibiotic efficacy. Through systematic analysis, we report that pooling nine isolates per sputum sample significantly decreased intrasample diversity in MIC and influenced clinical interpretation of antibiotic susceptibility tests compared to single isolate testing. Hence, pooling of isolates may offer a solution to obtain a consistent MIC test result and could lead to optimizing antibiotic therapy in pwCF and other infectious diseases where diversity in antibiotic resistance is observed.