ABSTRACTThe rapid acquisition of antibiotic resistance ofPseudomonas aeruginosahas been a complex problem in clinics. Two meropenem-resistantP. aeruginosaisolates were collected from the same patient on May 24, 2021, and June 4, 2021, respectively. The first was susceptible to aztreonam, while the second displayed resistance. This study aimed to identify the genetic differences between twoP. aeruginosaisolates and uncover alterations formed by the within-host bacterial evolution leading to aztreonam resistance during therapy. Strains were subjected to antimicrobial susceptibility testing using the broth microdilution method. Genomic DNAs were obtained to identify their genetic differences. The relative mRNA levels of β-lactam-resistance genes were determined by real-time PCR. Both isolates belonged to ST 773 high-risk clones with the same antibiotic resistance genes, eliminating the possibility of horizontally obtaining resistance genes. Reverse transcription (RT)-PCR results showed that theblaPDC-16mRNA level in the second one was about 1,500 times higher than that in the first one. When 3-aminophenyl boronic acid was added, the second strain recovered its susceptibility to aztreonam, which confirmed that the overexpression ofblaPDC-16was the main reason for the isolate’s resistance to aztreonam. Compared to the first strain, the second showed a single amino acid substitution in AmpR located upstream ofblaPDC-16, which may contribute to the upregulation ofblaPDC-16and lead to aztreonam resistance.AmpRplays an essential role in regulating antibiotic resistance inP. aeruginosa, and there is a need to be alert to clinical treatment failures associated with mutations inampR.IMPORTANCEPseudomonas aeruginosais notorious for being highly resistant to antimicrobial agents. In this study, twoP. aeruginosastrains isolated from the same patient with different susceptibility to aztreonam were used to illustrate the within-host resistance evolution process ofP. aeruginosa. Both isolates, which belonged to a ST773 high-risk clone, had the same β-lactam resistance genes (blaPDC-16,blaIMP-45,blaOXA-1, andblaOXA-395), which means the second isolate might have been derived from the first isolate by gaining aztreonam resistance via mutations associated with aztreonam resistance relative genes. Subsequently, we found that mutation inampRmay be the cause of aztreonam resistance in the second isolate. Mutation inampRleads to its loss of control overblaPDC-16, allowing overexpression ofblaPDC-16and further resistance to aztreonam. This study revealed thatampRplays an essential role in regulating antibiotic resistance inP. aeruginosa. There is a need to be alert to clinical treatment failures associated with mutations inampR.