Overproduction of a Metallo-β-Lactamase by a Strong Promoter Causes High-Level Imipenem Resistance in a Clinical Isolate of Pseudomonas aeruginosa

BACKGROUND: Metallo-beta-lactamase is one of the feared resistance mechanisms in Pseudomonas aeruginosa. METHODS: beta-Lactamase activity was determined using crude enzymes. Cloned plasmids were transformed in P. aeruginosa KG2505, PAO1 derivative without an AmpC beta-lactamase and a MexAB-OprM efflux pump. RESULTS: P. aeruginosa No. 20232 was highly resistant to imipenem (minimum inhibitory concentration >512 microg/ml). It possessed IMP-10 and the activity was 1.89 +/- 0.42 micromol/min/mg of protein, which was 5-8 times higher than other tested isolates. The bla(IMP-10) promoter was strong (-35 region, TTGACA; -10 region, TAAACT); on the other hand, bla(IMP) of other isolates had a hybrid promoter. Transformants with plasmid encoding bla(IMP-10) with a strong promoter had higher enzymatic activity and were less susceptible to imipenem than those encoding bla(IMP-10) with a hybrid promoter. CONCLUSIONS: The high metallo-beta-lactamase activity caused by a strong promoter was a major determinant for high-level imipenem resistance of P. aeruginosa No. 20232.