A novel plasmid-mediated β-lactamase that hydrolyzes broad-spectrum cephalosporins in a clinical isolate of Klebsiella pneumoniae

A new extended-spectrum beta -lactamase with an isoelectric point (pl) of 6.2 was detected in Klebsiella pneumoniae F161 that was isolated from a patient with infection. This strain was highly resistant to the third or fourth generation cephalosporins such as ceftazidime, ceftriaxone, cefoperazone, and cefpirome. Analysis of this strain by the double disk diffusion test showed synergies between amoxicillin-clavulanate (AMX-CA) and cefotaxime, and AMX-CA and aztreonam, which suggested that this strain produced a extended-spectrum beta -lactamase (ESBL). Genetic analysis revealed that the resistance was due to the presence of a 9.4-kb plasmid, designated as pKP161, encoding for new beta -lactamase gene (bla). Sequence analysis showed that a new bla gene of pKP161 differed from bla(TEM-1) by three mutations leading to the following amino acid substitutions: Val(84) --> Ile, Ala(184) --> Val, and Gly(238) --> Ser These mutations have not been reported previously in the TEM type beta -lactamases produced by clinical strains. The novel beta -lactamase was overexpressed in E. coli and purified by ion exchange chromatography on Q-Sepharose and CM-Sepharose, and then further purified by gel filtration on Sehadex G-200. The catalytic activity of the purified beta -lactamase was confirmed by the nitrocefin disk.