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A new extended-spectrum β-lactamase with an isoelectric point (pl) of 6.2 was detected inKlebsiella pneumoniae F161 that was isolated from a patient with infection, This strain was highly resistant to the third or fourth Beneration 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 cefotaxi me, and AMX-CA and aztreonam, which suggested that this strain produced a extended-spectrum β-lactamase (ISBL). Genetic analysis revealed that the resistance was due to the presence of a 9.4-kb plasmic, designated as pkpl 61, encoding for new β-lactamase gene (bla). Sequence analysis showed that a new bla gene of Pkp161 differed from blaTEM-1 by three mutations leading to the following amino acid substitutions: Val84 -> lle, Ala184 -> Val, and G1y238 -> Ser. These mutations have not been reported previously in the TEM type β-lactamases produced by clinical strains. The novel β-lactamase was overexpressed in E. coli and purified by ion exchange chromatofiraphy on Q-Sepharose and CM-Sepharose, and then further purified by gel filtration on Sehadex G-200. The catalytic activity of the purified β- lactamase was confirmed by the nitrocefin disk.
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