SHV-16, a beta-lactamase with a pentapeptide duplication in the omega loop

A clinical isolate of Klebsiella pneumoniae was found to be resistant to am picillin (MIC of 128 mug/ml), ticarcillin (MIC of 512 mug/ml), and ceftazid ime (MIC of 128 mug/ml) and susceptible to all other P-lactams; a synergist ic effect between clavulanate and ceftazidime suggested the presence of an extended-spectrum P-lactamase (ESBL). Transconjugants in Escherichia coli w ere obtained at low levels (10(-7) per donor cell) and exhibited a similar beta -lactam resistance pattern (resistant to ampicillin, ticarcillin, and ceftazidime at 64 mug/ml). The ESBL, pI 7.6, was encoded by a large plasmid (> 100 kb) which did not carry any other resistance determinant. The ESBL- encoding gene was amplified by PCR using bla(SHV)-specific primers and was sequenced. The deduced amino acid sequence of the SHV-16 ESBL showed that i t differed from SHV-1 by only a pentapeptide insertion (163DRWET167) corres ponding to a tandem duplication in the omega loop. The implication of the 1 63a-DRWET163b-DRWET sequence in ceftazidime resistance was confirmed by clo ning either bla(SHV-1) or bla(SHV-16) in the same vector, subsequently intr oduced in the same E. coli strain. Under these isogenic conditions, SHV-16 conferred a 32-fold increase in ceftazidime MIC compared to that with SHV-1 . Furthermore, site-directed mutagenesis experiments modifying either E166a A or E166bA revealed that the functional glutamic residue was that located in the first copy of the duplicated sequence. But surprisingly, the second E166b also conferred a low-level resistance to ceftazidime. This work is th e first description of a class A enzyme exhibiting an extended substrate sp ecificity due to an insertion instead of a nucleotide substitution(s) in a clinical isolate.