Between 1986 and 1988, multiresistant strains exhibiting high-level cefotaxime resistance were isolated from patient specimens particularly of the intensive care units of the Aachen Technical University Hospital. The resistance gene responsible was shown to be encoded on a conjugative 66 kb plasmid designated pZMPI. The MIC values for cefotaxime of the original isolates and the transconjugants were > 128 mg I and 64 mg I. respectively, Isoelectric focusing of protein preparations from the transconjugants showed a β-lactamase with a pl of 7.6. A 3.6 kb Hl fragment containing the β-lactamase gene was cloned into pLG339 resulting in the recombinant plasmid pZMP1-1. A restriction map of the cloned insert was established and l subfragments of the insert were further subcloned into pBGS18. The nucleotide sequence of the complete 3.6 kb fragment was determined. Within 3663 bp an open reading frame of 858 kb was found to show 99% homology to the SHV-2 and -3 nucleotide sequences. The deduced amino acid sequence differed in one and two positions, respectively, from these established SHV enzymes. The 3′ noncoding sequence exhibited nearly perfect homology to that of SHV-2, but the 5′ upstream sequence showed homology of less than 50% to the corresponding SHV-2 sequence, indicating an altered promoter region of the variant SHV-enzyme. Kinetic analysis of the β-lactamase revealed a 50-100% elevated hydrolytic effectivity on cefotaxime in comparison to other SHV enzymes. Cefoxitin, ceftazidime, aztreonam and imipenem were not hydrolysed by the enzyme. The variant enzyme was inhibited by commonly available β-lactamase inhibitors. Clavulanic acid had the highest affinity for the enzyme and the greatest effectivity in blocking its action. Based on the genetic and kinetic data we propose to classify the enzyme as a new variant β-lactamase of the SHV-type and name it SHV-2a.


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