Study of a Natural Mutant SHV-Typeβ-Lactamase, SHV-104, fromKlebsiella pneumoniae

Abstract

Klebsiella pneumoniaeML2011, a multiresistant isolate, was isolated from the Military Hospital of Tunis (Tunisia). The determination of the minimal inhibitory concentrations exhibited byK. pneumoniaeML2011 was performed by Etest. The crude extract of the isolates contains four differentβ-lactamases with pI 5.5, 7.3, 7.6, and 8.6. Only theβ-lactamases with pI 7.3 and pI 8.6 were transferred by transformation and conjugation experiment. Molecular characterization of these genes was performed by PCR and sequencing. The chromosomalβ-lactamases are TEM (pI 5.5) and SHV-1 (7.6). CTX-M-28 (pI 8.6) and the novel variant of SHV named SHV-104 (pI 7.3) were encoded byblagene located on a 50 kb highly conjugative plasmid. The SHV-104β-lactamase was produced inE. coliand purified. Its profile of activity was determined. Compared to SHV-1, SHV-104 contains one mutation, R202S. Their kinetic parameters were similar except for cefotaxime. The analysis of the predicted structure of SHV-104 indicated that the R202S mutation suppresses a salt bridge present in SHV-1. Therefore, the overall flexibility of the protein increased and might improve the hydrolysis of cefotaxime. We can conclude that the multiresistant phenotype ofK. pneumoniaeML2011 strain is mainly linked to the production of CTX-M-28 since SHV-104 possesses a narrow spectrum of activity.