Klebsiella pneumoniae ML2011, amultiresistant isolate,was isolated fromtheMilitaryHospital ofTunis (Tunisia).Thedetermination\nof the minimal inhibitory concentrations exhibited by K. pneumoniae ML2011 was performed by Etest. The crude extract of the\nisolates contains four different �Ÿ-lactamases with pI 5.5, 7.3, 7.6, and 8.6. Only the �Ÿ-lactamases with pI 7.3 and pI 8.6were transferred\nby transformation and conjugation experiment.Molecular characterization of these genes was performed by PCR and sequencing.\nThe chromosomal �Ÿ-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-\n104 (pI 7.3) were encoded by bla gene located on a 50 kb highly conjugative plasmid. The SHV-104 �Ÿ-lactamase was produced\nin E. coli and purified. Its profile of activity was determined. Compared to SHV-1, SHV-104 contains one mutation, R202S. Their\nkinetic parameters were similar except for cefotaxime.The analysis of the predicted structure of SHV-104 indicated that the R202S\nmutation suppresses a salt bridge present in SHV-1. Therefore, the overall flexibility of the protein increased and might improve the\nhydrolysis of cefotaxime. We can conclude that the multiresistant phenotype of K. pneumoniae ML2011 strain is mainly linked to\nthe production of CTX-M-28 since SHV-104 possesses a narrow spectrum of activity.
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