Role of SHV β-lactamase variants in resistance of clinical Klebsiella pneumoniae strains to β-lactams in an Algerian hospital

Three clinical Klebsiella pneumoniae strains, KpARG74, KpARG220 and KpARG185, isolated from a hospital in Algeria, carried the novel β-lactamases SHV-98, SHV-99 and SHV-100, respectively, and co-expressed TEM-1 and either CTX-M-3 or CTX-M-15. In contrast, transformed cells possessing the genes for these novel β-lactamases, i.e. EcDH5α-SHV-98, EcDH5α-SHV-99 and EcDH5α-SHV-100, respectively, carried unique sequence features of bla(SHV) gene variants, enabling oxyimino-cephalosporin susceptibility and confirming that none of the transformants exhibited extended-spectrum β-lactamase (ESBL) properties. SHV-100 is apparently functional, despite differing from the SHV-1 sequence by duplication of 13 amino acids. The SHV-99 enzyme differed from the parental SHV-1 by the amino acid substitution Asp104→Gly, which is an important position in the development of the ESBL phenotype in TEM β-lactamases. This is the first time, to our knowledge, that this mutation has been reported in clinically occurring isolates. Thus, kinetic characterization of the SHV-99 enzyme was performed. The SHV-99 enzyme showed higher affinity (K(m) of 196 µM), catalytic activity (k(cat) of 0.5 s⁻¹) and catalytic efficiency (k(cat)/K(m) of 0.003 µM⁻¹ s⁻¹) than SHV-1 β-lactamase against aztreonam. These results showed that the neutral glycine at residue 104 increased the affinity of the enzyme to aztreonam, but was unable to develop the ESBL phenotype in SHV enzymes. As the emergence of new threatening combinations of resistance determinants among nosocomial pathogens is further possible, this study has highlighted the need to reverse the spread of initial mutations.