Molecular Characterization of a Cephamycin-Hydrolyzing and Inhibitor-Resistant Class A β-Lactamase, GES-4, Possessing a Single G170S Substitution in the Ω-Loop

Abstract

The nosocomial spread of six genetically related Klebsiella pneumoniae strains producing GES-type β-lactamases was found in a neonatal intensive care unit, and we previously reported that one of the six strains, strain KG525, produced a new β-lactamase, GES-3. In the present study, the molecular mechanism of cephamycin resistance observed in strain KG502, one of the six strains described above, was investigated. This strain was found to produce a variant of GES-3, namely, GES-4, which was responsible for resistance to both cephamycins (cefoxitin MIC, >128 μg/ml) and β-lactamase inhibitors (50% inhibitory concentration of clavulanic acid, 15.2 ± 1.7 μM). The GES-4 enzyme had a single G170S substitution in the Ω-loop region compared with the GES-3 sequence. This single amino acid substitution was closely involved with the augmented hydrolysis of cephamycins and carbapenems and the decreased affinities of β-lactamase inhibitors to GES-4. A cloning experiment and sequencing analysis revealed that strain KG502 possesses duplicate bla _GES-4 genes mediated by two distinct class 1 integrons with similar gene cassette configurations. Moreover, the genetic environments of the bla _GES-4 genes found in strain KG502 were almost identical to that of bla _GES-3 in strain KG525. From these findings, these two phenotypically different strains were suggested to belong to a clonal lineage. The bla _GES-4 gene found in strain KG502 might well emerge from a point mutation in the bla _GES-3 gene harbored by its ancestor strains, such as strain KG525, under heavy antibiotic stress in order to acquire extended properties of resistance to cephamycins and carbapenems.