Genetic, biochemical, and structural characterization of a novel KLUS subgroup of CTX-M-type extended-spectrum β-lactamases reveals an evolutionarily distinct lineage with unique functional properties

Extended-spectrum β-lactamases (ESBLs), particularly the CTX-M family, have progressively undermined the clinical utility of β-lactam antibiotics. Herein, we identified and characterized a novel CTX-M subgroup designated KLUS, and investigated its taxonomic position and functional properties. Phylogenetic analysis combined with genetic distance calculations supported KLUS as a distinct lineage, with inter-subgroup distances significantly exceeding intra-subgroup distances and distinct clustering (bootstrap support >95%), consistent with the previously established family-wide CTX-M classification. Recombinant plasmids harboring each of the four bla KLUS alleles were constructed in E. coli BL21 (DE3), with optimal expression achieved using 1.0 mM IPTG at 30 °C for 5 h. Minimum inhibitory concentration (MIC) determinations revealed that KLUS-1 through KLUS-3 conferred high-level resistance to ampicillin and ceftriaxone, moderate resistance to cefotaxime, and minimal resistance to ceftazidime (MIC < 0.125 μg/mL). KLUS-4 exhibited enhanced ceftazidime resistance (MIC = 8 μg/mL) and a reduced inhibitor response at the phenotypic level (a 16-fold reduction of the cefotaxime MIC with clavulanic acid, in contrast to KLUS-1, where clavulanic acid reduced the MIC to <0.015 μg/mL). β-lactamase inhibitors reduced cefotaxime MICs far exceeding 16-fold in KLUS-1 through KLUS-3, consistent with ESBL phenotypes. Steady-state kinetic analyses demonstrated subtype-dependent catalytic efficiencies that were highest for ampicillin and penicillin G in KLUS-2/3/4 and for ceftriaxone in KLUS-1, broadly consistent with MIC results. The substantially lower catalytic efficiency for ceftazidime may explain its reduced resistance phenotype. Structural modeling revealed that KLUS enzymes comprise 11 α-helices and 5 β-sheets, with a conserved catalytic cavity and SDN loop. Twenty-three unique amino acid substitutions distinguish KLUS from CTX-M-2. V77I (pre-protein positions 106) is located proximal to the Ω-loop region, and any functional contribution of this conservative substitution remains speculative. These findings expand the known diversity of CTX-M β-lactamases; the clinical significance of KLUS requires further investigation in clinical isolates.