Characterization of GUA-1, a chromosomally encoded ESBL from Pseudomonas guariconensis-like

Putative novel β-lactamase-encoding genes are increasingly identified in whole-genome sequencing (WGS) data, often without phenotypic or biochemical characterization. Here, we characterized the chromosome-encoded GUA-1 Ambler class A β-lactamase from the clinical isolate Pseudomonas guariconensis-like 65411. Among the 40 P. guariconensis genomes available in the GenBank database, only 8 carried a bla_GUA-like gene, whereas the others encoded an AmpC β-lactamase, suggesting the existence of two distinct subspecies. Notably, these eight isolates, despite originating from diverse geographical regions, all harbored the bla_GUA-like gene inserted at the same chromosomal locus. Heterologous expression in Pseudomonas aeruginosa and Escherichia coli revealed a clavulanic-acid-inhibited cefotaximase-type ESBL. GUA-1 shared 72% amino acid sequence identity with putative Ambler class A β-lactamases from Pseudomonas fulva, Pseudomonas mosselii, and Pseudomonas soli, and 57% with LUT-1, a class A cephalosporinase from Pseudomonas luteola. Kinetic analyses showed that GUA-1 hydrolyzed cefotaxime but not ceftazidime, similarly to LUT-1 and CTX-M-1-type ESBLs. IC₅₀ measurements indicated strong inhibition by clavulanic acid, tazobactam, and avibactam, but not by vaborbactam. Molecular modeling supported these findings, showing favorable binding of cefotaxime in the active site, whereas steric hindrance from Trp274 and electrostatic repulsion from Asp240 likely prevent ceftazidime binding. The bla_GUA-1 gene was chromosomally located, with no obvious promoter identified in the immediate upstream region. RT-PCR experiments revealed expression levels comparable to other β-lactamase genes present (bla_DHA, bla_CMY, bla_OXA-1, and bla_OXA-204) and to the upstream-located Na+/H antiporter gene. 5' RACE experiments suggested that the bla_GUA-1 gene is co-transcribed along with the Na+/H gene. Overall, this study highlights the diversity of β-lactamases within Pseudomonas species.