Phenotypic and genotypic characterization of HMB-3, a metallo-beta-lactamase from Pseudomonas asiatica.

objectivesWe describe the identification and characterization of the HMB-3 variant, produced by a P. asiatica isolate from a patient in Switzerland. MATERIALS AND

methodsA carbapenem-resistant P. asiatica isolate was sent to the Swiss National Reference Center for Emerging Antibiotic Resistance for investigation. Antibiotic susceptibility testing was performed according to CLSI guidelines. WGS was performed on Illumina and Oxford Nanopore platforms. The blaHMB alleles were cloned into the pCR-Blunt II-TOPO plasmid. Site-directed mutagenesis was performed on blaHMB-1 and blaHMB-3 inserted into the pTOPO plasmids. Purified HMB-1, HMB-3, NDM-1 and IMP-1 were used for steady state kinetic measurements of hydrolysis of selected beta-lactams. RESULTS AND DISCUSSION: The isolate was obtained from a tracheostomy wound swab. Susceptibility testing showed that it was resistant to all beta-lactams, except aztreonam; however, no classical carbapenemase genes were identified by routine testing. WGS produced a complete chromosome of 6.1 Mb but no plasmids, and identified a gene encoding a novel MBL, namely HMB-3, differing from HMB-1 by 23 amino acid substitutions. HMB-3 was chromosomally encoded and located within a 25.6 kb genomic island. HMB-3 conferred resistance to most beta-lactam antibiotics, except piperacillin (MIC 4 mg/L), aztreonam (0.125 mg/L) and cefiderocol (2 mg/L). Site-directed mutagenesis of blaHMB-1 and blaHMB-3 revealed that a single amino acid change, E181H/H181E, in active site loop 10, could significantly alter the MIC of cefiderocol. HMB-3 demonstrated increased hydrolytic activity against cefiderocol compared with HMB-1 and NDM-1.

conclusionsHere we described a novel MBL enzyme responsible for acquired resistance to carbapenems and reduced susceptibility to cefiderocol in Pseudomonas spp.