Molecular, Genetic, and Biochemical Characterization of OXA-484 Carbapenemase, a Difficult-to-Detect R214G Variant of OXA-181

• Published in: Microorganisms (Basel) Vol. 12; no. 7; p. 1391
• Main Authors: Gonzalez, Camille, Oueslati, Saoussen, Rima, Mariam, Nermont, Réva, Dortet, Laurent, Hopkins, Katie L, Iorga, Bogdan I, Bonnin, Rémy A, Naas, Thierry
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 09.07.2024; MDPI
• Subjects: antibiotic resistance; Antibiotics; Antimicrobial agents; Bacteriology; beta-lactamase; Carbapenemase; Carbapenems; Citrobacter; Clinical isolates; Cloning; detection; E coli; Enzymes; Food; Gene flow; Genes; Genomes; Genomics; Hydrolysis; Imipenem; Immunoassay; Immunoassays; Life Sciences; Microbiology and Parasitology; Molecular modelling; OXA-181; oxacillinase; Plasmids; Selective media; Substrates; Temocillin; United Kingdom > UK; France; United States > US; Germany; oxacillinase; detection; OXA-181; beta-lactamase; carbapenemase; antibiotic resistance
• ISSN: 2076-2607; 2076-2607
• DOI: 10.3390/microorganisms12071391

OXA-244, an R214G variant of OXA-48, is silently spreading worldwide likely because of difficulties in detection using classical screening media. Here, we characterized two clinical isolates of and that displayed reduced susceptibility to carbapenems but were lacking significant carbapenemase activity as revealed by negative Carba NP test results. However, positive test results were seen for OXA-48-like enzymes by lateral flow immunoassays. WGS revealed the presence of a OXA-181-like gene that codes for OXA-484, an R214G variant of OXA-181. OXA-484 gene was located on a 58.4-kb IncP1-like plasmid (pN-OXA-484), that upon transfer into HB4 with impaired permeability, conferred carbapenem and temocillin resistance (MICs > 32 mg/L). TOP10 (pTOPO-OXA-484) revealed reduced MICs in most substrates as compared to TOP10 (pTOPO-OXA-181), especially for imipenem (0.25 mg/L versus 0.75 mg/L) and temocillin (16 mg/L versus 1028 mg/L). Catalytic efficiencies of OXA-484 were reduced as compared to OXA-181 for most ß-lactams including imipenem and temocillin with 27.5- and 21.7-fold reduction, respectively. Molecular modeling confirmed that the salt bridges between R214, D159, and the R1 substituent's carboxylate group of temocillin were not possible with G214 in OXA-484, explaining the reduced affinity for temocillin. In addition, changes in active site's water network may explain the decrease in hydrolysis rate of carbapenems. OXA-484 has weak imipenem and temocillin hydrolytic activities, which may lead to silent spread due to underdetection using selective screening media or biochemical imipenem hydrolysis confirmatory tests.