First Report of bla_OXA-677 with Enhanced Meropenem-Hydrolyzing Ability in Pseudomonas aeruginosa in China

Abstract

Purpose

OXA-10-type class D β-lactamases have shown their evolutionary potential of enhancing carbapenem resistance. This study aimed to elucidate the role of OXA-10 variants in clinical isolated multidrug resistant (MDR) Pseudomonas aeruginosa and characterize the first appearance of OXA-677 in China.

Methods

Six bla_OXA-10-like-positive strains were screened by PCR from 41 P. aeruginosa strains, which were resistant to both carbapenems and ceftazidime-avibactam, collected across China in 2018. The minimum inhibitory concentrations (MIC) were determined with the broth microdilution method. The resistance-associated genes and genetic environment were investigated by whole-genome sequencing (WGS). The function and mechanism of OXA-677 β-lactamase were identified by molecular cloning and protein structure modeling.

Results

All the bla_OXA-10-like-positive Pseudomonas aeruginosa were MDR strains. They also had outer membrane porin defects and produced β-lactam resistance gene bla_PER-1, fluoroquinolone-resistant gene crpP, aminoglycoside-resistance gene aph(3ʹ)-IIb, aph(6)-Id, aacA and aadA, fosfomycin-resistance gene fosA, sulfamethoxazole-resistance gene sul1, and chloramphenicol-resistance gene catB7. All bla_OXA-10 variants were located in a Tn1403-related transposon, containing aacA4-12-bla_OXA-677-aadA1, aacA4-12-bla_OXA-101-aadA5, and bla_OXA-246-aacA3-aadA13 gene cassette arrays, respectively. Notably, the bla_OXA-677 producer showed a high MIC level of meropenem (MIC>64 mg/L). Compared to bla_OXA-10, bla_OXA-677 was found a G-to-T transversion at position 350, leading to a phenylalanine-for-valine substitution in position 117, which is closer to leucine155 in the omega loop of the active site. MIC of meropenem for E. coli DH5α with the recombinant plasmid pHSG398 carrying bla_OXA-677 was elevated by 8 times.

Conclusion

We speculate that the OXA-10-like enzymes and the decrease of membrane permeability confer carbapenem resistance, and the V117 substitution in OXA-677 might lead to a higher resistance level of meropenem.

Keywords:

• Pseudomonas aeruginosa
• bla _OXA-677
• bla _{OXA-10-like gene}
• CHDL

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81871690 and 81902101), and China Antimicrobial Surveillance Network (2020QD049).

Ethics Approval

The study protocol was approved by the Institutional Review Board of Huashan Hospital, Fudan University (No. 2020-041).

Disclosure

The authors report no conflicts of interest in this work.