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(3')-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: CHDL; Pseudomonas aeruginosa; blaOXA-10-like gene; blaOXA-677.

Figure 1

The genomic context of the bla_OXA-677 (A), bla_OXA-101 (B), bla_OXA-246 (C). Genes are denoted by arrows. Genes, MGEs, and other features are colored based on their functional classification.

Figure 2

Amino acid alignment of OXA-10 and three OXA-10-like enzymes identified in this study. A dash indicates synonymous substitution without amino acid change. Key amino acid motifs conserved among OXA-10 are highlighted in red.

Figure 3

Superposition of OXA-10/OXA-101/ OXA-246 (blue), OXA-655 (yellow), and OXA-677 (red) with detailed view of active site. Leu155 and the omega loop are shown and labeled by the three mixed colors. Meropenem is depicted in green as a substrate. In the OXA-677 enzyme, the phenylalanine117 is closer to leucine155 in the omega loop.