Increased Plasmid Copy Number Contributes to the Elevated Carbapenem Resistance in OXA-232-Producing .

A total of 345 unique (one isolate per patient) clinical carbapenem-resistant isolates were recovered in our hospital over the past 3 years (2016-2018), with 325, 14, and 6 isolates carried , , and , respectively. The six OXA-232-Kp isolates were recovered in neurosurgery intensive care unit in 2018. All OXA-232-Kp belonged to ST15, differed by one or two pulsed-field gel electrophoresis (PFGE) bands, and belonged to the same clone. However, three isolates (RJ18-04-6) displayed elevated carbapenem resistance, with imipenem minimum inhibitory concentration (MIC) of 16-32 μg/mL. In contrast, other three isolates (RJ18-01-3) had imipenem MIC of 0.5-1 μg/mL. S1-PFGE revealed two different plasmid profiles and all strains had at least four plasmids. Strain RJ18-01 and RJ18-06 were selected for whole-genome sequencing, and a ColE2-type 6,141 bp -carrying plasmid was identified. No gene was located on chromosome or other plasmids. Furthermore, no β-lactamase resistance gene other than was identified. Polymerase chain reaction-based sequencing of -carrying plasmid was performed, and all OXA-232-Kp shared identical -carrying plasmid sequences. The expression and copy number of in RJ18-04-6 were significantly higher than those of other isolates. The hydrolysis activity of nitrocefin and carbapenems were about six-fold higher in RJ18-04-6. Since only one copy of gene was spotted on the plasmid, the elevated carbapenem resistance could be attributed to the increased copy number of -carrying plasmids. OmpK35 porin deficiency was observed in one isolate with decreased carbapenem susceptibility and two isolates with elevated carbapenem resistance, suggesting that OmpK35 deficiency did not significantly alter carbapenem MICs in OXA-232-Kp.