A Novel Plasmid-Mediated Efflux Pump Gene Confers Transferable Resistance to Tigecycline in <i>Klebsiella pneumoniae</i> from Human and Animals

Abstract

Background: Tigecycline is considered one of the last-resort antimicrobials for carbapenem-resistant K. pneumoniae. Plasmid-mediated tigecycline resistance remains largely unclear except for the recently reported tet (X3) and tet (X4). Methods: To investigate the unknown plasmid-mediated mechanism, comparative analysis of HiSeq and PacBio sequencing data were performed on 5 isolates of K. pneumoniae with similar genetic background but different resistance profile to tigecycline. Conjugation assay and subcloning were utilized to confirm the function of the novel plasmid-mediated gene in tigecycline resistance. The prevalence of this gene was screened in 257 tigecycline-non-susceptible K. pneumoniae (TNSKP) from a nationwide collection of 10536 Gram-negative bacilli. Plasmid characteristics were further evaluated by growth curve assay, whole genome sequencing and phylogenetic analysis. Finding: A novel plasmid-mediated tigecycline resistance gene, pmexAB-oprY, a 6, 489bp RND family efflux pump was identified from K. pneumoniae in both patients and animals. Conjugation and transformation experiments indicated that pmexAB-oprY increased tigecycline MICs by 8-16-fold in the recipient without fitness cost. The pump gene was mainly present in IncFIA, IncFIB and IncFII type plasmids, the size of which ranging from 110-kb to 200-kb. Importantly, 7 animal and 3 inpatient isolates carried both pmexAB-oprY and mcr ( mcr-1, mcr-8 · 1, mcr-3 · 11 and a new mcr-8 variant mcr-8 · 5 ), conferring resistance to tigecycline and colistin. Among them, pmexAB-oprY was present with mcr-8 · 1 or mcr-8 · 5 in one single transferable plasmid in 4 animal isolates. Alarmingly, the plasmid-mediated pmexAB-oprY and mcr co-existed with blaNDM -harboring IncX3 plasmid in the same host, resulting in pandrug resistance. Phylogenetic analysis suggested that pmexAB-oprY originated from the chromosome of Aeromonas spp. through Tn5393-mediating translocation to the plasmid. Both plasmid-harbored pmexAB-oprY gene and mcr-8 likely originated from animal isolates and then spread to human. The prevalence of pmexAB-oprY in TNSKP were 52·4% in animals and 2·5% in patients, respectively. Interpretation: Our findings reveal a novel plasmid-mediated mechanism in TNSKP, highlighting a substantial threat of the pmexAB-mcr8 co-harboring IncFIA/ IncFII plasmid to public health due to their mobile resistance. We emphasize the urgent need for a ‘one-health’ strategy and suggest that further global surveillance on the distribution and dissemination of this plasmid is in great need to assess its clinical significance impact on public health. Funding Statement: National Natural Science Foundation for Distinguished Young Scholars of China Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Patients information were not included in this study; thus, ethical approval was waived (Acceptance number of Ethics Review Committee of Peking University People's Hospital: 2016-PHB-135).