Emergence of a Plasmid-Encoded Resistance-Nodulation-Division Efflux Pump Conferring Resistance to Multiple Drugs, Including Tigecycline, in Klebsiella pneumoniae

Abstract

Transporters belonging to the chromosomally encoded resistance-nodulation-division (RND) superfamily mediate multidrug resistance in Gram-negative bacteria. However, the cotransfer of large gene clusters encoding RND-type pumps from the chromosome to a plasmid appears infrequent, and no plasmid-mediated RND efflux pump gene cluster has yet been found to confer resistance to tigecycline. Here, we identified a novel RND efflux pump gene cluster, designated tmexCD1-toprJ1, on plasmids from five pandrug-resistant Klebsiella pneumoniae isolates of animal origin. TMexCD1-TOprJ1 increased (by 4- to 32-fold) the MICs of tetracyclines (including tigecycline and eravacycline), quinolones, cephalosporins, and aminoglycosides for K.pneumoniae, Escherichia coli, and Salmonella TMexCD1-TOprJ1 is closely related (64.5% to 77.8% amino acid identity) to the MexCD-OprJ efflux pump encoded on the chromosome of Pseudomonas aeruginosa In an IncFIA plasmid, pHNAH8I, the tmexCD1-toprJ1 gene cluster lies adjacent to two genes encoding site-specific integrases, which may have been responsible for its acquisition. Expression of TMexCD1-TOprJ1 in E. coli resulted in increased tigecycline efflux and in K. pneumoniae negated the efficacy of tigecycline in an in vivo infection model. Expression of TMexCD1-TOprJ1 reduced the growth of E. coli and Salmonella but not K. pneumoniaetmexCD1-toprJ1-positive Enterobacteriaceae isolates were rare in humans (0.08%) but more common in chicken fecal (14.3%) and retail meat (3.4%) samples. Plasmid-borne tmexCD1-toprJ1-like gene clusters were identified in sequences in GenBank from Enterobacteriaceae and Pseudomonas strains from multiple continents. The possibility of further global dissemination of the tmexCD1-toprJ1 gene cluster and its analogues in Enterobacteriaceae via plasmids may be an important consideration for public health planning.IMPORTANCE In an era of increasing concerns about antimicrobial resistance, tigecycline is likely to have a critically important role in the treatment of carbapenem-resistant Enterobacteriaceae, the most problematic pathogens in human clinical settings-especially carbapenem-resistant K.pneumoniae Here, we identified a new plasmid-borne RND-type tigecycline resistance determinant, TMexCD1-TOprJ1, which is widespread among K. pneumoniae isolates from food animals. tmexCD1-toprJ1 appears to have originated from the chromosome of a Pseudomonas species and may have been transferred onto plasmids by adjacent site-specific integrases. Although tmexCD1-toprJ1 still appears to be rare in human clinical isolates, considering the transferability of the tmexCD1-toprJ1 gene cluster and the broad substrate spectrum of TMexCD1-TOprJ1, further dissemination of this mobile tigecycline resistance determinant is possible. Therefore, from a "One Health" perspective, measures are urgently needed to monitor and control its further spread. The current low prevalence in human clinical isolates provides a precious time window to design and implement measures to tackle this.

Keywords: Enterobacteriaceae; antimicrobial agents; efflux pumps; mechanisms of resistance; multidrug resistance; plasmid-mediated resistance.

FIG 1

The activity of TMexCD1-TOprJ1 on tigecycline in vitro and in vivo. (a) Tigecycline accumulation by E. coli DH5α carrying pHSG575 or pHSG575-tnfxB1-tmexCD1-toprJ1. Each bar and error bar show the mean value and standard deviation of three replicates. Student’s t tests were performed to analyze data. *, P < 0.05; **, P < 0.01. (b) In vivo effects of plasmid pHNAH8I-1 carrying tmexCD1-toprJ1 on the efficacy of tigecycline treatment in a neutropenic-mouse thigh infection model. Error bars represent the standard deviations of the means (n = 6). ***, P < 0.001.

FIG 2

Growth curves of K. pneumoniae YX94, E. coli DH5α, S. Typhimurium HN227, and their transformants. Values represent the means ± standard deviations obtained from three independent repeated experiments. (a) Optical densities of K. pneumoniae YX94 and its transformants at 600 nm (OD₆₀₀) measured at hourly intervals. (b) Optical densities of E. coli DH5α and its transformants. (c) Optical densities of S. Typhimurium HN227 and its transformants. A repeated measures two-way analysis of variance (ANOVA) with Tukey’s multiple comparison was used to evaluate statistical significance.

FIG 3

Phylogenetic tree of TMexD1 and TMexD1-like proteins. Neighbor-joining tree based on the amino acid sequences of RND family proteins related to TMexD1 (53 sequences with >90% amino acid identity) generated using MEGA X with 1,000 bootstrap replicates. All amino acid sequences were obtained from the NCBI databases. The tree was visualized using iTOL. The locations of genes encoding the proteins are represented in different colors as follows: orange (chromosome); green (plasmid); blue (chromosome); and black (could not be identified as chromosome or plasmid); the latter two with adjacent mobile genetic elements and showing <90% amino acid sequence identity to the chromosomally encoded MexD protein from the same Pseudomonas species, suggesting that these are acquired rather than intrinsic genes.

FIG 4

Plasmid structures and stability of pHNAH8I-1. (a) Plasmid pHNAH8I-1. Circles from outside to inside represent sequence positions in base pairs, the locations of predicted forward coding sequences (CDS), %GC plot, and GC skew [(GC)/(G+C)], respectively. The extents and positions of Tn5393 and the nfxB1-tmexCD1-toprJ1 insertion are shown. Functions encoded by different genes are shown in different colors as indicated in the key. (b) Stability of pHNAH8I-1 in K. pneumoniae AH8I and transconjugants of E. coli J53 and K. pneumoniae AH58I. Error bars represent standard deviations (n = 3).

FIG 5

Comparison of the genetic context of tmexCD1-toprJ1 with those of closely related sequences. The extents and directions of genes are shown by arrows labeled with gene names, with tmexCD1-toprJ1 genes shown as black or blue arrows and int indicating genes predicted to encode site-specific integrases. ISBvi1 is shown as a green arrow, with the red vertical arrow indicating the insertion point. Tall bars represent the inverted repeats (IRs) of Tn5393 and other transposons. Horizontal dotted lines represent the plasmid backbone or chromosome. In pHNAH8I-1 and some other sequences, possible 5-bp direct repeats (DRs) were identified flanking the insertion, but comparison with uninterrupted versions of the flanking sequences indicated that DRs are not consistently found adjacent to the same point and the creation of DRs is not generally characteristic of site-specific integration, so these are not shown. The context in p18-29-MDR suggests IS26-mediated movement. Additional contigs matching part of the p18-29-MDR context (accession numbers QFMF01000058, QFRE01000039, and QFMA01000080, from K. pneumoniae) or the pHNAH8I context (accession numbers QWTW01000047 and QWIX01000043, from R. ornithinolytica) were also identified, all from chicken cloacae in China. P. putida, Pseudomonas putida.

FIG 6

Prevalence and distribution of tmexCD1-toprJ1-positive isolates. (a) Prevalence of tmexCD1 in human clinical K. pneumonia isolates, 2016 to 2018. (b) Geographical distribution of isolates harboring tmexCD1-toprJ1 in China (collected between November 2018 and July 2019). Colors indicate the numbers of positive isolates and symbols indicate the sources of samples as shown in the key. Provinces that were not sampled are shown in white. The human tmexCD1-toprJ1-positive isolate from Henan Province was identified in GenBank (accession number MK262712.1). Map was generated by R version 3.5.2. (c) Prevalence of tmexCD1-toprJ1-positive isolates in food animal and retail meat samples from China, 2018 to 2019.