Emergence of high-level colistin resistance mediated by multiple determinants, including mcr-1.1, mcr-8.2 and crrB mutations, combined with tigecycline resistance in an ST656 Klebsiella pneumoniae

Abstract

Colistin and tigecycline are usually regarded as the last resort for multidrug-resistant Klebsiella pneumoniae infection treatment. Emergence of colistin and tigecycline resistance poses a global healthcare challenge and is associated with high mortality due to limited therapeutic options. Here, we report the ST656 extensively drug-resistant K. pneumoniae strain KP15-652, which was isolated from a patient's urine in China. Antimicrobial susceptibility testing showed it to be resistant to tigecycline, amikacin, levofloxacin, ciprofloxacin, and high-level colistin resistance (> 2048 mg/L). Whole-genome sequencing revealed that it harbors one chromosome and seven plasmids, including four plasmids carrying multiple acquired resistance genes. Transformation/conjugation tests and plasmid curing assays confirmed that mcr-1.1, mcr-8.2 and crrB mutations are responsible for the high-level colistin resistance and that a series of efflux pump genes, such as tmexCD1-toprJ1, tet(A) and tet(M), contribute to tigecycline resistance. mcr-1.1 and tet(M) are located on an IncX1 plasmid, which has conjugation transfer potential. mcr-8.2 and tet(A) are located on a multireplicon IncR/IncN plasmid but unable to be transferred via conjugation. Moreover, another conjugable and fusion plasmid carries the tmexCD1-toprJ1 gene cluster, which may have arisen due to IS26-mediated replicative transposition based on 8-bp target-site duplications. Importantly, a complex class 1 integron carrying various resistance genes was detected on this fusion plasmid. In conclusion, it is possible that the high-level of colistin resistance is caused by the accumulated effect of several factors on the chromosome and mcr-carrying plasmids, combined with many other resistances, including tigecycline. Effective surveillance should be performed to prevent further dissemination.

Keywords: co-integration; colistin; mcr; tigecycline; tmexCD1-toprJ1.

Figure 1

The distribution of resistance determinants on chromosome or plasmids and corresponding MIC manners in clinical isolate, plasmid curing isolate, transformants, conjugants and their recipient controls. For the distribution of resistance determinants, the red cell represents the original chromosome or any positive plasmid, and the green cell represents none. For MICs of the antimicrobial agents, the fold change between any acquired clone in the transferability assay and its relative control is illustrated with blue cells for no change or less than 2 times, light purple cells for change of 4 or 8 times and dark purple cells for change of 16 times or more. The light gray cell indicates MICs of the clinical isolate or recipient controls.

Figure 2

Circular map of plasmid p652-4 harboring mcr-1.1 and comparison with other IncX1 plasmids. (A) Blue arrows indicate the replicon. Red arrows indicate antimicrobial resistance genes. Yellow arrows represent IS elements. The blue arrows indicate the vir operon that plays a role in the Type IV secretion system (T4SS). The black arrows indicate other open reading frames (ORFs). (B) Plasmid comparison with p869Rt_IncX1 (CP080086) and pD72-IncX1 (CP035315). Near 100% identical sequences are bridged by gray shading. Red filled boxes indicate resistance genes. Yellow filled boxes indicate insertion sequences (ISs). Purple filled boxes indicate replicons. Green filled boxes represent the T4SS region.

Figure 3

Circular map of plasmid p652-3 harboring mcr-8.2 and plasmid comparison with pMCR8_095845, pD120-1 and pMCR8_020135. (A) Circular map of the p652-3 plasmid. Replicon (green), antimicrobial resistance genes (red), IS elements (yellow), T4SS (blue) and other ORFs (black) are shown. Another thin red arrow indicates the oriT region for the origin of transfer. (B) Linear structure of the p652-3 plasmid compared with pMCR8_095845 (CP031883), pD120-1 (CP034679) and pMCR8_020135 (CP037964). Near 100% identical sequences are bridged by gray shading. Red filled boxes indicate mcr-8.2 genes. Blue, green, and purple filled boxes represent IS903B, ISKpn26 and ISEcl1, respectively. Eight IS26 copies are also shown in pMCR8_020135 in light green.

Figure 4

Circular proposed map of plasmid p652-1 harboring the tmexCD1-toprJ1 gene cluster and the genetic structure of two multidrug resistance (MDR) regions. (A) Violet- and yellow-filled boxes indicate IncFIB and IncHI1B replicons, respectively. Two MDR regions are also shown in different positions. (B) The genetic structure of MDR region 1. IS26 copies are shown as green filled boxes, with black arrows indicating the direction. A circular intermediate form of the tmexCD1-toprJ1 gene cluster is shown. Resistance genes are labeled red in the circular intermediate form. The 5’ conserved segment (5’ CS) and 3’ CS of the class 1 integron are labeled. Various kinds of resistance genes in the integron are also shown as different colors and the names labeled above with the orientation indicated by thin black arrows. (C) The genetic structure of MDR region 2. IS elements are shown as colored boxes. Various kinds of resistance genes are labeled with red.

Figure 5

Proposed diagram for the formation of the co-integrate p652-1 plasmid. (A) The ancestral IncFIB plasmid and IncHI1B plasmid. Replicons are shown as purple and yellow filled boxes, respectively. IS26 copies are shown as green filled boxes. (B) A co-integrate containing IncFIB-IncHI1B sequences via an IS26-mediated transposition event. Positions of the 8-bp target site duplication (TSD) adjacent to two IS26 are indicated by a black line with a hollow circle. Directions of IS26 are indicated via black arrows. (C) Plasmid comparison between p652-1 (this study, CP097191) and pHNAH8I-1 (MK347425). Plasmid backbones are shown as thick, blue and red lines, with one copy of IS26 shown as green filled boxes with directions indicated by black arrows. Thick lines indicate the tmexCD1-toprJ1 RND efflux pump-encoding gene cluster. Gray shading indicates the region with 99.98% identity.