Characterization of a mcr-1 and CRISPR-Cas System Co-harboring Plasmid in a Carbapenemase-Producing High-Risk ST11 Klebsiella pneumoniae Strain

Abstract

We set out to study the prevalence of the mcr-1 gene in carbapenemase-producing Klebsiella pneumoniae (CPKP) strains, and to determine whether its presence is associated with a fitness cost. A total of 234 clinical CPKP isolates were collected from a tertiary medical center in Taiwan from January 2018 to January 2019. The mcr-1 and carbapenemase genes were detected by polymerase chain reaction (PCR) followed by Sanger sequencing. The mcr-1-positive carbapenemase-producing strain was characterized by whole genome sequencing, a plasmid stability test and a conjugation assay. In vitro growth rate and an in vivo virulence test were compared between the parental mcr-1-positive strain and its mcr-1 plasmid-cured strain. We identified only one mcr-1 positive strain (KP2509), co-harboring bla _{KPC- 2} and bla _{OXA- 48}, among 234 (1/234, 0.43%) CPKP strains. KP2509 and its Escherichia coli mcr-1 transconjugant showed moderate colistin resistance (MIC = 8 mg/L). The mcr-1 is located on a large conjugative plasmid (317 kb), pKP2509-MCR, with three replicons, IncHI, IncFIB, and IncN. Interestingly, a complete Type IV-A3 CRISPR-Cas system was identified in pKP2509-MCR. Plasmid pKP2509-MCR was highly stable in KP2509 after 270 generation of passage, and the pKP2509-MCR cured strain PC-KP2509 showed similar growth rate and in vivo virulence in comparison to KP2509. The prevalence of mcr-1 in CPKP strains remains low in our center. Notably, we identified a large plasmid with multiple replicons containing both the mcr-1 and the Type IV-3A CRISPR-Cas genes. The further spread of this highly stable plasmid raises concern that it may promote the increase of mcr-1 prevalence in CPKP.

Keywords: carbapenemase; colistin; fitness cost; high-risk clone; mcr-1.

FIGURE 1

(A) Plasmid structure of mcr-1 and CRISPR-Cas co-harboring plasmids. Colored arrows indicate open reading frames, with purple, orange, green, red, teal, and blue arrows representing replication genes, mobile elements, plasmid transfer genes, the antimicrobial and heavy metal resistance gene, CRISPR-Cas, and plasmid backbone genes, respectively. (B) The CRISPR-Cas IV-A3 region in pKP2509-MCR. (C) The mcr-1-harboring Tn6330 and neighboring genes in pKP2509-MCR. Blue shading denotes regions of shared homology among different elements. hp, hypothetical protein gene.

FIGURE 2

Genomes of E. coli J53 (lane 2), transconjugant (lane 3), KP2509 (lane 4) and PC-KP2509 (lane 5) were treated with S1-nuclease, and the resulting products were subsequently analyzed by PFGE. The profile showed an approximately 300 kbps plasmid in the transconjugant, that is consistent with the mcr-1-harboring plasmid from KP2509. Only four plasmids of KP2509 were detected in the gel, because the small size (10,060 bp) of the undetectable plasmid, pKP2509-5, was below the lower limit of markers.

FIGURE 3

The stability of pJET-TC and pJET-TC-MCR-1 plasmids in PC-KP2509 was compared after consecutive passage. Without antibiotic pressure, pJET-TC-MCR-1 was lost from PC-KP2509 within 7 days (i.e., ∼ 70 generations), and only 24.7% PC-KP2509 population carried the mcr-1 recombinant plasmid on Day 28. In contrast, 88% PC-KP2509 population kept pJET-TC on Day 28. The percentage of mcr-1-bearing colony was performed from the three independent experiments. The statistical difference was analyzed by two-way ANOVA.

FIGURE 4

(A) The growth curves of KP2509 and PC-KP2509 strains were observed during 0–6 h, respectively, and the tendency of KP2509 was similar that of PC-KP2509 (P = 0.96). Each value was performed from the three independent experiments, and the statistical difference was analyzed by Mann–Whitney U test. (B) KP2509 and PC-KP2509 strains with 2.1 × 10⁶ cfu were intraperitoneal inoculated into mice, and survival rates were compared during 2 weeks. No statistical difference in Kaplan–Meier analysis with a log-rank test (P = 0.57).