Dissemination of pKPI-6
All blaIMP-positive CRE isolates of Escherichia coli (n = 135) and Klebsiella pneumoniae (n = 95) were classified into seven groups based on the results of S1-PFGE followed by Southern blot hybridization with probes for the blaIMP and repA genes (Figure 1). Ninety-nine of the 135 E. coli isolates (73%) and 88 of the 95 K. pneumonia isolates (93%) carried plasmids classified as Group pKPI-6 based on plasmid size and replicon type (Supplementary Figure S1). pKPI-6 was the predominant plasmid responsible for the transmission of blaIMP-6 (187 out of 230 blaIMP-positive CRE isolates, 85.6%). Next, we compared the similarity between pKPI-6 and 39 representative plasmids categorized as Group pKPI-6 based on WGS data. The overall sequence identity was 99 ± 0.28%, and the sequence coverage was 98 ± 4.0% (mean ± standard deviation) (Supplementary Figure S1). This analysis confirmed that pKPI-6 is the predominant plasmid carried by CRE isolates in the study area.
Genomic Analysis of Derivatives of the Predominant Plasmid, pKPI-6
During the characterization of the blaIMP-6 plasmids mentioned above, nine E. coli isolates and three K. pneumoniae isolates possessed blaIMP-6 plasmids categorized as Group IncN (Figure 1). Group IncN blaIMP-6 plasmids were characterized by replicon type IncN and ranged from 35 to 264 kbp in size, which was different from the pKPI-6 plasmid of 50 kbp (Supplementary Figure S1). The complete sequences of these plasmids indicated that they had preserved the nearly complete locus of pKPI-6 and typically were multi-replicon plasmids that had integrated IncF-type plasmids framed by insertion sequences (Supplementary Figure S2 and Table S1). Additionally, two isolates (E208 and E328) of K. pneumoniae harboured plasmids categorized as Group Non-IncN KP (Figure 1B). These plasmids comprised a cassette carrying blaIMP-6 without IncN-type repA of the pKPI-6 plasmid integrated into another plasmid (Supplementary Figure S3). Interestingly, E. coli isolate E119 and K. pneumoniae isolate E206 coharboured two distinct blaIMP-6-encoding plasmids of different sizes and were categorized as Group double blaIMP-6 (Figure 1 and Supplementary Figure S4). Barring occasional isolations of strains coharbouring different carbapenemase genes [12, 13], few studies have shown the coexistence of two identical carbapenemase genes on different plasmids within an isolate [14]. WGS revealed that isolate E119 carried pKPI-6 and an IncF-type plasmid (pEC743_1) that had a blaIMP-6 cassette from pKPI-6 integrated (Supplementary Figure S5).
Characterization of IncF Plasmids Encoding blaIMP-6
In addition to the K. pneumoniae isolates carrying Group Non-IncN KP plasmids, E. coli isolates carrying plasmids without IncN replicon were found in a single hospital (hospital D; Figure 1A). WGS of these isolates revealed that they harboured nearly identical blaIMP-6-encoding plasmids with an IncFIA-type replicon (categorized as Group IncF) (Supplementary Figure S6A and Table S1). These plasmids were generated by integration of a cassette carrying blaIMP-6 on pKPI-6 into another IncF plasmid at IS26. This IncF plasmid (pEC302/04; Supplementary Figure S6B) has been reported to transmit antimicrobial resistance since 1965 [15].
The minimum inhibitory concentrations (MICs) of meropenem for the E. coli isolates carrying Group IncF plasmids were low when compared with those of E. coli isolates harbouring other blaIMP-6-encoding plasmids, such as pKPI-6 (Supplementary Figure S7). Mutations or deletions in the porin (OmpF) gene in E. coli have been reported to enhance resistance to β-lactams [16]. However, all E. coli isolates carrying Group IncF plasmids had a premature termination codon within ompF, whereas the other isolates carried wild-type ompF (Supplementary TableS2 and S3). MICs of meropenem were low for these Group IncF plasmid-carrying isolates, despite them being OmpF-deficient. To investigate carbapenem resistance in the same genetic background, plasmids from representative isolates in each blaIMP-6 carriage group were transformed into the E. coli TOP10 strain and MICs for the transformants were determined. Transformant T305 carrying pE305_IMP6single of Group IncF from E. coli isolate E305 was more susceptible to meropenem than transformants carrying blaIMP-6-harbouring plasmids of groups (Supplementary Table S4). blaIMP-6 transcription in the pE305_IMP6single transformant was significantly lower than that in the pKPI-6 transformant (Supplementary Figure S8A), although the plasmid copy numbers in the bacterial cells were comparable (Supplementary Figure S8B). These results indicated that the lower MICs of meropenem in E. coli isolates carrying Group IncF plasmids were due to reduced transcription of blaIMP-6.
Heteroresistance to Carbapenems: Enhanced Resistance Through Gene Amplification
E. coli isolates E305 and E318 were found to carry Group IncF plasmids, and WGS revealed that their chromosomes were nearly identical (query: E318, identity 100%, coverage 100%; query: E305, identity 100%, coverage 98% in BLASTN). Isolate E318 harboured genes encoding extended-spectrum β-lactamases (ESBLs), such as blaCTX-M-14 and blaTEM-1B, on a plasmid other than pE318_IMP6, whereas isolate E305 did not have these genes (Supplementary TableS5). IMP-6 confers resistance to cephalosporins and meropenem, but it hydrolyses penicillins very poorly [17]. Therefore, isolate E318 exhibited broader antimicrobial resistance than isolate E305. In contrast, the MIC of meropenem for E305 was higher than that for E318.
WGS of E305 and E318 revealed the complete sequence of pE318_IMP6; however, it failed to determine the complete sequence of pE305_IMP6. Therefore, to analyse the structure of pE305_IMP6, we used a combination of WGS, Southern blotting, and qPCR analysis. The length and depth of each contig of pE305_IMP6 deduced from WGS are shown in the de-novo assembly graphs generated using the Bandage software [18] in Figure 2A. The total length of pE305_IMP6 deduced from WGS data was approximately 149 kbp. However, according to Southern blotting results, pE318_IMP6 and pE305_IMP6 were ~145 kbp and ~200 kbp in size, respectively (Figure 2B). Based on the depth of each contig, the copy number of each contig was predicted as follows: Contig3, 1 copy; Contig2 and Contig5, 6 copies; Contig1 and Contig6, 3 copies; Contig4, 5 copies (Figure 2A). Therefore, pE305_IMP6 was predicted to have a ~19-kbp repeat region consisting of triplication of Contig1 and Contig6, sextuplication of Contig2 and Contig5, and quintuplication of Contig4 (Figure 2C). Except for the repeat region, pE305_IMP6 and pE318_IMP6 exhibited high sequence similarity (identity; 99.27%, coverage; 100%) (Figure 2D). The blaIMP-6 gene was located on Contig6 and was predicted to be triplicated. qPCR analysis corroborated that pE305_IMP6 carried three copies of blaIMP-6, whereas pE318_IMP6 harboured a single copy (appendix p10). blaIMP-6 transcription was significantly higher in isolate E305 than in isolate E318 (Figure 2E), even though the blaIMP-6-carrier plasmid copy numbers in the cells of these isolates were not significantly different (Supplementary Figure S9B). Triplication of blaIMP-6 in tandem resulted in a higher transcription level in E305, resulting in a higher level of resistance to meropenem.
Subculture of the clonal isolate E305 in broth medium revealed a mixture of subpopulations of bacteria carrying a plasmid with multiple blaIMP-6 copies (which represented the majority) and bacteria carrying a plasmid with a single blaIMP-6 copy. In Southern blotting for blaIMP-6, a faint band at ~145 kbp was observed in addition to the major band at ~200 kbp (Figure 2B). It was also found that T305 (transformant of pE305_IMP6 single extracted from E305) carried a ~145-kbp plasmid without blaIMP-6 amplification due to recA deficiency in the recipient E. coli TOP10 strain (Supplementary Figure S10) [19]. qPCR analysis confirmed that T305 carried one blaIMP-6 copy on its plasmid (Supplementary Figure S9C). These results indicated the existence of a subpopulation carrying a plasmid with one blaIMP-6 copy within E. coli isolate E305, whereas the majority of the population carried a plasmid harbouring three copies of blaIMP-6.
Comparison of CRE Isolates Carrying pKPI-6 with Those Carrying Other Groups of Plasmids Harbouring blaIMP-6
blaCTX-M-2, which is an ESBL gene located distant from blaIMP-6 on pKPI-6, compensated for the narrow range of hydrolysis of β-lactams by IMP-6 [11, 17]. However, toweverhhhhese two β-lactamase genes were not always transferred together from pKPI-6 to another plasmid. Plasmids categorized as Group Non-IncN KP and Group IncF did not carry ESBL genes (Supplementary Table S6) and rarely conferred resistance to penicillins, in contrast to pKPI-6, which confers broad resistance to β-lactams (Figure 1). We next measured the conjugation efficiency of representative plasmids in each group (Supplementary Table S7). pKPI-6 plasmids and Group IncN plasmids, which had the entire pKPI-6 plasmid incorporated, showed a higher conjugation efficiency than Group Non-IncN KP/IncF plasmids. These characteristics may have facilitated the vast horizontal dissemination of pKPI-6 in the study area.
Compared with the chromosomal diversity among E. coli isolates bearing pKPI-6, K. pneumoniae isolates carrying pKPI-6 exhibited higher clonality as indicated by XbaI-PFGE analysis (Figure 1). This may be explained by the presence of the kikA gene on pKPI-6, the product of which reportedly promotes cell death of K.pneumoniae following conjugation [20]. The conjugation efficiency of pKPI-6 into K. pneumoniae ATCC13883 was considerably lower than that into E. coli TUM3456 (3.3 × 10–4 and 3.7 × 10–1, respectively). Maybe only “kikA-resistant” K. pneumoniae are able to acquire pKPI-6, leading to clonal similarity among the K. pneumoniae isolates bearing pKPI-6.
Chromosomal Integration of blaIMP-6
Unlike most CRE isolates, which carried the predominant pKPI-6 or other blaIMP-6-encoding plasmids, three out of 135 E. coli isolates (E138, E300, and E302) harboured blaIMP-6 on their chromosomes as indicated by S1-PFGE followed by Southern blotting with blaIMP-6 probes (Figure 1A and Figure 3A). I-CeuI-PFGE followed by Southern blotting with probes for the blaIMP-6 and 16S rRNA genes confirmed chromosomally located blaIMP-6 (Figure 3B). WGS revealed that the chromosome of isolate E138 had a cassette harbouring blaIMP-6 integrated, framed by a set of IS15 (Figure 3C). The chromosomes of E300 and E302 had IncFIA plasmids carrying blaIMP-6 integrated (Figure 3D,E). While these plasmids were essentially identical to pE301_IMP6 (E. coli, Group IncF), these isolates were phylogenetically distinct on the XbaI-PFGE phylogenetic tree (Figure 1).
Emergence of pKPI-6-like Plasmid Harbouring blaIMP-1
One K. pneumoniae isolate, E105, harboured blaIMP-1, which is a single-nucleotide variant of blaIMP-6, within a clonal cluster of pKPI-6 carriers (Figure 1B). Due to this mutation, E105 was resistant to imipenem, whereas most isolates carrying blaIMP-6 were susceptible to this antibiotic. WGS revealed that plasmids pKPI-6, pE013_IMP6 (plasmid group pKPI-6), and pE105_IMP1 were 99.8% identical, with a coverage of 100% (query: pE013_IMP6) (Figure 4). The only difference was the presence of a 714-bp region bracketed by a set of homologous regions in pE013_IMP6.