Characterization of the plasmid of incompatibility groups IncFIIpKF727591 and IncpKPHS1 from Enterobacteriaceae species

Background Multiple incompatibility (Inc) groups of plasmids have been identified in Enterobacteriaceae species, but there are still quite a few sequenced plasmids that could not be assigned to any known Inc groups. Methods One IncFIIpKF727591β plasmid p205880-qnrS and two IncpKPHS1 plasmids p11219-CTXM and p205880-NR1 were fully sequenced in this work. Detailed genomic comparison was applied to all available sequenced plasmids of IncFIIpKF727591 or IncpKPHS1 group. Results p205880-qnrS carried a novel transposon Tn6396, which was an ISKpn19-compsite transposon and represented a prototype transposable element carrying a minimum core qnrS1 module. p11219-CTXM harbored a novel transposon Tn6559, which was generated from integration of a truncated IS903D–blaCTX-M-14–ISEcp1 unit into the Tn3-family cryptic unit transposon Tn1722. Two Inc groups, IncFIIpKF727591 and IncpKPHS1, of plasmids from Enterobacteriaceae species were proposed, and IncFIIpKF727591 was further grouped into two subgroups IncFIIpKF727591α and IncFIIpKF727591β. Each of the 11 IncFIIpKF727591 plasmids carried multiple accessory modules including at least one resistance module, and the relatively small IncFIIpKF727591 backbones could acquire a wealth of foreign genetic contents. The modular structures of plasmid backbones were conserved within each of IncFIIpKF727591α and IncFIIpKF727591β subgroups but dramatically different, although with similar gene organizations, between these two subgroups. The IncpKPHS1 backbones were conserved with respect to modular structures, and only four of the 14 IncpKPHS1 plasmids carried accessory modules, two of which contained resistance genes. Conclusion A genomic comparison of sequenced IncpKPHS1 or IncFIIpKF727591 plasmids provides insights into modular differences and genetic diversification of these plasmids, some of which carries antimicrobial resistance genes.


Introduction
Plasmid is a small DNA molecule within a bacterial cell and capable of replicating independently from the host's chromosomal DNA. Plasmids are mobile genetic elements that commonly carry antimicrobial resistance genes and other genetic factors such as virulence genes. Plasmid-mediated transmission of antimicrobial resistance genes among Enterobacteriaceae and other bacteria imposes a major public health concern.
The original replicon-based scheme to classify plasmids into different incompatibility (Inc) groups was developed in 1970s, which is based on the experimental observations that plasmids with similar replication machinery are often unable to stably co-exist within the same host cell and thus the plasmid shows incompatibility with the same Inc group plasmid. 1 Nowadays, Inc classification is always based on replication initiation protein (Rep) sequences, and it is not necessarily confirmed by conventional conjugation-based incompatibility experiments. 1 At least 27 Inc groups have been identified in Enterobacteriaceae species, 1 but there are quite a few sequenced plasmids that could not be assigned to any known Inc groups.
This study presented three sequenced plasmids (p205880-qnrS carrying a novel ISKpn19-compsite transposon Tn6396, p11219-CTXM harboring a novel Tn1722-derivated unit transposon Tn6559, and p205880-NR1 containing no resistance genes) and proposed two novel Inc groups (IncFII pKF727591 and Inc pKPHS1 ). p205880-qnrS belonged to Inc pKPHS1 , while p11219-CTXM and p205880-NR1 could be assigned to IncFII pKF727591 . Further detailed genomic comparison of all sequenced plasmids of Inc pKPHS1 or IncFII pKF727591 indicated considerable modular differences and genetic diversification of each group of plasmids.

Bacterial strains and genome sequencing
Klebsiella pneumoniae 205880 and 11219 were recovered from the sputum specimens of two different patients with pneumonia in two different Chinese hospitals in 2012 and 2013, respectively. For each strain, genomic DNA isolation, genome sequencing, and sequence assembly and annotation were carried out as described previously. 2 An unrooted neighbor-joining tree was generated from the aligned repA sequences of indicative plasmids. 2 Plasmids p205880-qnrS, p11219-CTXM and p205880-NR1 had GenBank accession numbers MF190368, MF133442 and MF144193, respectively.

Phenotypic assays
Plasmid conjugal transfer was carried out, as described previously, 2 with Escherichia coli EC600 as a recipient and the 205880 or 11219 isolates as a donor, for selecting an E. coli transconjugant that carried bla CTX-M-14 (p11219-CTXM) or qnrS1 (p205880-qnrS), respectively. Electroporation of plasmid p11219-CTXM from the 11219 isolate into E. coli TOP10 was performed, as described previously, 2 to obtain an E. coli electroporant carrying bla CTX-M-14 (p11219-CTXM). Double-disk synergy test was performed to detect the activity of extended-spectrum β-lactamase (ESBL) in indicative bacterial strains. 3 BioMérieux VITEK 2 was used to test bacterial antimicrobial susceptibility, which was interpreted as per the Clinical and Laboratory Standards Institute (CLSI) guidelines. 4 Results and discussion Diversification of IncFII pKF727591 plasmids One new plasmid p205880-qnrS was fully sequenced (Table 1) and could be transferred from the wild-type 205880 isolate into EC600, through conjugation, giving a qnrS-positive transconjugant p205880-qnrS-EC600. As expected, these two strains were resistant to ciprofloxacin and levofloxacin with minimum inhibitory concentration (MIC) values ≥4.
A collection of 11 plasmids including p205880-qnrS (Table S1), which had homologous repA (replication initiation) genes and similar backbone gene organizations, were assigned into a novel Inc group designated IncFII pKF727591 (Inc reference plasmid ), because all these RepA proteins had an IncFII super-family domain. The phylogenetic tree ( Figure 1) based on repA sequences indicated that these 11 plasmids could be divided into two separately clustering subgroups IncFII pKF727591 α (n=8) and IncFII pKF727591 β (n=3). As shown by pairwise comparison of repA nucleotide sequences, plasmids within each subgroup showed 100% identity, while those from different subgroups displayed ≥79% identity (Table S2A). Predicted RepA-binding iterons were located from 245 bp to 365 bp downstream of repA for IncFII pKF727591 α plasmids, but upstream from 366 bp to 460 bp for IncFII pKF727591 β plasmids, and three copy numbers of iteron were found for all IncFII pKF727591 plasmids (Table S1). Plasmids within each subgroup shared a conserved iteron motif, but those from different subgroups had dramatically different iteron motifs ( Figure 1). pKF727591 (the first sequenced IncFII pKF727591 plasmid) and pKp_Goe_414-4 (the first sequenced IncFII pKF727591 β plasmid) were identified as the references for IncFII pKF727591 α and IncFII pKF727591 β, respectively. p205880-qnrS belonged to IncFII pKF727591 β.
The modular structure (Table 1 and Figure S1) of each plasmid could be divided into one or more accessory modules (defined as acquired DNA regions associated or bordered with mobile elements) and the remaining IncFII pKF727591 backbone regions (responsible for plasmid replication, maintenance and conjugal transfer). The eight IncFII pKF727591 α plasmids shared ≥88% of their backbone sequences with ≥99% nucleotide identity, and the three IncFII pKF727591 β plasmids showed ≥99% nucleotide identity over ≥98% of their backbone sequences; by contrast, the backbones of IncFII pKF727591 α and IncFII pKF727591 β had ≤92% nucleotide identity across ≤70% of their backbone sequences (Table S2B). The modular structures of plasmid backbones were conserved within each of IncFII pKF727591 α and IncFII pKF727591 β subgroups but dramatically different between two subgroups. Integration of accessory modules at various sites of IncFII pKF727591 backbones led to the interruption of relevant backbone genes (eg, umuC), the disruption of the maintenance or conjugal transfer regions, or the deletion of surrounding backbone regions (eg, 5.3-kb deletion containing mtsM) (Figure 2). The IncFII pKF727591 replicons and the conjugal transfer regions (encoding an F-type type IV secretion system) were found in all 11 plasmids and thus represented the core IncFII pKF727591 backbone. An 11-kb maintenance region carrying parAB (partition) and ccdBA (toxin-antitoxin) was found in all IncFII pKF727591 α plasmids, while another distinct 15-kb maintenance region containing stbAB (mediator of plasmid stability) and resD (resolvase) in all IncFII pKF727591 β plasmids.
All the three IncFII pKF727591 β plasmids contained a single IncFII pKF727591 β replicon, which six of the eight IncFII pKF727591 α plasmids contained a second Inc pA1763-KPC replicon beside the master IncFII pKF727591 α replicon ( Figure 2). Notably, the Inc pA1763-KPC replicon was located within a 9.7-or 8.1-kb backbone region [carrying maintenance Figure 1 A neighbor-joining phylogenetic tree for IncFII pKF727591 plasmids. The degree of support (percentage) for both cluster of associated taxa, as determined by bootstrap analysis, is shown next to each branch. The bar corresponds to the scale of sequence divergence. The triangles indicate the reference plasmids, while the square denotes the plasmid sequenced in this study. genes such as parA and resA; as observed in pA1763-KPC (GenBank accession number MH909340], which was a part of relevant accessory modules (see below; Figure 3). Two coexistent replicons IncFII pKF727591 α and Inc pA1763-KPC , together with their supporting maintenance genes, will promote relevant plasmids to overcome incompatibility barrier with incoming plasmids. All the above replicons belonged to the iteron-regulated replicon, for which Rep monomers specifically bound to iterons. 5 Accessory resistance modules of IncFII pKF727591 plasmids A large accessory module was integrated at a site between the two maintenance genes mtsM and ccdA in each of the eight IncFII pKF727591 α plasmids (Figure 3). These eight modules had some common regions but showed considerable modular differences across the whole modules, indicating their sole evolutionary origin followed by parallel mosaic diversification. The 9.7-or 8.1-kb backbone region from Inc pA1763-KPC (see above) was found in six IncFII pKF727591 α plasmids except for pKpn235-BG and pKpn240-BG.The accessory modules from seven IncFII pKF727591 α plasmids, except for pCAV1217-71, carried resistance loci (Figure 3 and Table S3): i) a truncated Tn125 transposon carrying bla NDM−1 6 was harbored in pB-3002cz, pKpn235-BG and pKpn240-BG, while another truncated version of Tn125 in pKF727591 and pEh1A; ii) the ars (arsenical resistance) locus was found in pKF727591, pEh1A and pCP020050 and, notably, the first two plasmids showed coexistence of bla NDM−1 and ars; and iii) qnrB1 and dfrA14-carrying In191 were identified in pLN824135. A 24.7-kb MDR region, another 26.9-kb MDR region and Tn6396 (Figure 4) were inserted at the same site within the umuC gene of the three IncFII pKF727591 β plasmids p675920-2, pKp_Goe_414-4 and p205880-qnrS, respectively. The 24.7-kb MDR region, carrying multiple resistance genes (Table S3), was generated from integration of an IS26-ΔTn6346-ΔGIsul2-IS26 unit 7 into Tn1721, 8 which was further connected with a truncated IS26-bla LAP-2 -qnrS1-IS26 unit. 7 The 26.9-kb MDR region was highly similar to the 24.7-kb MDR region but differed from it mainly by inversion of IS26-ΔTn6346-ΔGIsul2-IS26 and further upstream insertion of an IS26pdk-catA2-IS26 unit. Tn6396 was a novel ISKpn19compsite transposon, which carried the qnrS1-ΔtnpR region and bracketed by 7-bp direct repeats (DRs: target site duplication signals for transposition) at both ends. Tn6396 represented a prototype transposable element carrying a minimum core qnrS1 module. Different Tn6396 derivatives with distinct terminal truncations were found in various plasmids including pKp_Goe_414-4 and p675920-2 ( Figure S2).

Characterization of Inc pKPHS1 plasmids
Two additional new plasmids p11219-CTXM (carrying bla CTX-M-14 ) and p205880-NR1 (containing no resistance genes) were fully sequenced (Table 1 and Figure S1). p11219-CTXM could not be transferred from the wildtype 11219 isolate into EC600 through conjugation, but could be transferred into TOP10 through electroporation, generating a bla CTX-M-14 -positive electroporant 11219-CTXM-TOP10. These two wild-type and electroporant strains had ESBL activity (data not shown) and were resistant to cefazolin, cefuroxime and ceftazidime with MIC values ≥64.
A total of 14 plasmids including p11219-CTXM and p205880-NR1 (Table S1), each of which carried a single repA gene with >96% nucleotide identity to repA pKPHS1 (Table S4A) and had a backbone gene organization similar to pKPHS1 (Figure 5), were assigned into a novel Inc group named as Inc pKPHS1 ( Figure S3). These 14 RepA proteins did not any of known domain super-families. Four copy numbers of a conserved iteron motif ( Figure S3) were found 48 bp to 218 bp downstream of repA for all Inc pKPHS1 plasmids (Table S1). All plasmids carried a single iteron-regulated Inc pKPHS1 replicon. pKPHS1, the first sequenced Inc pKPHS1 plasmid, was identified as the Inc pKPHS1 reference. These 14 plasmids had >96% nucleotide identity over >75% coverage of their backbone sequences (Table S4B). Modular differences were found at multiple sites of the maintenance regions ( Figure 5). None of conjugal transfer genes was found in all plasmids, which was consistent to the non-conjugative nature of p11219-CTXM. Remarkably, all these plasmids carried ɸpKPHS1 regions resembling SSU5 phage. 9 Only four Inc pKPHS1 plasmids had accessory modules, including the two resistance modules: Tn6558 from pKPHS1 and Tn6559 from p11219-CTXM (Table 1 and Figure 5). The highly similar Tn6558 and Tn6559 ( Figure 6) were novel Tn3family unit transposons generated from integration of truncated IS903D-bla CTX-M-14 -ISEcp1 units (representing the master prototype bla CTX-M-14 genetic environments in China) 10 into the mcp gene of cryptic Tn1722, 8 and they slightly differed from one another by distinct truncations occurred within IS903D-bla CTX-M-14 -ISEcp1 unit or mcp.

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