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-bla CTX-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 (IncFIIpKF727591 and IncpKPHS1). p205880-qnrS belonged to IncpKPHS1, while p11219-CTXM and p205880-NR1 could be assigned to IncFIIpKF727591. Further detailed genomic comparison of all sequenced plasmids of IncpKPHS1 or IncFIIpKF727591 indicated considerable modular differences and genetic diversification of each group of plasmids.

Materials and methods

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 blaCTX-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 blaCTX-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 IncFIIpKF727591 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.

Table 1

Major features of plasmids analyzed

Inc group	Plasmid	Accession number	Total length (bp)	Total number of open reading frames	Mean G+C content, %	Length of the backbone (bp)	Accessory module (s)	Reference
IncFIIpKF727591α	pKF727591@	KF727591	94,790	112	53.1	51,559	blaNDM-1 region#, and ΔISEcl6	NA
	pKpn235-BG	KT852336	76,360	90	53.9	51,749	blaNDM-1 region#, and ΔISEcl6	NA
	pKpn240-BG	KT852335	76,980	92	53.8	51,594	blaNDM-1 region#, and ΔISEcl6	NA
	pB-3002cz	KJ958926	97,650	122	53.2	51,558	blaNDM-1 region#, and ΔISEcl6	11,12
	pEh1A	KR822246	96,120	105	53.1	51,559	blaNDM-1 region#, and ΔISEcl6	13
	pCP020050	CP020050	113,430	129	52.5	51,559	ars region#, and ΔISEcl6	NA
	pLN824135	LN824135	118,320	142	52.5	46,102	dfrA14-qnrB1 region#, ISEc10, and ΔISEcl6	NA
pCAV1217-71	CP018674	70,610	95	52.4	46,100	glgC region, ISEc27, and ΔISEcl6	NA
IncFIIpKF727591β	pKp_Goe_414-4@	CP018341	81,641	88	53.9	54,186	MDR region#	NA
	p205880-qnrS$	MF190368	65,110	75	52.9	54,649	IS26–ΔTn1696, and Tn6396#	This study
p675920-2	MF133496	79,370	83	54.1	54,726	MDR region#	7
IncpKPHS1	pKPHS1@	CP003223	122,800	131	49.5	113,828	Tn6558 region#	14
	pRJA166c	CP019050	111,080	117	49	111,080	None	NA
	pPMK1-B	CP008931	111,690	117	49.2	111,690	None	15
	pSg1-1	CP012427	126,470	134	49.2	126,470	None	16
	p11219-CTXM	MF133442	122,080	128	50	110,993	Tn6559 region#, and ISKpn24	This study
	pCP020063	CP020063	109,020	119	49.2	107,422	ISKpn38	NA
	pCP015755	CP015755	109,350	125	49.3	109,350	None	NA
	pCP016161	CP016161	109,350	123	49.3	109,350	None	NA
	pUCLAOXA232-4	CP012565	112,060	117	49	112,060	None	NA
	pUCLAOXA232-4.X	CP012570	111,240	115	49	111,240	None	NA
	pIncFIB_DHQP1400954	CP016924	111,540	116	49.3	111,540	None	NA
	pKPN-04f	CP014756	121,030	118	49.3	109,640	IS3000, and ISKpn25	NA
	p205880-NR1	MF144193	108,040	117	49.3	108,040	None	This study
pKP301b	KY354306	110,253	111	48.3	110,653	None	NA

Notes: p205880-qnrS, p11219-CTXM and p205880-NR1 were sequenced in this study, while all the other plasmids were derived from GenBank (last accessed May 28, 2017). @Reference plasmids. #Carrying resistance gene.

Abbreviation: NA, not applicable.

A collection of 11 plasmids including p205880-qnrS (), which had homologous repA (replication initiation) genes and similar backbone gene organizations, were assigned into a novel Inc group designated IncFIIpKF727591 (Increference 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 IncFIIpKF727591α (n=8) and IncFIIpKF727591β (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 (). Predicted RepA-binding iterons were located from 245 bp to 365 bp downstream of repA for IncFIIpKF727591α plasmids, but upstream from 366 bp to 460 bp for IncFIIpKF727591β plasmids, and three copy numbers of iteron were found for all IncFIIpKF727591 plasmids (). Plasmids within each subgroup shared a conserved iteron motif, but those from different subgroups had dramatically different iteron motifs (Figure 1).

Figure 1

A neighbor-joining phylogenetic tree for IncFIIpKF727591 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.

pKF727591 (the first sequenced IncFIIpKF727591 plasmid) and pKp_Goe_414-4 (the first sequenced IncFIIpKF727591β plasmid) were identified as the references for IncFIIpKF727591α and IncFIIpKF727591β, respectively. p205880-qnrS belonged to IncFIIpKF727591β.

The modular structure (Table 1 and ) 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 IncFIIpKF727591 backbone regions (responsible for plasmid replication, maintenance and conjugal transfer). The eight IncFIIpKF727591α plasmids shared ≥88% of their backbone sequences with ≥99% nucleotide identity, and the three IncFIIpKF727591β plasmids showed ≥99% nucleotide identity over ≥98% of their backbone sequences; by contrast, the backbones of IncFIIpKF727591α and IncFIIpKF727591β had ≤92% nucleotide identity across ≤70% of their backbone sequences (). The modular structures of plasmid backbones were conserved within each of IncFIIpKF727591α and IncFIIpKF727591β subgroups but dramatically different between two subgroups.

Integration of accessory modules at various sites of IncFIIpKF727591 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 IncFIIpKF727591 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 IncFIIpKF727591 backbone. An 11-kb maintenance region carrying parAB (partition) and ccdBA (toxin-antitoxin) was found in all IncFIIpKF727591α plasmids, while another distinct 15-kb maintenance region containing stbAB (mediator of plasmid stability) and resD (resolvase) in all IncFIIpKF727591β plasmids.

Figure 2

Linear comparison of complete sequences of IncFIIpKF727591 plasmids. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on function classification. Shading regions denote homology of plasmid backbone regions (light blue: ≥90% nucleotide identity; light red: <90% nucleotide identity) but not accessory modules.

All the three IncFIIpKF727591β plasmids contained a single IncFIIpKF727591β replicon, which six of the eight IncFIIpKF727591α plasmids contained a second IncpA1763-KPC replicon beside the master IncFIIpKF727591α replicon (Figure 2). Notably, the IncpA1763-KPC replicon was located within a 9.7- or 8.1-kb backbone region [carrying maintenance 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 IncFIIpKF727591α and IncpA1763-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

Figure 3

Organization of selected accessory modules from IncFIIpKF727591α plasmids and comparison with related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on function classification. Shading denotes regions of homology (>95% nucleotide identity). Numbers in brackets indicate e nucleotide positions within corresponding plasmids. The accession numbers of Tn125,6 Tn625617 and Tn133118 for reference are JN872328, KP851978 and KC354802, respectively.

Accessory resistance modules of IncFIIpKF727591 plasmids

A large accessory module was integrated at a site between the two maintenance genes mtsM and ccdA in each of the eight IncFIIpKF727591α 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 IncpA1763-KPC (see above) was found in six IncFIIpKF727591α plasmids except for pKpn235-BG and pKpn240-BG.The accessory modules from seven IncFIIpKF727591α plasmids, except for pCAV1217-71, carried resistance loci (Figure 3 and ): i) a truncated Tn125 transposon carrying blaNDM−16 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 blaNDM−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 IncFIIpKF727591β plasmids p675920-2, pKp_Goe_414-4 and p205880-qnrS, respectively. The 24.7-kb MDR region, carrying multiple resistance genes (), was generated from integration of an IS26–∆Tn6346–∆GIsul2–IS26 unit7 into Tn1721,8 which was further connected with a truncated IS26–blaLAP-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–∆Tn6346–∆GIsul2–IS26 and further upstream insertion of an IS26–pdk–catA2–IS26 unit. Tn6396 was a novel ISKpn19-compsite 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 4

Organization of accessory resistance modules from IncFIIpKF727591β plasmids and comparison with related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on function classification. Shading denotes regions of homology (>95% nucleotide identity). Numbers in brackets indicate e nucleotide positions within corresponding plasmids. The accession numbers of GIsul2,19 Tn6346,20 Tn1728 and IS26–blaLAP-2–qnrS1–IS26 unit21 for reference are CP001918, EU696790, X61367 and HF545433, respectively.

Characterization of IncpKPHS1 plasmids

Two additional new plasmids p11219-CTXM (carrying blaCTX-M-14) and p205880-NR1 (containing no resistance genes) were fully sequenced (Table 1 and ). p11219-CTXM could not be transferred from the wild-type 11219 isolate into EC600 through conjugation, but could be transferred into TOP10 through electroporation, generating a blaCTX-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 (), each of which carried a single repA gene with >96% nucleotide identity to repApKPHS1 () and had a backbone gene organization similar to pKPHS1 (Figure 5), were assigned into a novel Inc group named as IncpKPHS1 (). These 14 RepA proteins did not any of known domain super-families. Four copy numbers of a conserved iteron motif () were found 48 bp to 218 bp downstream of repA for all IncpKPHS1 plasmids (). All plasmids carried a single iteron-regulated IncpKPHS1 replicon. pKPHS1, the first sequenced IncpKPHS1 plasmid, was identified as the IncpKPHS1 reference. These 14 plasmids had >96% nucleotide identity over >75% coverage of their backbone sequences (). 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

Figure 5

Linear comparison of complete sequences of IncpKPHS1 plasmids. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on function classification. Shading regions denote homology of plasmid backbone regions (≥90% nucleotide identity) but not accessory modules.

Only four IncpKPHS1 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 Tn3-family unit transposons generated from integration of truncated IS903D–blaCTX-M-14–ISEcp1 units (representing the master prototype blaCTX-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–blaCTX-M-14–ISEcp1 unit or mcp.

Figure 6

Organization of Tn6558 and Tn6559 and comparison with related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on function classification. Shading denotes regions of homology (>95% nucleotide identity). Numbers in brackets indicate the nucleotide positions within the corresponding plasmids. The accession numbers of ISEcp1–blaCTX-M-14–IS903D unit22 and Tn17228 are KX646543 and X61367, respectively.