Comparative analysis of an IncR plasmid carrying armA, bla_DHA-1 and qnrB4 from Klebsiella pneumoniae ST37 isolates

Abstract

Objectives

The objective of this study was to conduct a comparative analysis with reported IncR plasmids of a Klebsiella pneumoniae IncR plasmid carrying an MDR region.

Methods

MDR K. pneumoniae isolates were serially identified from two inpatients at a hospital in the USA in 2014. MDR plasmid pYDC676 was fully sequenced, annotated and compared with related plasmids. Antimicrobial susceptibility testing, PFGE and MLST were also conducted.

Results

The K. pneumoniae isolates were identical by PFGE, belonged to ST37 and harboured an identical ∼50 kb IncR plasmid (pYDC676). pYDC676 possessed the backbone and multi-IS loci closely related to IncR plasmids reported from aquatic bacteria, as well as animal and human K. pneumoniae strains, and carried an MDR region consisting of armA, bla_DHA-1 and qnrB4, a combination that has been reported in IncR plasmids from K. pneumoniae ST11 strains in Europe and Asia. A plasmid with the identical IncR backbone and a similar MDR region containing bla_DHA-1 and qnrB4 has also been reported in ST37 strains from Europe, suggesting potential dissemination of this lineage of IncR plasmids in K. pneumoniae ST37.

Conclusions

K. pneumoniae ST37 strains with an MDR IncR plasmid carrying armA, bla_DHA-1 and qnrB4 were identified in a hospital in the USA, where these resistance genes remain rare. The IncR backbone may play a role in the global dissemination of these resistance genes.

Introduction

Klebsiella pneumoniae, especially epidemic clones such as ST258 and ST11, have accumulated multiple resistance genes on plasmids with various backbones in recent years.^1–3 In addition to these well-recognized global MDR clones, other MDR lineages, such as ST37, have been reported worldwide.^1,2,4–6 The earliest ST37 strains were identified in the USA in 1996 and in Italy in 1997.^2,7 Since then, ST37 strains have been reported mainly from Europe, China and India.^1,4,5 For example, ST37 strains carrying bla_CTX-M-15 and bla_DHA-1 were reported to circulate in a hospital in Italy and in a hospital in France, respectively.^5,6 Furthermore, an outbreak of carbapenem- and colistin-resistant K. pneumoniae ST37 strains has occurred in a hospital in Spain.⁸ In the USA, K. pneumoniae ST258 strains are predominant among carbapenem-resistant strains, with ST37 strains occurring sporadically. In our hospital, carbapenem-resistant ST37 strains carrying bla_KPC-3 have been identified from transplant patients but with a much lower frequency than the epidemic ST258 strains.⁹

16S ribosomal RNA methyltransferases conferring aminoglycoside resistance have spread worldwide in Enterobacteriaceae, as well as Acinetobacter species and Pseudomonas aeruginosa.¹⁰ armA is the most common 16S rRNA methyltransferase gene and is typically located within composite transposon Tn1548.¹⁰ Together with bla_DHA-1, which encodes an AmpC β-lactamase conferring cephalosporin resistance, armA is widespread in Asia and Europe,^10–12 but remains rare among Enterobacteriaceae in the USA.^10,13

Since the initial characterization of the IncR incompatibility group,¹⁴ IncR plasmids have been reported worldwide, mainly in clinical isolates of the family Enterobacteriaceae harbouring various resistance genes.^{11,12,15–18} Here we report an MDR IncR plasmid carrying armA, bla_DHA-1 and qnrB4 identified in K. pneumoniae ST37 and present a comparative analysis of related IncR plasmids.

Materials and methods

Strains

The two K. pneumoniae isolates of interest were identified a week apart from two inpatients in different units at a hospital in Pennsylvania, USA, in 2014. The first case was a male patient with alcoholic cirrhosis and the second case was a male patient with end-stage liver disease from hepatitis C and alcohol use. Both patients had peritonitis with growth of the K. pneumoniae strains reported in this study (YD760 and YDC676, respectively).

Antimicrobial susceptibility testing and molecular typing

MICs were determined with Etest (bioMérieux, France). PFGE was carried out according to the procedure described by the CDC's PulseNet program (http://www.cdc.gov/pulsenet/pathogens/index.htm).³ MLST was conducted as described previously.⁷ The quinolone resistance-determining regions (QRDRs)^3,19 of the gyrA and parC genes were sequenced using primers gyrA-1-F (5′-ATGAGCGACCTTGCGAGAGA-3′), gyrA-760-R (5′-ATTTCGCGTCCACTTCCACT-3′), parC-1-F (5′-ATGAGCGATATGGCAGAGCG-3′) and parC-638-R (5′-GGCCCCTGTACGATATCCA-3′).

Plasmid analysis and bioinformatics

Conjugation and transformation experiments were performed using Escherichia coli J53Azi^R and E. coli TOP10 as the recipients, respectively.³ The K. pneumoniae isolates and their transformants were investigated for the presence of repB of IncR, bla_TEM, bla_SHV, bla_CTX-M, plasmid-mediated ampC genes, armA, rmtB, aac(6′)-Ib, qnrA, qnrB and qnrS by PCR amplification and sequencing.^3,14,20 Plasmid fingerprint analysis of the MDR plasmids pYDC676 and pYD760 was conducted using the restriction enzymes HindIII and EcoRI.

pYDC676 was extracted from the transformant of YDC676 and sequenced on a PacBio RSII sequencing instrument at Yale Center for Genomics. The average depth of coverage was 2096×. Assembly and annotation were conducted as previously described (GenBank accession number KT225462).²¹ Easyfig 2.0 was used to compare pYDC676 with related plasmids.

Results and discussion

K. pneumoniae clinical isolates and their transformants

K. pneumoniae YDC676 and YD760 had identical antimicrobial susceptibility patterns (Table 1) and PFGE patterns and belonged to ST37 by MLST. The isolates had an identical plasmid profile (a single ∼50 kb IncR plasmid) and resistance genotype (positive for bla_TEM-1, bla_SHV-11, bla_DHA-1, armA and qnrB4). The QRDRs of gyrA and parC genes showed 100% identity to those in MDR ST37 strains from Spain,⁸ with mutations corresponding to amino acid substitutions Ser83Ile in GyrA and Ser80Ile in ParC implicated in fluoroquinolone resistance.

Table 1.

MICs for K. pneumoniae isolates and their transformants

	MIC (mg/L)
	CTX	CAZ	FEP	ETP	DOR	IPM	AMK	NAL	CIP	SXT	CST
K. pneumoniae YDC676	4	>32	0.19	0.5	0.47	0.38	>256	>256	>32	>32/608	0.125
K. pneumoniae YD760	4	>32	0.19	0.5	0.47	0.38	>256	>256	>32	>32/608	0.094
E. coli TOP10 (pYDC676)	0.38	3	0.94	0.006	0.032	0.38	>256	1.5	0.032	0.047/0.89	0.064
E. coli TOP10 (pYD760)	0.38	3	0.64	0.006	0.032	0.38	>256	1.5	0.032	0.047/0.89	0.047
E. coli TOP10	≤0.25	≤0.5	0.47	0.004	0.016	0.19	1.5	1.0	0.002	0.047/0.89	0.032

CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; ETP, ertapenem; DOR, doripenem; IPM, imipenem; AMK, amikacin; NAL, nalidixic acid; CIP, ciprofloxacin; SXT, trimethoprim/sulfamethoxazole; CST, colistin.

Conjugation experiments were unsuccessful for both K. pneumoniae isolates, as reported for other IncR plasmids,¹⁵ but transformation was successful using 50 mg/L amikacin for selection. The plasmids were identical in size and restriction profile, and contained an IncR repB replicon as well as resistance genes armA, bla_DHA-1 and qnrB4.

Comparative analysis of the backbone of MDR IncR plasmids

pYDC676 is 50 182 bp in size and shares the common backbone of IncR plasmids (Table S1, available as Supplementary data at JAC Online). A total of 38 IncR plasmids were included in this analysis based on >75% repB gene identity to that of YDC676 and the presence of four or more out of six loci (repB, resD, parAB, umuCD, retA and vagDC) in the backbone as the inclusion criteria. The repB and resD genes of pYDC676, involved in plasmid replication and maintenance, exhibited 100% identity to those on 20 IncR plasmids (Table S1). Downstream of repB were parAB and umuCD operons followed by retA, likely playing a role in the partitioning, compatibility and SOS mutagenesis of the plasmid.¹⁵ This part of pYDC676 is almost identical (>99%, 5366/5371) to that of 12 reported IncR plasmids. The vagDC operon was first characterized on a Salmonella Dublin plasmid pG19 in the UK and encodes a toxin–antitoxin system involved in plasmid maintenance.²² This operon constituted another part of the pYDC676 backbone and had 99% (643/644) to 100% identity to that in nine IncR plasmids (Table S1). In total, six plasmids (pKQ57, pKPS30, pIS04_68, pIMP-HB623, pDMC1097-218.836 kb and pCAV1596-41) share a nearly identical backbone (2–5 nt differences) with pYDC676. These plasmids were reported from ST37, ST11 or ST258 strains in Europe, China and the USA, indicating a widespread distribution of this lineage of IncR plasmids.^8,15

Upstream of vagDC, a multiple IS (multi-IS) locus (locus A) was located, comprising an IS102 (IS903-like) remnant truncated by an IS2-like element and an IS2-like remnant truncated by ISEc15 (Figure 1). A similar multi-IS locus A with intact or partial ISEc15 is shared by 10 IncR plasmids (Table S1), including the six plasmids mentioned above and one IncFII/IncFIB plasmid p1658/97 (KJ484639.1), which was identified in SHV-5-producing E. coli during a clonal outbreak in a Polish hospital in 1997.²³ Moreover, a vagDC fragment identical to that in pYDC676, with an IS102 remnant, has also been found on IncI plasmids (pC23-89, KJ484639.1; pESBL-117, CP008734.1), suggesting mobilization of vagDC among plasmids.

Figure 1.

Comparative analysis of pYDC676 and related IncR plasmids generated by Easyfig. pYDC676 (K. pneumoniae ST37, KT225462), pTR2 (K. pneumoniae, KJ187752.1, inversely displayed over 20 kb) and pKP048 (K. pneumoniae ST11, FJ628167.2, partially displayed over 26 kb) have similar MDR regions of Tn1548. pYDC676 shares a common backbone with pIS04_68 (E. coli, HG963476.1, rearranged to compare the structure) and pKPS30 (K. pneumoniae ST11, KF793937.1). pKQ57 (K. pneumoniae ST37, NZ_JH930402.1) is nearly identical to pKPS30 both in the backbone and the MDR region. Blue arrows indicate replication-associated genes. Orange arrows denote genes associated with plasmid stability and maintenance. Red and yellow arrows indicate antimicrobial resistance genes and mobile genetic elements, respectively. Two regions that contain fragments identical to pB1025 (K. pneumoniae ST11) or p1658/97 (E. coli, AF550679.1) are denoted by large brackets towards pKP048 and pIS04_68, respectively. Paired filled squares represent direct repeats (DR, CAAGC) of ISKpn21. Multi-IS loci A and B are indicated by vertical arrows. Tall bars represent the inverted repeats (IR) of ISs and transposons. Unfilled gaps in pKQ57 are indicated by vertical arrows.

pYDC676 contains a contiguous segment of 17 476 bp (nt 43 603–50 182 and 1–10 895) with >99% identity (11 nt differences) to that of IncR plasmid pIS04_68, including the backbone and an identical multi-IS locus A. pIS04_68 was identified in E. coli from a healthy swine in Denmark. pIS04_68 in turn contains a fragment (IS903B-like-orfs-lon-vagDC) with >99% identity to that in p1658/97 (Figure 1).²³ The orf-lon sub-region shows 98% identity to that in the chromosome of Vibrio cholerae (CP001485.1), indicating its possible origin from aquatic bacteria. Intact or truncated lon genes are also found in IncR plasmids pEFER (CU928144.1) and pYDC676. Interestingly, pQ19-1, a plasmid recovered from a seawater bacterium in China, shares a near identical fragment (including the repB-parAB-umuCD-retA module) with both pIS04_68 and pYDC676, and another identical fragment (5′ end of IS903B-like-ΔIS1X3-like-ΔISEcl1-orf1-4-Δorf5) with pIS04_68 (Figure 1), suggesting a close phylogenetic relationship among these plasmids. pQ19-1 has been partially sequenced as pQ19-1rep, pQ19-1str and pQ19-1tet (Table S1).¹⁶ The reason for the absence of information on the lon-vagDC fragment is that the full sequence of plasmid pQ19-1 has not been made available.

Another multi-IS locus is located on pYDC676, composed of six intact or truncated mobile genetic elements (IS1R-ΔISEcp1-ΔTn5393-ΔISSen4-ΔISEc15-like-IS903-like) on the proximal side of Δlon (Figure 1). Although many IS903 variants are registered in GenBank, an identical IS903-like element was only found in p1658/97 and pQ19-1 (100%),^16,23 while the others had no more than 98% identity (1032/1057). This further supports co-evolution of p1658/97, pQ19-1 and pYDC676. pQ19-1 is the only plasmid in GenBank containing the ΔISSen4-ΔISEc15-like-IS903-like module (multi-IS locus B) identical to that in pYDC676. Therefore, pQ19-1 may be the intermediary plasmid between pIS04_68 and pYDC676 in the evolution of IncR plasmids.

Comparative analysis of the MDR region of pYDC676

The MDR region of pYDC676, bracketed by IS903B-like and IS903-like elements, contains a 22 088 bp fragment that is nearly identical to part of pKP048, except for the apparent recent insertion of ISKpn21 in pYDC676, supported by the presence of 5 bp direct repeats (Figure 1). pKP048 was identified from a K. pneumoniae ST11 strain in China and harbours bla_KPC-2, as well as IncR and IncFII_K5 replicons.¹¹ armA in pYDC676 is flanked by ISEc29 and ΔISEc28, located adjacent to mph(E)-msr(E) macrolide resistance genes and ISCR1-sul1-qacEΔ1. Besides pKP048, this module is conserved in plasmids of various Inc types, including pMUR050 (IncN, NC_007682.3), pXD1 (IncFII, NG_041448.1), pNDM-1_Dok01 (IncA/C, AP012208.1), pCTX-M3 and pNDM-HK (IncL/M, AF550415.2 and NC_019063.1), and is always flanked by two direct repeats of IS26, thus constituting composite transposon Tn1548.^10,24 bla_DHA-1 was first discovered in Salmonella Enteritidis and likely originated from the chromosome of Morganella morganii.²⁵ bla_DHA-1 has always been found in association with ISCR1 as part of a complex class 1 integron.^13,26 qnrB4 is flanked by psp (phage shock protein) and sap (ABC transporter) operons and incorporated downstream of bla_DHA-1, possibly mobilized by ISCR1.^26,27 In plasmids pMUR050, pCTX-M3 and pNDM-1_Dok01, armA is associated with an intact class 1 integron, while in pYDC676 and pKP048, the class 1 integron is replaced at the 5′ extremity by ampR-bla_DHA-1 and the psp operon linked with qnrB4, leaving the 3′ conserved sequence (3′-CS, sul1-qacEΔ1), thus generating the armA-bla_DHA-1-qnrB4 cluster. This may also be a result of rolling circle transposition by ISCR1²⁷ and then insertion of IS26, which mediated partial deletion of the integron as described by Verdet et al.²⁶ Furthermore, IS26-mediated insertion and deletion have probably generated various combinations of bla_DHA-1, qnrB4 or other resistance genes in different plasmids, promoting their worldwide spread in Enterobacteriaceae.^11,26 The armA-bla_DHA-1-qnrB4 cluster in Tn1548 of pKP048 is bracketed by two copies of IS26, while in pYDC676 the open reading frame downstream of cinA was truncated by IS1R. An identical armA-bla_DHA-1-qnrB4 cluster was also located on an IncR plasmid, pB1025, found in five K. pneumoniae ST11 strains isolated from pets in Spain (Figure 1)¹² and IncFII plasmids from China.^3,28 More recently, a bla_NDM-1-bearing plasmid, pTR2, was reported from Taiwan containing IncR, IncFIA and IncA/C replicons exhibiting high gene synteny to pYDC676 from IS26 to bla_DHA-1 (nt 13 839–30 707, 1 nt difference), except for the truncation of bla_DHA-1 and its replacement by a ΔTn125 module (Figure 1). Furthermore, pKPN101-IT, a bla_KPC-2-bearing plasmid, has a gene array from IS26 to armA that is identical to pYDC676 but has lost the subsequent genes, including bla_DHA-1-qnrB4, due to the replacement of ΔISEc28 by IS26.²⁹ Like pYDC676 and pKPN101-IT, pTR2 has the same ISKpn21 insertion event, which suggests that the acquisition of the armA-bla_DHA-1-qnrB4 cluster was likely a single recombinant event that preceded the acquisition of bla_NDM-1 by pTR2. A similar genetic structure (armA-Δbla_DHA-1-bla_NDM-1) without the ISKpn21 insertion has also been found in two IncL/M plasmids (pNDM-HK, pNDM-OM, NC_019889) and IncFII plasmid pNDM-1saitama01 (NC_021180).

armA encodes 16S rRNA methyltransferase, which was first documented on plasmid pIP1204 in France and confers high-level aminoglycoside resistance.²⁴ bla_DHA-1 encodes plasmid-mediated AmpC β-lactamase DHA-1, conferring cephalosporin resistance.¹³ qnrB4 encodes a pentapeptide protein with affinity to gyrA, conferring low-level fluoroquinolone resistance.³⁰ Therefore, acquisition of this cluster results in simultaneous resistance to the three key classes of agents that are otherwise active against Gram-negative bacteria.

Dissemination of IncR plasmids

Although IncR was characterized relatively recently as a new incompatibility group, IncR plasmids have been associated with diverse species of Enterobacteriaceae, especially in K. pneumoniae (Table S1), frequently encoding resistance to fluoroquinolones, aminoglycosides, β-lactams (sometimes including carbapenems) and other groups of antibiotics.^{8,11,12,14–16,18} Besides armA, qnrB, bla_DHA-1 and carbapenemase genes, bla_CTX-M-bearing IncR plasmids have been identified from strains in different European countries and the USA.^17,18 IncR plasmids have disseminated especially well in K. pneumoniae strains represented by ST11 and ST258 (Table S1), while ST37 may be an emerging ST of K. pneumoniae carrying MDR IncR plasmids. Among IncR plasmids, pYDC676 is genetically closest to pKQ57 carried by K. pneumoniae ST37 isolates that caused an outbreak at a hospital in Spain,⁸ sharing an identical multi-IS locus A and truncated retA in the backbone. pKQ57 is in turn similar to pKPS30, an IncR plasmid identified from K. pneumoniae ST11 in France (Figure 1).¹⁵

Conclusions

An increasing number of IncR plasmids have been associated with various antibiotic resistance genes, including armA, qnr, bla_DHA-1, bla_CTX-M, bla_KPC, bla_VIM and bla_NDM, in recent years.^{8,11,15,17,18,31} Here, we identified two ST37 clinical isolates harbouring an MDR IncR plasmid that had a close genetic relationship with those recovered from seawater bacteria, as well as animal and human strains of Enterobacteriaceae in Asia and Europe, and had captured the armA-bla_DHA-1-qnrB resistance gene cluster. In our hospital, a bla_KPC-3-carrying ST37 strain was identified earlier,⁹ and a bla_KPC-2-carrying, colistin-resistant K. pneumoniae ST37 strain has also been found (data not shown). Taking these results together, ST37 appears to be a potential high-risk MDR K. pneumoniae clonal lineage that is emerging.

Funding

The study procedures were conducted through internal funding. Q. G. was supported by grant no. 81102509 and no. 81120108024 from the National Natural Science Foundation of China. Y. D. was supported in part by research grants R21AI107302 and R01AI104895 from the National Institutes of Health.

Transparency declarations

Y. D. has served on advisory boards for Shionogi, Meiji and Tetraphase, consulted for Melinta Therapeutics and received research funding from Merck for a study unrelated to this work. All other authors: none to declare.

Supplementary data

Table S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).