Molecular characteristics of clinical IMP-producing Klebsiella pneumoniae isolates: novel IMP-90 and integron In2147

Abstract

Background

Since the first report of carbapenem-resistant Klebsiella pneumoniae isolates in China in 2007, the prevalence of CRKP and CRE has increased significantly. However, the molecular characteristics of IMP-producing Klebsiella pneumoniae (IMPKp) are rarely reported.

Methods

A total of 29 IMPKp isolates were collected from a Chinese tertiary hospital from 2011 to 2017. Clinical IMPKp were identified by VITEK^®MS, and further analyzed by whole-genome DNA sequencing with HiSeq and PacBio RSII sequencer. Sequencing data were analyzed using CSI Phylogeny 1.4, Resfinder, PlasmidFinder and the MLST tool provided by the Centre for Genomic Epidemiology. The analysis results were visualized using iTOL editor v1_1. The open reading frames and pseudogenes were predicted using RAST 2.0 combined with BLASTP/BLASTN searches against the RefSeq database. The databases CARD, ResFinder, ISfinder, and INTEGRALL were performed for annotation of the resistance genes, mobile elements, and other features. The types of bla_IMP in clinical isolates were determined by BIGSdb-Pasteur. Integrons were drawn by Snapgene, and the gene organization diagrams were drawn by Inkscape 0.48.1.

Results

Four novel ST type, including ST5422, ST5423, ST5426 and ST5427 were identified. The IMP-4 and IMP-1 were the dominant IMP type. The majority of bla_IMP-carrying plasmids belonged to IncN and IncHI5. Two novel bla_IMP-carrying integrons (In2146 and In2147) were uncovered. A novel variant bla_IMP-90 presented in novel integron In2147 has been identified.

Conclusions

IMPKp showed low prevalence in China. Novel molecular characteristics of IMPKp have been identified. Continuous monitoring of IMPKp shall also be carried out in the future.

Introduction

The spread of carbapenem-resistant Enterobacteriaceae (CRE) has become a major public health problem. Carbapenems are the most effective drugs for the treatment of Gram-negative bacterial infections. In China, since the first report of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in 2007, the prevalence of CRKP and CRE has increased significantly [1].

Resistance to carbapenems involves multiple mechanisms, and the production of various carbapenemases is the most common mechanism. Based on their dependency on actions for enzyme activity, carbapenemases can be divided into two different groups: serine/non-metallo- (zinc-independent; classes A, C, and D) and metallo-carbapenemases (MBLs; zinc-dependent; class B) [2]. MBLs include various clinically and epidemiologically important enzymes, such as IMP, VIM and NDM. Since the first report of IMP-1 from Pseudomonas aeruginosa in Japan, IMP-type enzymes have been reported globally. Compared with NDM, IMP exhibited a relatively low prevalence among clinical K. pneumoniae isolates [3]. The CRKP harboring the bla_IMP-1, bla_IMP-4, bla_IMP-22 and bla_IMP-26 have been recovered from East Asia, Europe, Australia, or North America [3]. In China, the main IMP variants include IMP-4, IMP-8, and IMP-26 [1].

The plasmid-mediated dissemination of carbapenemase-coding genes plays an important role in emergence and spread of CRKP. The most encountered bla_IMP genes located in plasmids with diverse incompatible groups include HI2/HI5, FI/FII, L/M, A/C, P and N [4]. Various integrons that contain bla_IMP genes had been characterized such as In1763, In809/In823, In722, In73 and In687, and these integrons harbored bla_IMP-1, bla_IMP-4, bla_IMP-6, bla_IMP-8 and bla_IMP-14, respectively [5, 6].

Few studies have reported the complete sequence of bla_IMP-harbouring plasmids, which limited our understanding of the transmission mechanism of bla_IMP genes. In this study, 29 clinical IMP-producing K. pneumoniae (IMPKp) isolates were analyzed by whole-genome sequencing and further molecular analysis. A novel IMP-type enzyme (IMP-90) and novel bla_IMP-harbouring integron (In2147) were identified.

Material and methods

Bacterial strains and antimicrobial susceptibility testing

A total of 29 IMPKp isolates were collected from July 2011 to November 2017 from our hospital (Additional file 1: Table S1). All isolates were identified by VITEK^®MS (bioMérieux SA, Marcy-l’Étoile, France). At first, the MICs of antimicrobial agents [ceftazidime (CAZ), cefepime (CFP), ceftazidime-avibactam (CAZ-AVI), imipenem (IPM), meropenem (MEM), ertapenem (ETP), amikacin (AK), ciprofloxacin (CIP) and sulfamethoxazole/trimethoprim (SXT)] were measured by BioMérieux VITEK 2 AST-GN09 and AST-GN13 according to the manufacturer’s instructions. Then, were measured with broth microdilution method by using the Biofosun R Gram-negative panels (Biofosun Biotechnology Corporation Ltd., Shanghai, China). If the MICs of carbapenem antibiotics exceeded the upper limit of detection [IPM (MICs > 8 mg/L), ETP (MICs > 8 mg/L) or MEM (MICs > 4 mg/L)], the dry strip method was performed to correct the results by using E-test (Autobio Diagnostics Corporation Ltd., Zhengzhou, China). All susceptibility results were interpreted according to the 2021 CLSI guidelines [7].

Whole-genome DNA sequencing

All isolates were subjected to draft-genome sequencing using a paired-end library with an average insert size of 350 bp (ranged from 150 to 600 bp) on a HiSeq sequencer (Illumina, CA, USA). All sequences were assembled using SOAPdenovo (SOAP Version 2.21). The N₅₀, N₉₀, coverage rate and scaffold number were used to identify de novo characteristics. In addition, one or two strains from each subclade were selected for subsequent long PacBio reads sequencing using a sheared DNA library with average size of 15 kb (ranged from 10 to 20 kb) on a PacBio RSII sequencer (Pacific Biosciences, Menlo Park, CA, United States). The paired-end short Illumina reads were used to correct the long PacBio reads utilizing proofreads, and then the corrected PacBio reads were assembled de novo utilizing SMARTdenovo.

Data analysis

WGS data for all isolates were analyzed using CSI Phylogeny 1.4, Resfinder, PlasmidFinder and the MLST tool provided by the Centre for Genomic Epidemiology (https://cge.cbs.dtu.dk/services). Determination of capsular type for strains was conducted by using Kleborate (https://github.com/katholt/Kleborate). The tree file was visualized by iTOL (https://itol.embl.de), and annotated information were edited by iTOL editor v1_1.

Sequence annotation

The open reading frames and pseudogenes were predicted using RAST 2.0 combined with BLASTP/BLASTN searches against the RefSeq database as previous described [8]. Plasmid replicon types were identified using PlasmidFinder and corrected by the RAST 2.0. The databases CARD, ResFinder, ISfinder, and INTEGRALL were performed for annotation of the resistance genes, mobile elements, and other features. The types of bla_IMP in clinical isolates were determined by BIGSdb-Pasteur. Integrons were drawn by Snapgene, and the gene organization diagrams were drawn by Inkscape 0.48.1.

Transformation experiments

As previously described [9], we cloned the open reading frame of bla_IMP-90 and bla_IMP-8 into the chloramphenicol-resistant pHSG398 vector (Takara Bio, Shiga, Japan) at the EcoRI-KpnI site and used it to transform Escherichia coli DH5α cells (Takara Bio).

Nucleotide sequence accession numbers

All genome sequences in this study were submitted to GenBank under the accession BioProject No. PRJNA862666.

Results

Clinical characteristics of IMPKp isolates

A total of 29 IMPKp isolates were collected from July 2011 to November 2017 (Fig. 1). The sputum (n = 11, 37.9%) and urine (n = 6, 20.7%) were the main sources of these isolates. The strains were mainly isolated from the departments of hepatobiliary (n = 9), neurology (n = 6), surgical care unit (n = 4) and respiratory medicine (n = 4). In this study, four novel STs (ST5422, ST5423, ST5426 and ST5427) with the allelic profile (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) 3-3-1-1-1-1-9, 42-22-25-96-115-20-712, 16-24-21-31-47-257-760 and 17-73-90-39-541-18-148, respectively, have been identified. The ST5422 (n = 5), ST5423 (n = 3), ST48 (n = 3), ST2391 (n = 3) outbreaks may occur in our hospital (Fig. 1, Additional file 1: Table S1). The antimicrobial susceptibility results were listed in Table S1. About 55.2% (n = 16) of IMPKp isolates carried various bla_CTX-M, while nine isolates (24.1%) carried armA which was related to the amikacin resistance.

Fig. 1

Genetic characteristics of IMP-producing K. pneumoniae. All the bla_IMP-carrying plasmid were analyzed by the HiSeq + PacBio RSII, except that in IR5739 isolates was identified by HiSeq (marked with asterisks)

The plasmids and integrons related to known bla _IMP

The complete genome sequences of eighteen strains were analyzed. All bla_IMP gnes were located on the plasmids (Fig. 1 and Additional file 1: Table S1). In total, three known bla_IMP variants have been identified, including bla_IMP-1, bla_IMP-4 and bla_IMP-26. The gene bla_IMP-4 was distributed in 10 different ST types. Besides the ST761, other isolates harboring the bla_IMP-1 were novel ST types, including ST5422, ST5423, and ST5427 (Fig. 1). The bla_IMP-1-carrying plasmids belonged to IncN3 and IncHI5 with a size ranging from 43 to 258 kb. Plasmids harboring bla_IMP-4 were IncN1 and IncHI5 with a size ranging from 53 to 289 kb. The bla_IMP-26 located on a 311 kb IncHI5 plasmid (Fig. 1 and Additional file 1: Table S1). All bla_IMP-carrying plasmids carried various resistance genes (Additional file 1: Table S1).

The bla_IMP-1 was presented in the In994 and In1223, the bla_IMP-4 was presented in the In809, In823, and novel identified In2146, while the bla_IMP-26 was presented in the In837 (Fig. 2 and Additional file 1: Table S1). The integrons carrying the bla_IMP-4 and bla_IMP-26 exhibited a certain degree of similarity. Compared with the bla_IMP-4-carrying In809, the novel In2146 (bla_IMP-4-attCDΔ::Kl.pn.I3-aacA4'-attC-3'CS) seems like inserted the Kl.pn.I3 and lost the resistance genes qacG2 and catB3. Meanwhile, there was a possibility that the In2146 was derived from the In823 by inserting the aacA4 and bla_IMP-4 mutation (Fig. 2). The In809, In823 and In2146 contained 3'-conserved segment (CS) structures immediately downstream of the gene cassettes. The majority contained In4-like structures consisting of 3'-CS-IS6100 (with or without partial deletion of 3'-CS and chrA-padR insertion).

Fig. 2

Comparative analysis of the bla_IMP-carrying integrons. A The In994, In1223 and In2147 shown the similarity. B The In809, In2146, In837 and In823 shown the similarity

Novel metallo-β-lactamase IMP-90 and integron In2147

In this study, a novel IMP-type MBL, bla_IMP-90, was identified. An IMPKp ST48 emerged and caused an outbreak in 2011 (Fig. 1). Whole-genome sequencing revealed that the isolate carried a novel bla_IMP variant, which was designated as the bla_IMP-90. IMP-90 differed from IMP-8 by an amino acid substitution (Ser262Gly). The bla_IMP-90 was located on a 72905-bp IncM1 plasmid (Fig. 3), and was presented in a novel integron In2147 (bla_IMP-90-attC-aacA4'-3-attCD), which showed similarity with In994 and In1223 (Fig. 2). The IR5012 was susceptible to imipenem (MIC = 1 ug/mL), suggesting that the IMP-90 had a weaker imipenem hydrolyzing activity compared with other IMP enzymes (Additional file 1: Table S1). Meanwhile, the transformation experiments of bla_IMP-90 and bla_IMP-8 demonstrated that the IMP-90 exhibited similar carbapenem hydrolyzing activity with IMP-8.

Fig. 3

Backbone structure of the bla_IMP-90-encoding plasmid pIMP-90-IR5012

Discussion

The prevalence of IMPKp varies by regions and often induces outbreaks in clinical departments. A multicenter study showed that the MBLs were not equally common, with more than 60% of MBL-positive CRE isolates carrying the bla_NDM, while only 9% of the isolates produced the IMP [3]. In China, the predominance of MBL among Enterobacteriaceae was NDMs, and the IMP has been sporadically detected [10]. We have reported the molecular characterization of some clinical IMPKp isolates [11]. In this study, a larger collection (n = 29) of IMPKp were collected and analyzed by more accurate methods such as whole-genome sequencing, and thus limited results in previous study are revised and supplemented. The high homology among some strains indicated the existence of small-scale outbreaks caused by various IMPKp clones (Fig. 1). Most IMPKp isolates exhibited low-level resistance or even susceptible to various carbapenems (Additional file 1: Table S1), indicating the low hydrolysis capacity of IMP, and may leading to a low epidemic level of IMPKp. In this study, IMP-4 (14/29, 48.28%) was the most popular enzyme type, followed by IMP-1 (10/29, 34.48%), and this was consistent with previous finding [12].

The ST type of IMPKp isolates in this study was relatively dispersed (Fig. 1). The K. pneumoniae CG258 are the most common carbapenem-resistant isolates reported worldwide, including the ST11 and the ST5422 in this study. The tonB allele of ST11 (tonB-4) is widely distributed in approximately 80 unrelated STs of CG258, but the tonB-79 allele of ST258 has only been observed among less than 10 STs (http://www.pasteur.fr/mlst). Some researchers considered that the tonB-79 was probably derived from tonB-4 by acquisition of site substitutions [13]. In this study, among the four novel STs (ST5422, ST5423, ST5426 and ST5427), only ST5422 [allelic profile (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) was 3-3-1-1-1-1-9] may arise from ST11 (allelic profile: 3-3-1-1-1-1-4) by substituting tonB-4 with tonB-9. However, although it has high homology with ST11, completely different characteristics of IMP-coding genes suggested the independent evolutionary path of this novel identified clone (Fig. 1).

Consistent with previous study [14], we found that all known IMP-coding genes were located on IncN and IncHI5 plasmids in this study (Fig. 1). IncN and IncHI plasmids are both conjugative plasmids and have been reported to be part of a broad-host-range group [15]. Based on the nucleotide sequence homology over the backbones, the IncN group can be divided into three subgroups, including IncN1, IncN2 and IncN3 [16]. In this study, the predominant bla_IMP-carrying plasmids belonged to N1 with a smaller size (Fig. 1). The bla_IMP-1-carrying IncN3 plasmid was identified in UK [5], and this was the first report to identify this plasmid in China, showing the necessity of investigating prevalence and evolutionary history of IncN3 plasmids. The IncHI plasmids are important vectors in the dissemination of heavy metal resistance genes and antimicrobial resistance genes, and usually have a size larger than 200 kb [17]. In this study, all bla_IMP-carrying IncHI5 plasmids carried conserved IncHI5 backbones, including repHI5B and a repFIB-like, parABC, and tra1, mediating replication, partition and conjugal transfer, respectively. Further studies are needed to continuously monitor the prevalence of bla_IMP-carrying IncN and IncHI5 plasmids in different sources, especially among clinical settings [11, 14].

To date, various genetic context of bla_IMP-4 has been identified, e.g. bla_IMP-4-qacG-aacA4-aphA15, bla_IMP-4-Kl.pn.I3-mobC and bla_IMP-4-Kl.pn.I3-qacEΔ-sul1. Of note, the structure of bla_IMP-4-Kl.pn.I3 seems unique in isolates from China revealed by blasting in GenBank, thus it could be used as an epidemiological marker for bla_IMP-4 detection in China. In823 harboring bla_IMP-4 has been identified in IncHI5 plasmid [18]. In this study, all In823 harboring the bla_IMP-4 was presented in the IncN1 plasmid, and the bla_IMP-4-attCDΔ::Kl.pn.I3 was the most common cassette. Meanwhile, the integrons in IncHI5 were In809 and the novel integron In2146 (Figs. 1 and 2). In809 with four cassettes (bla_IMP-4-qacG2-aacA4-catB3) is widely disseminated in Enterobacteriaceae and Acinetobacter spp. in Asia–pacific region [19]. In2146 may be derived from In809, and is considered as an In809-like integron (bla_IMP-4-Kl.pn.I3-qacG2-aacA4-catB3Δ), of which a group II intron Kl.pn.I3 was inserted into the attC site of the bla_IMP-4 cassette (Fig. 2). The bla_IMP-1-carrying In994 and In1223 has been identified in previous studies [16], and it has also been found in this study (Fig. 1). In addition, the integron In837 (intI1-bla_IMP-26-attCDΔ::Kl.pn.I3-3'CS, Fig. 2) showed low homology with other bla_IMP-26-carrying integrons, and had different genetic structure such as intI1-bla_IMP-26-ltrA-qacEΔ1-sul1, intI1-bla_IMP-26-qacG-aacA4-aac(6ʹ)-orf-catB3, and intI1-bla_IMP-26-qacG-aac(6ʹ)-Ib-aac(6ʹ)-orf3-orf4-catB3-dfrA1-tnpA-istB-orf5, respectively [20]. In general, country-specific bla_IMP subtypes corresponded to the specific integron types previously characterized in that country, i.e., bla_IMP-4-carrying In809 in Australia [21]; bla_IMP-6-carrying In722 in Japan [22]; bla_IMP-8-carrying In73 in Philippines [23]; and bla_IMP-14-carrying In687 in Thailand [24]. In this study, we identified 7 different bla_IMP-carrying integron types, including 2 novel cassette combinations (In2146 and In2147; Fig. 2). All the four class I integrons have a complete set of IRi/IRt, intI1, and attI1. All integrons carried the strong promoters PcS (In2147 and In809) or the PcW_TGN-10 (In837, In994, In823, In1223 and In2146) (Additional file 1: Table S1), and these promoters can drive the high-level expression of cassette-borne genes [25, 26].

IMP-90 is a novel variant with an amino acid substitution at Ser262Gly compared with IMP-8. The IMP-90-producing isolates showed a sensitive feature to imipenem (MIC = 1 mg/L), while the bla_IMP-90-carrying E. coli DH-5α (competent cell) showed a reduced resistance to imipenem, compared with the bla_IMP-8 (data not published). Previous studies show that isolates producing the IMP-type metallo-beta-lactamases with Ser262Gly substitution all exhibited susceptibility to imipenem, including the IMP-6 and IMP-68, which were the Ser262Gly substitution variants of IMP-1 and IMP-11, respectively [9, 27]. These results indicate that the Ser262Gly substitution may affect the hydrolysis ability of IMP variants to imipenem. The bla_IMP-90 presented in the novel integron In2147 located on a IncM1 plasmid (Figs. 2 and 3). Plasmids belonging to the IncL/M are the important mobile genetic platforms for dissemination of clinically important resistance genes, e.g. bla_CTX-M and bla_OXA-48 [28]. Although L and M plasmids showed high level of DNA homology (approximately 94% overall nucleotide identity), the ExcA, TraY, and TraX proteins exhibited evident division (35%, 59%, and 75% amino acid identity, respectively). IncM plasmids have a wide host range, while IncM1 and IncM2 showed the 99% amino acid identity of the entry exclusion ExcA and TraY proteins [29]. This was the first report about the bla_IMP-carrying IncM1 plasmid. Conjugation between isolates carrying various IncM plasmids is compromised by inhibitory interactions of the exclusion system, which is highly conserved across all IncM plasmids (Fig. 3) [29]. Therefore, more attention should be paid to the transmission of drug resistance mediated by the IncM-type plasmids, and continuous monitoring shall also be carried out.

Conclusions

In conclusion, we collected and analyzed 29 IMPKp isolates from a Chinese tertiary hospital during the year 2011 to 2017. In this study, we identified four novel ST type, including the ST5422, ST5423, ST5426 and ST5427. The dominant IMP type was IMP-4 and IMP-1. The majority of bla_IMP-carrying plasmids belonged to IncN and IncHI5. Two novel bla_IMP-carrying integrons (In2146 and In2147) were uncovered. A novel variant bla_IMP-90 presented in novel integron In2147 has been identified.