Distribution of β-lactamase Gene and Genetic Context of blaKPC-2 in Clinical Carbapenemase-producing Klebsiella Pneumoniae Isolates

doi:10.21203/rs.3.rs-96880/v1

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Distribution of β-lactamase Gene and Genetic Context of blaKPC-2 in Clinical Carbapenemase-producing Klebsiella Pneumoniae Isolates

https://doi.org/10.21203/rs.3.rs-96880/v1

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published 26 Jan, 2021

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Background: This study is designed to characterize the dissemination mechanism and genetic context of Klebsiella pneumoniae carbapenemase (KPC) genes in carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates.

Methods: A retrospective analysis was performed on CRKP isolates from a teaching hospital of Wenzhou Medical University from 2015-2017. Polymerase chain reaction (PCR)-based amplification and whole-genome sequencing (WGS) were used to analyze the genetic context of the bla_KPC-2 gene. Conjugation experiments were performed to evaluate the transferability of bla_KPC-2-bearing plasmids. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to investigate the clonal relatedness of bla_KPC-2-producing strains.

Results: The bla_KPC-2 gene was identified in 13.61% (40/294) of clinical K. pneumoniae isolates. Three different sequence types (ST11, ST15 and ST656) and 5 PFGE subtypes (A to E) were classified among them. ST11 was the dominant sequence type (92.50%, 37/40). Plasmid-oriented antibiotic resistance genes such as extended spectrum-β-lactamases (ESBLs) and other antimicrobial resistance genes were also found in KPC-positive K. pneumoniae (KPC-Kp). Mapping PCR and genomic sequencing revealed that the bla_KPC-2-bearing sequence regions related to different mobile elements, including Tn1721- and IS26-based transposons, were mainly located in but not restricted to IncFII-like plasmids and were structurally divergent.

Conclusions: The bla_KPC-2 genes related to divergent mobile genetic elements encoded on transferable plasmids may transfer easily and widely, resulting in the spread of resistance among bacteria of different species or genera. Co-infection of KPC-Kp plasmids carrying additional non-β-lactam antibiotic resistance genes simultaneously further limits the antibiotics available to treat infections with KPC-producing pathogens.

Infectious Diseases

General Microbiology

Carbapenemase

CRKP

blaKPC-2

KPC-Kp

ST11

Tn1721

Carbapenems represent a critical class of β-lactam antimicrobials that are used against multidrug-resistant (MDR) gram-negative bacteria, especially strains expressing a high level of AmpC cephalosporinase or extended spectrum-β-lactamases (ESBLs)^[1]. Though Klebsiella pneumoniae is considered an opportunistic pathogen, the prevalence of carbapenem-resistant K. pneumoniae (CRKP) associated with severe nosocomial and systemic infections has become an alarming threat to public health in recent years^[2]. CRKP may serve as a reservoir for antimicrobial resistance because of its propensity to accumulate and transfer ESBLs and other antibiotic inactivating enzymes, mostly in the form of transferable plasmids and transposons, leading to MDR bacterial infections^[3].

By far, the CRKP pandemic is primarily driven either by the widespread dissemination of clonal complex (CC) 258, more specifically to multilocus sequence type (ST) 258, or the horizontal dissemination of mobile genetic elements, especially insertion sequences and transposons encoded on the plasmids^{[4, 5]}. ST258 is the international hyper-epidemic lineage clone contributing to the spread of K. pneumoniae carbapenemase (KPC)-positive K. pneumoniae (KPC-Kp), which has disseminated throughout the USA and European countries^[6]. However, ST11, a single locus variant of ST258, is frequently reported as the dominant clone of CRKP in Asia due to its association with multidrug resistance determinants, hypervirulence and high transmission ability^[7].

The most frequent plasmid-carried Ambler class A carbapenemase is KPC, and the bla_KPC−2 gene is the main cause of a prolonged outbreak^[3]. The bla_KPC−2 gene is carried frequently within the conserved Tn3 family transposon Tn4401 on transferable plasmids in Europe and the United States^{[8, 9]}. However, non-Tn4401 mobile elements, especially Tn1721-like transposons among ST11 K. pneumoniae, are mainly responsible for the effective spread of the bla_KPC−2 gene in China^[10].

Despite the description of several novel structures and detailed data obtained in previous research, little is known about the genetic process of the conversion that accounts for the genetic differences in the bla_KPC−2 gene. The mechanism behind the transmission of this gene warrants further exploration. This research aimed to provide a molecular epidemiology investigation and the genetic features of retrospectively collected bla_KPC−2-harboring K. pneumoniae in a Chinese tertiary hospital.

Bacterial strains and antibiotic susceptibility testing

Between January 2015 and December 2017, 294 clinical isolates of K. pneumoniae were obtained from a hospital in Wenzhou, China, and identified as K. pneumoniae by a VITEK 2 automated microbiology analyzer (BioMerieux Corporate, Craponne, France). Antimicrobial susceptibility tests were determined by the agar dilution method for 14 antibiotics and by the broth microdilution method for tigecycline, colistin and ceftazidime/avibactam (CAZ/AVI). The results were interpreted by the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2018)^[11], while for colistin, tigecycline, and CAZ/AVI, the breakpoints were defined according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org/)^[12]. Escherichia coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as quality controls. Isolates resistant to either or both carbapenems (imipenem and meropenem) tested were classified as CRKP^[13]. The carbapenemase phenotype was further confirmed by the Carba NP test^[14]. MDR, extensively drug-resistant (XDR) and pandrug-resistant (PDR) phenotypes were defined according to a previous report^[15].

PCR amplification and sequencing

Bacterial DNA was extracted using the AxyPrep Bacterial Genomic DNA Miniprep Kit (Axygen Scientific, Union City, CA, United States) from a single colony subcultured on MacConkey agar plates at 37 °C for 18 hours. Carbapenemase genes (bla_KPC, bla_GES, bla_IMP, bla_VIM, bla_NDM, and bla_OXA−48), plasmid-mediated AmpC β-lactamase genes (bla_MOX, bla_CIT, bla_DHA, bla_ACC, bla_EBC, and bla_FOX) and broad-spectrum β-lactamase genes (bla_CTX−M, bla_TEM, and bla_SHV) were analyzed by PCR as previously described^[16]. PCR amplicons were sequenced and compared with sequences available in the National Center for Biotechnology Information (NCBI) database using BLAST searches.

Molecular typing

All CRKP isolates were classified by XbaI (Takara, Dalian, China)-digested pulsed-field gel electrophoresis (PFGE) using a CHEF Mapper System (Bio-Rad) as previously described^[13]. A phylogenetic dendrogram of PFGE profiles was constructed using UPGMA clustering under appropriate tolerance (2%) settings. Cutoff lines at 85% and 95% were used to delineate PFGE clusters^[17]. Multilocus sequence typing (MLST) of the K. pneumoniae isolates was performed with PCR and PCR products of seven housekeeping genes, namely, gapA, infB, mdh, pgi, phoE, rpoB, and tonB, were sequenced^[10]. All the sequences were compared with information in the MLST database (http://bigsdb.web.pasteur.fr/klebsiella).

Conjugation experiment

Plasmid conjugation experiments were carried out by filter mating using rifampicin-resistant E. coli EC600 as a recipient^[18]. The transformants were selected on MacConkey agar supplemented with rifampicin (1,024 mg/L) and meropenem (2 mg/L). Putative transconjugant colonies were selected, further verification was performed based on 16S rRNA sequencing and PCR-amplified bla_KPC−2 detection of the transconjugant. PCR-based replicon typing was conducted and replicons were sequenced using previously reported primers^[19].

Genetic environment analysis of the bla_KPC−2 gene

A series of primers based on bla_KPC-surrounding sequences were designed. Mapping PCR was performed to compare the genetic context of the bla_KPC gene according to previously reported conditions^{[10, 20]}. When a standard primer pair failed to yield a product, alternative outer primers were used to span the region of variation and all amplification products obtained were sequenced.

WGS and bioinformatic analyses

Genomic DNA was extracted using the aforementioned method. DNA was sequenced with an Illumina HiSeq 2500 and PacBio RS II instrument (Pacific Biosciences) at Personalbio Technology Co., Ltd. (Shanghai, China). The PacBio long reads were initially assembled by Canu v1.6^[21] to obtain contigs of the genome sequences, and then two FASTQ sequence files generated using the Illumina HiSeq 2500 platform were mapped onto the primary assembly to control assembly quality. The potential open reading frames (ORFs) were predicted using Glimmer software (http://ccb.jhu.edu/software/glimmer) and annotated against UniProt/Swiss-Prot. BlastX (https://blast.ncbi.nlm.nih.gov) was used to annotate the predicted protein-coding genes against a non-redundant protein database with an e-value threshold of 1e-5. Annotation of resistance genes was performed using ISfinder, INTEGRALL and ResFinder with default parameters^[22].

Comparative genomics analysis of the bla_KPC−2-bearing plasmids and bla_KPC−2 gene-related regions

The plasmid and chromosome genome sequences used for comparative genomics analysis were downloaded from the NCBI database. The sequences with an identity and coverage of ≥ 80% with pKPC3020-124 were retained for later ortholog analysis using BlastP and InParanoid^[23]. A map of the plasmid with GC content and GC skew was drawn using the online CGView Server and local GView 1.7 with a visual interface^[22]. For the linear comparison of the bla_KPC−2 gene-related fragments, wild-type KPC-Kp isolates were mixed, and the plasmids were extracted using a Qiagen Plasmid Mega Kit (Qiagen, Valencia, CA, USA). Sequences containing the bla_KPC−2 gene were also obtained from the NCBI Nucleotide database using bla_KPC−2, Tn1721, ISKpn6, and ISKpn8 as the key words. The results were filtered, and only sequences containing a complete bla_KPC−2 gene and more than 20 kb in length were retained. Multiple sequence alignments were performed by MAFFT using the 11 kb bla_KPC−2 gene-related fragment of pKPC3020-124 as a reference, and the sequences were clustered with an identity of 80%^[24]. The sequence sharing the greatest similarity to the other sequences in each cluster was chosen as the candidate for ortholog analysis.

Nucleotide Accession Number

The nucleotide sequences reported in this paper have been deposited in GenBank nucleotide database, and the accession numbers for the genome sequences of K. pneumoniae KP3020 are CP061354 (chromosome), CP061355 (pKPC3020-124), CP061356 (pKPC3020-54), CP061357 (pKPC3020-4) and CP061358 (pKPC3020-2), while CP061347, CP061346 and CP061348 were for the bla_KPC−2-bearing plasmid pKPC1880 and sequences contig1846 and 41_pilon, respectively.

Prevalence of carbapenemase and distribution of the β-lactamase genes

Ten β-lactamase genes were predicted through the sequencing of the pooled genomic DNA of 294 strains and were confirmed by the PCR screening method. Regardless of the intrinsic genes bla_SHV, bla_OKP, and bla_LEN encoded in the chromosome, bla_CTX−M−65 and bla_TEM−1 were the most prevalent genes with positive rates of 27.21% and 18.71%, respectively, while bla_OXA−10 and bla_OXA−1 showed the lowest positive rates of 1.02% and 0.68%, respectively (Table S1). The bla_KPC gene was identified in 13.61% (40/294) of the strains, all of which were verified to be carbapenemase producers. Sequencing results of the PCR products revealed identical bla_KPC−2 alleles (NG_049253) among these carbapenemase-producing K. pneumoniae. No other carbapenemase gene was identified. The KPC-Kp isolates also carried at least one other β-lactamase gene, such as the ESBL gene bla_CTX−M (90%, 36/40) and the narrow-spectrum bla_TEM (70%, 28/40) (Fig. 1). The bla_SHV variants included bla_SHV−2 (n = 2), bla_SHV−12 (n = 10), bla_SHV−28 (n = 1) and bla_SHV−11 (n = 37), while bla_CTX−M−65 and bla_TEM−1 were the only bla_TEM genotypes observed.

Antimicrobial susceptibility and conjugation results of the CRKP isolates

The minimum inhibitory concentrations (MICs) of 17 antimicrobial agents against all forty CRKP isolates showed multidrug resistance profiles, particularly to β-lactams (including all carbapenems, ertapenem, meropenem and imipenem detected), quinolones and aminoglycosides (Table 1 and Table S1). Notably, all isolates were susceptible to CAZ/AVI, except for KP3034, which exhibited low level resistance to this combination with the MIC of 16/4 mg/L. Colistin was shown to be the most active agent (MIC₉₀ = 1 mg/L, 100% susceptible), followed by tigecycline (MIC₉₀ = 8 mg/L, 80% susceptible). The MIC values of aminoglycoside antibiotics were variable: 62.5% (25/40) of isolates showed resistance to gentamicin, and 57.5% (23/40) of isolates showed resistance to both tobramycin and amikacin. Chloramphenicol and fosfomycin exhibited low sensitivity (< 20%). In general, all isolates were not sensitive to at least one agent in three or more antimicrobial categories, while 8 isolates (20%, 8/40) were not sensitive to at least one agent in all categories except colistin, exhibiting MDR and XDR phenotypes.

Table 1

Antimicrobial susceptibility testing results of carbapenem-resistant *Klebsiella pneumoniae* isolates from 2015 to 2017 (mg/L)
Antibiotics	Range	MIC₅₀	MIC₉₀	MIC Interpretation
Antibiotics	Range	MIC₅₀	MIC₉₀	S (%)	I (%)	R (%)
Ceftazidime^a	16–256	64	256	0	0	100
Ceftazidime/avibactam^b	0.5–16	2	2	97.5	0	2.5
Cefotaxime^a	8-512	256	512	0	0	100
Cefepime^a	16–256	256	> 256	0	0	100
Aztreonam^a	128-1,024	1,024	> 1,024	0	0	100
Imipenem^a	8-512	32	64	0	0	100
Meropenem^a	4-256	32	64	0	0	100
Ertapenem^a	32 − 1,024	512	512	0	0	100
Gentamicin^a	0.25-1,024	128	512	37.5	0	62.5
Tobramycin^a	0.25-1,024	128	512	37.5	5	57.5
Amikacin^a	0.125-1,024	1,024	> 1,024	42.5	0	57.5
Ciprofloxacin^a	16–256	32	128	0	0	100
Levofloxacin^a	8-128	32	64	0	0	100
Fosfomycin^a	32 − 1,024	512	1,024	5	10	85
Chloramphenicol^a	2-512	512	512	20	2.5	77.5
Tigecycline^b	0.5–32	2	8	80	12.5	7.5
Colistin^b	0.25-2	1	1	100	0	0
S, susceptible; I, intermediate; R, resistant.
^aThe antimicrobial susceptibility test of these antibiotics was conducted by the agar diffusion method and interpreted by CLSI criteria.
^bFor colistin, tigecycline and ceftazidime/avibactam, the antimicrobial susceptibility test was performed using broth microdilution method and interpreted by EUCAST criteria.

To localize the bla_KPC gene and determine the transferable ability of bla_KPC-bearing plasmids in the K. pneumoniae strains, a conjugation experiment was performed. Despite repeated attempts, only 5 of 40 isolates consisting of different ST types successfully transferred carbapenem-resistant plasmids to E. coli EC600 by conjugation. The transconjugants exhibited a phenotype of resistance or reduced susceptibility to carbapenems compared with the recipient E. coli EC600 (Table 2).

Table 2

Resistance genes and carbapenem MIC levels (mg/L) of wild strains and transconjugants
Strains	β-lactam genes	ETA	MEM	IMP	Replicon types	ST types
KP1878	bla_KPC−2, bla_TEM−1, bla_CTX−M−65, bla_SHV−11	512	256	64	IncFII	11
pKPC1878/EC600	bla_KPC−2, bla_TEM−1, bla_CTX−M−65	16	8	4	IncFII	-
KP1880	bla_KPC−2, bla_TEM−1, bla_CTX−M−65, bla_SHV−11	256	128	32	IncFII, IncFII_K	11
pKPC1880/EC600	bla_KPC−2, bla_TEM−1, bla_CTX−M−65	4	2	2	IncFII	-
KP2159	bla_KPC−2, bla_TEM−1, bla_CTX−M−6, bla_SHV−2	32	8	8	IncFIA, IncFII_K	656
pKPC2159/EC600	bla_KPC−2, bla_TEM−1	8	2	2	IncFII_K	-
KP3020	bla_KPC−2, bla_TEM−1, bla_CTX−M−65, bla_SHV−28	64	64	32	IncFII_K	15
pKPC3020-124/EC600	bla_KPC−2, bla_TEM−1	8	2	2	IncFII_K	-
KP4603	bla_KPC−2, bla_TEM−1, bla_CTX−M−65, bla_SHV−11, bla_SHV−12	512	256	64	IncFII, IncFII_K	11
pKPC4603/EC600	bla_KPC−2, bla_CTX−M−65, bla_SHV−12	16	4	4	IncFII_K	-
EC600	-	0.0075	0.03	0.015	-	-
ETA, ertapenem; MEM, meropenem; IMP, imipenem.

Genetic relatedness of the CRKP isolates

The results of MLST and PFGE analyses divided 40 carbapenemase producers into three different ST types (ST11, ST15 and ST656) and five PFGE pulsotypes (A to E) (Fig. 1). ST11 (n = 37) was the most predominant sequence type, consisting of all strains of PFGE pulsotypes C (n = 11), D (n = 10) and E (n = 16), while ST15 (n = 1) and ST656 (n = 2) consisted of isolates of PFGE pulsotypes A and B, respectively. All the CRKP isolates harbored one or more other β-lactamase genes along with the bla_KPC−2 gene, especially bla_CTX−M−65 or/and bla_TEM−1 in most ST11 K. pneumoniae isolates. The bla_TEM−1 gene was not identified in any strains of PFGE cluster C.

Comparative genomics analysis of the plasmid pKPC3020-124 with homologous plasmids

Sequencing analysis of the conjugative bla_KPC-bearing IncFII_K plasmid pKPC3020-124 from KP3020 showed that the plasmid was 124,452 bp in length with an average GC content of 53.2% and was predicted to encode 152 ORFs. Using the plasmid sequence as a query, 15 sequences with the highest similarities (> 80% coverage and > 80% identity) with pKPC3020-124 were retrieved from the NCBI Nucleotide database. 14 of them were IncFII_K plasmid sequences, including 2, 10 and 2 sequences from the Enterobacteriaceae species of E. coli, K. pneumoniae and Serratia marcescens, respectively, and one was from the chromosome sequence of K. pneumoniae 91a83dc8-b809-11e8-aae5-3c4a9275d6c8 (Table S2). These bla_KPC-bearing plasmids could mainly be divided into two functional regions: a variable region and a conserved region (Fig. 2). The conserved region could be further divided into two fragments, with one fragment encoding genes related to the type IV secretion system (conjugative transfer region) and the other encoding backbone genes related to plasmid replication and stability (plasmid maintenance region). The variable region is full of insert sequences (such as ISKpn14, IS5075, ISStma11, and ISEc38) and some resistance genes (MDR region), including bla_KPC−2, bla_TEM−1 and four mercury resistance genes (merACPT). The difference between pKPC3020-124 and the other plasmids is that former has a unique region of approximately 15.5 kb in length encoding 5 insert sequences and four DNA metabolism enzyme genes (resA, agp, nqrC and lold) (Fig. 2, unknown function region).

Comparison of genetic environments of bla_KPC−2-encoding regions

Based on the results of the mapping PCR approach, four divergent forms of bla_KPC−2-harboring structures were identified among these carbapenem-resistant plasmids (Table S3). The primers used and their positional counterparts are summarized in Table S4 and Fig. S1. The mapping PCR results were confirmed by subsequent sequencing and aligning with WGS and plasmid sequencing results. To analyze the structure of the bla_KPC−2 encoding region, all the sequences (including four obtained in this work) of approximately 5–20 kb in length (with the bla_KPC−2 gene at the center) were retrieved from the NCBI Nucleotide database. According to the similarities of the core sequences adjacent to the bla_KPC−2 gene, 10 sequences were chosen as representatives and are illustrated in Fig. 3, revealing that an approximately 4-kb fragment encoding tpnR-ISKpn8-bla_KPC−2-ΔISKpn6 was conserved and present in the majority of the sequences. According to whether a Tn1721 (or its derivative) was adjacent to the bla_KPC−2-encoding fragment, these sequences were roughly categorized into two groups. The first group consisted of the sequences without Tn1721 (or its derivative), while the second group contained Tn1721 (or its derivative) next to the bla_KPC−2-encoding fragment. The sequences of the second group were mainly from K. pneumonia, but those of the first group existed in various species such as K. pneumonia, E. coli, and Citrobacter freundii.

In this study, all carbapenem-resistant strains were confirmed to be carbapenemase producers and were highly resistant to the most common antimicrobial agents. All isolates showed MDR phenotypes, with 20% of them resistant to agents of all antimicrobial categories tested except for colistin, which could be evaluated as XDR pathogens according to a previous report^[15]. The data showed that CAZ/AVI displayed potent activity against clinical KPC-Kp isolates with an MIC₉₀ of 2/4 mg/L. Intriguingly, in addition to the high level of carbapenem resistance with a meropenem MIC of 1,024 mg/L, the strain KP3034 exhibited a higher MIC level for CAZ/AVI (16/4 mg/L) than the other strains detected. As no history of previous antimicrobial exposure to CAZ/AVI treatment or specific mutations were observed in the bla_KPC−2 gene, hyper-expression of the bla_KPC−2 gene associated with porin deficiency in the outer membrane, or other unknown mechanisms might account for this low level of CAZ/AVI resistance^[25].

In addition to the bla_KPC−2 gene, one or more other β-lactamase genes (such as bla_CTX−M, bla_SHV and bla_TEM) were identified in these KPC-producing K. pneumoniae strains, with 90% (36/40) of the strains carrying the ESBL gene bla_CTX−M. bla_CTX−M−65 identified in this work belongs to the group 9 CTX-M β-lactamase gene, which mediates cephalosporin resistance^[26], while bla_SHV, a core chromosomal gene in K. pneumonia, mainly mediates ampicillin resistance^[27]. CTX-M-65 is a variant of CTX-M-14 that differs by 2 amino acid substitutions (Ala78Val and Ser273Arg)^[28] and was identified in the variable regions of F33:A-:B- plasmids from food-producing animals in China^[29]. In contrast to bla_CTX−M−14 reported by Yang et al^[14], bla_SHV−11, bla_CTX−M−65, and bla_TEM−1 were shown to be the predominant genotypes. The co-existence of these multiple broad-spectrum β-lactamase genes simultaneously in KPC-Kp confers resistance to 1st-, 2nd- and 3rd-generation cephalosporins, leading to difficulties in treating the corresponding bacterial infections. Other resistance genes, such as rmtB and oqxA/B, were also identified in these CRKP strains, corroborating the published literature that both the plasmid-mediated quinolone resistance genes (PMQRs) and 16S-RMTase-encoding genes were found in the KPC-Kp strain due to the selection pressure of antimicrobial agents, making it a potential reservoir for the spread of multidrug resistance^[30].

MLST results revealed that these 40 KPC-producing K. pneumoniae strains were composed of three ST types (ST11, ST15 and ST656), and most of these strains were ST11 (92.5%, 37/40). Consistent with previous reports, ST11 was the predominant ST attributed to the spread of carbapenem resistance in CRKP isolates^[12], while ST15 and ST656 appeared sporadically. Different bla_SHV variants were found in CRKP strains of different ST types. bla_SHV−11 was the predominant subtype in ST11 K. pneumoniae, while bla_SHV−2 and bla_SHV−28 were restricted to ST656 and ST15 K. pneumoniae in this work. bla_SHV−28 was previously found in ST15 K. pneumoniae isolated from dogs in a veterinary clinic, but these strains produced OXA-48 instead of KPC type carbapenemase^[31]. Transmission of IncX plasmids co-producing bla_KPC−2 and bla_SHV−12 genes have been reported in many CRKP isolates^[32]. In this study, bla_SHV−12 was located downstream of the Tn1721 transposons in the KPC-bearing IncFII plasmids among 10 strains in PFGE plusotype C (Fig. 3, variant 2). We also found a substantial proportion of ST11 K. pneumoniae from various samples sharing identical PFGE profiles and similar MIC values, indicating a nosocomial transmission and outbreak of ST11-type KPC-Kp in the hospital. ST11 was proven to be a successful clone that contributed to the outbreak of KPC-2-producing K. pneumoniae, leading to severe nosocomial infections^{[7, 33]}.

Our results are consistent with those from previous studies in which the bla_KPC−2 gene is associated with the core bla_KPC platform (ΔISKpn6-bla_KPC−2-ISKpn8) and can be broadly classified as the NTE_KPC−II (bla_KPC-bearing non-Tn4401 elements type II) group due to the absence of bla_TEM between ISKpn8 and bla_KPC−2^{[27, 34]}. Sequencing data revealed that the bla_KPC−2 gene was embedded in divergent IncFII-like plasmids, which had a superiority to capture bla_KPC−2 by the mobile gene elements, resulting in gradual acquisition or accumulation of carbapenem resistance in ST11 K. pneumoniae^[35]. Tn1721 transposon is located adjacent to bla_KPC−2 in the plasmid pKPC3020-124. Tn1721 was a result of an integration of the Tn3-based transposon and the partial Tn4401 segment reported in the USA and European countries and was confirmed to transport the carbapenem resistance gene bla_KPC−2 in China^[36]. Interestingly, the Tn1721-based transposons are more flexible than expected when the IS26 was inserted into Tn1721-tnpA in p44-2^[10], and the insertion of the fosA3 gene sandwiched by two IS26 was found in a transferable bla_KPC−2-carrying plasmid pHS102707 at the same position^[37], resulting in additional fosfomycin resistance (Fig. 3). Moreover, a 5-bp target site duplication (TSD), a characterized signature of the transposition event of the Tn1721-like transposon, was also confirmed in this study^[36]. An identical bla_KPC−2-bearing region was found in 15 sequences investigated in comparative genomics analysis, suggesting that spread of the bla_KPC−2 gene could occur between species such as K. pneumoniae, E. coli, and even S. marcescens due to horizontal gene transfer mediated by the conjugative plasmids.

However, the bla_KPC−2-carrying plasmids were transferable and did not appear to be restricted to a certain ST or PFGE. We observed the identical Tn1721-ISKpn6-bla_KPC−2-ISKpn8-ΔTn3-IS26 configuration located in conjugative plasmids within ST656 K. pneumoniae strain KP2159 and ST15 strain KP3020. Congruent with previous reports^[36], the bla_KPC−2 gene can be transferred from one strain to another, even if not embedded in Tn1721 transposons. The bla_KPC−2 gene was detected in the ST656 K. pneumoniae isolate FK2181 in China, as determined previously in the same hospital^[38]. Moreover, while KP2171 exhibited the identical ST type, PFGE profile and similar susceptibility patterns with KP2159, we speculate that the sporadic spread of the bla_KPC gene among these bla_KPC-producing ST656 and ST15 K. pneumoniae is presumably caused by the transmission of conjugative plasmids or mobilization of genetic elements, such as Tn1721 transposons and the insertion sequence IS26.

In summary, carbapenemase-producing K. pneumoniae isolates in this study differed in clonal backgrounds, mainly consisting of ST11 or PFGE pulsotypes C, D and E. The mobile genetic elements related to the bla_KPC-2 gene that are accompanied by multiple resistance genes, such as ESBLs, PMQRs and 16S-RMTase encoded on the transferable plasmid, may indicate the widespread resistance among bacteria of different species. Moreover, vigilance should be heightened with regard to the occurrence of CAZ/AVI resistance without previous exposure. Effective prevention and control measures should be adopted to control nosocomial infection and reduce the spread of CRKP strains in hospitals.

CRKP: Carbapenem-resistant Klebsiella pneumoniae; PCR: Polymerase chain reaction; WGS: Whole-genome sequencing; MLST: Multi-locus sequence typing; PFGE: Pulsed-field gel electrophoresis; KPC-Kp: KPC-producing K. pneumoniae; ESBLs: extended spectrum-β-lactamases; CLSI: Clinical and Laboratory Standards Institute; MIC: minimum inhibitory concentration; CAZ/AVI: ceftazidime/avibactam; MDR: Multidrug-resistant; XDR extensively drug-resistant; PDR: pandrug-resistant.

Acknowledgements

The authors thank all the colleagues and the reviewers who helped this work.

Authors’ contributions

Conceived and designed the experiments: YH, TX QB and HZ. Performed the experiments: HLiu, HLin, XZhu, XZhang, QL, JL and XL. Analyzed the data: ZS, LL, KL, MZ and TX. Wrote the paper: HLiu, QB and HZ.

Funding

This study was supported by the National Natural Science Foundation of China [81973382, 81960381 and 81700011]; the Natural Science Foundation of Zhejiang Province, China [LY19C060002, LQ17H190001 and LQ17H010003]; the Science & Technology Project of Wenzhou City, China [Y2020112, Y20170205 and 2019Y0358] and the Science & Technology Project of Inner Mongolia Autonomous Region, China (201802125).

Availability of data and materials

The datasets used and analyzed during the current study are included in this published article.

Ethics approval and consent to participate

Not required for this type of work without individual patient data.

Consent for publication

All authors read and approved the final manuscript and gave consent for publication.

Competing interests

The authors have no competing interests.

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TableS1.xls
The MICs and resistant genes in 294 clinical K. peumoniae isolates.
TableS2.xlsx
Comparative genomic analysis of pKPC3020-124 with 15 sequences obtained from NCBI database.
TableS3.doc
Mapping PCR results of 40 KPC-Kp isolates.
TableS4.doc
Primers used in mapping PCR approach.
FigureS1.doc
Position of the primers used in the mapping PCR for the backbones of the blaKPC-2 region.

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Distribution of β-lactamase Gene and Genetic Context of blaKPC-2 in Clinical Carbapenemase-producing Klebsiella Pneumoniae Isolates

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Bacterial strains and antibiotic susceptibility testing

PCR amplification and sequencing

Molecular typing

Conjugation experiment

Genetic environment analysis of the bla_KPC−2 gene

WGS and bioinformatic analyses

Comparative genomics analysis of the bla_KPC−2-bearing plasmids and bla_KPC−2 gene-related regions

Nucleotide Accession Number

Results

Prevalence of carbapenemase and distribution of the β-lactamase genes

Antimicrobial susceptibility and conjugation results of the CRKP isolates

Genetic relatedness of the CRKP isolates

Comparative genomics analysis of the plasmid pKPC3020-124 with homologous plasmids

Comparison of genetic environments of bla_KPC−2-encoding regions

Discussion

Conclusion

Abbreviations

Declarations

Acknowledgements

Authors’ contributions

Funding

Availability of data and materials

Ethics approval and consent to participate

Consent for publication

Competing interests

References

Supplementary Files

Status:

Journal Publication

Version 1

Distribution of β-lactamase Gene and Genetic Context of blaKPC-2 in Clinical Carbapenemase-producing Klebsiella Pneumoniae Isolates

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Bacterial strains and antibiotic susceptibility testing

PCR amplification and sequencing

Molecular typing

Conjugation experiment

Genetic environment analysis of the blaKPC−2 gene

WGS and bioinformatic analyses

Comparative genomics analysis of the blaKPC−2-bearing plasmids and blaKPC−2 gene-related regions

Nucleotide Accession Number

Results

Prevalence of carbapenemase and distribution of the β-lactamase genes

Antimicrobial susceptibility and conjugation results of the CRKP isolates

Genetic relatedness of the CRKP isolates

Comparative genomics analysis of the plasmid pKPC3020-124 with homologous plasmids

Comparison of genetic environments of blaKPC−2-encoding regions

Discussion

Conclusion

Abbreviations

Declarations

Acknowledgements

Authors’ contributions

Funding

Availability of data and materials

Ethics approval and consent to participate

Consent for publication

Competing interests

References

Supplementary Files

Status:

Journal Publication

Version 1

Genetic environment analysis of the bla_KPC−2 gene

Comparative genomics analysis of the bla_KPC−2-bearing plasmids and bla_KPC−2 gene-related regions

Comparison of genetic environments of bla_KPC−2-encoding regions