Various arrangements of mobile genetic elements among CC147 subpopulations of Klebsiella pneumoniae harboring blaNDM-1: a comparative genomic analysis of carbapenem resistant strains

Background Certain clonal complexes (CCs) of Klebsiella pneumoniae such as CC147 (ST147 and ST392) are major drivers of blaNDM dissemination across the world. ST147 has repeatedly reported from our geographical region, but its population dynamics and evolutionary trajectories need to be further studied. Methods Comparative genomic analysis of 51 carbapenem-nonsusceptible strains as well as three hypervirulent K. pneumoniae (hvKp) recovered during 16-months of surveillance was performed using various bioinformatics tools. We investigated the genetic proximity of our ST147 strains with publicly available corresponding genomes deposited globally and from neighbor countries in our geographic region. Results While IncL/M plasmid harboring blaOXA-48 was distributed among divergent clones, blaNDM-1 was circulated by twenty of the 25 CC147 dominant clone and were mostly recovered from the ICU. The NDM-1 core structure was bracketed by a single isoform of mobile genetic elements (MGEs) [ΔISKpn26-NDM-TnAs3-ΔIS3000-Tn5403] and was located on Col440I plasmid in 68.7% of ST392. However, various arrangements of MGEs including MITESen1/MITESen1 composite transposon or combination of MITESen1/ISSen4/IS903B/IS5/ISEhe3 on IncFIb (pB171) were identified in ST147. It seems that ST392 circulated blaNDM-1 in 2018 before being gradually replaced by ST147 from the middle to the end of sample collection in 2019. ST147 strains possessed the highest number of resistance markers and showed high genetic similarity with four public genomes that harbored blaNDM-1 on the same replicon type. Mainly, there was a convergence between clusters and isolated neighboring countries in the minimum-spanning tree. A conserved arrangement of resistance markers/MGEs was linked to methyltransferase armA which was embedded in class 1 integron in 8 isolates of ST147/ST48 high-risk clones. Conclusion Our findings highlight the dynamic nature of blaNDM-1 transmission among K. pneumoniae in Iran that occurs both clonally and horizontally via various combinations of MGEs. This is the first analysis of Iranian ST147/NDM + clone in the global context. Supplementary Information The online version contains supplementary material available at 10.1186/s12929-023-00960-0.


Introduction
Klebsiella pneumoniae is a member of the Enterobacterales family with an increasing rate of antimicrobial resistance, owing primarily to the production of carbapenemases and extended-spectrum beta-lactamases (ESBLs).This has led to the "Centers for Disease Control and Prevention" (CDC) to classify this organism as an urgent threat [1].In addition, this bacterium is classified as a "priority pathogen" by "surveillance and infection control agencies" [2].Molecular analyses have shown that the spread of epidemic lineages (high-risk clones) such as clonal complexes CC15, CC101, CC147, and CC258 in different geographical regions promotes the increasing emergence of multidrug resistant (MDR) K. pneumoniae strains [3][4][5].
Global healthcare systems have experienced an increase in the emergence and spread of epidemic lineages of carbapenem resistant K. pneumoniae (CRKp) since 2001 [6,7].The CC147, including sequence types (ST) ST147 and ST392, has been identified as a successful high-risk clone in the spread of carbapenemases [8].In the years from 2010 to 2014, the ST147 clone was identified as a vehicle for the distribution of bla NDM worldwide using various mobile genetic elements (MGEs), including IncX3, IncFIIA and IncA/C plasmid types [9].According to the antimicrobial resistance reports of the "Center for Disease Dynamics, Economics, and Policy", Iran had a trend of 43-45% of CRKp during 2018 and 2019 [10].While the emergence of bla NDM has been described in Iran since 2013 [11], a deep genomic study based on whole genome sequencing (WGS) has been published recently [12].Sequence types ST11, ST268, ST893, and CC147 have been linked to bla NDM and bla OXA-48 distributed across the country, with the latter being common in different surveys [13,14].In view of the relatively widespread distribution of CRKP in Iran [15], it is necessary to analyze the population structure and resistance/virulence mechanisms of these strains.By combining new tools like WGS with information from epidemiological, clinical, and phenotypic sources, we can learn more about how bacteria spread, how dangerous they are, and how antimicrobials affect them.
In follow-up to our previous cross-sectional study describing the occurrence of CC147 strains co-harboring bla NDM /bla OXA-48 at Semnan province [16], carbapenem non-susceptible K. pneumoniae strains were subjected to WGS.The aim of this study was to focus on the CC147 subpopulation and mostly the ST147 in the context of a local hospital and on a worldwide scale to gain an understanding of the evolution, genomic content, and plasmid repertoire contributing to success of this high-risk clone.

Collection of bacterial population and antibiotic susceptibility patterns
The study was conducted in a single acute-care referral hospital in Semnan, Iran, that covered > 120,000 people in this province.During the 16-month surveillance study from March 2018 to June 2019, out of 123 Klebsiella spp.isolates were recovered from extra-intestinal specimens.Isolates were phenotypically identified by standard biochemical tests, including the sugar fermentation pattern on Triple Sugar Iron Agar, the pattern of SH2/Indole/ Motility reactions on SIM, Citrate consumption, and Urease production [17].
The disk diffusion test was applied for susceptibility testing of collected isolates, and the results were interpreted based on the Clinical and Laboratory Standard Institute (CLSI) guidelines [18].A resistant phenotype against one of the three or more antimicrobial classes was considered for the multidrug resistant (MDR) definition [19].Strains showing a non-susceptible phenotype against either of the study carbapenems (imipenem, meropenem, or ertapenem) were considered carbapenem non-susceptible and subjected to minimum inhibitory concentration (MIC) determination using E-test strips.The MIC criteria for considering of susceptibility against imipenem/meropenem and ertapenem were ≤ 1 μg/ml and ≤ 0.5 μg/ml, respectively [18].

Analyses of sequencing data
The first step of whole genome sequencing analysis was DNA extraction and an Invitrogen Easy-DNA kit was used for this purpose.After determination of DNA concentration using the Qubit dsDNA BR assay kit (Invitrogen), library preparation was conducted according to the Illumina protocol and sequencing was run on NextSeq and MiSeq platforms using 150 bp paired-end reads [20].
Evaluation of raw read quality was done by FastQC version 0.11.5 (https:// www.bioin forma tics.babra ham.ac.uk/ proje cts/ fastqc/), and TrimmomaticPE version 0.22 [21] was used for trimming with the following parameters: The minimum quality required for maintaining a baseline from the beginning and from the end of the read was Q30, and the average quality was based on a window size of 10 with Q20.De novo assembling of raw reads was performed by SPAdes version 3.11.0[20].The quality evaluation of generated assemblies was done with QUAST version 4.6.3[22].In silico bioinformatics tools were used for analyzing assembled sequences in order to confirm the sequence types (STs), plasmid replicons, acquired resistance markers, fluoroquinolone resistance associated mutations, and plasmid MLST (pMLST) using the following pipelines: MLST 2.0 [23], plasmid-Finder 2.1 [24], ResFinder 4.1 [25], and pMLST 2.0 [23].Single target analyses using the NCBI BLAST tool were performed with the BIGSdb-Kp database curated by the Pasteur Institute (https:// bigsdb.paste ur.fr/).Genomes were uploaded into the Pathogenwatch global platform in order to obtain information on capsular and O-antigen serotyping, and Integrative Conjugative Elements (ICEs) harboring aerobactin/salmochelin/yersiniabactins (https:// cgps.gitbo ok.io/ patho genwa tch/).The annotation of some related resistance carrying contigs was performed by the RAST online server and, gbk format files were downloaded.The MGEFinder 1.0 was used to find the association of resistance markers with MGEs [26], and the origin of chromosome, phage, and plasmid derived sequences was detected by SourceFinder 1.0 [27].The Geneious Prime 2021.1.1 was applied for graphical annotation of genetic environment of contigs harboring resistance genes and the position of insertion sequences on these contigs was manually curated using ISFinder BLAST results [28,29].The similarity determination of genomes to hvKp virulence content was done by using the Blast Ring Image Generator (BRIG) [30].
We also studied the ST147 strains in a global context.To do this, the raw-fastq.gzreads and assemblies were downloaded from public databases and subjected to two different platforms.First, the ST147/NDM + raw reads (n = 128) from the United States that were recently deposited in the European Nucleotide Archive (ENA) were downloaded [31].Furthermore, 46 ST147/NDM + raw reads from different studies of Europe, Southeast Asia, South-America, and Canada were also included [32].Totally, 174 ST147/NDM + short read data sets were subjected to the pipelines mentioned above and the CSI phylogeny (as described below) to draw a SNP-phylogenetic tree.Second, cgMLST analysis was used to study our ST147 strains and corresponding genomes (both bla NDM + ve or − ve) deposited from countries in our geographic region (n = 121) by downloading assemblies from the PATRIC database (https:// www.patri cbrc.org/, accessed on 29 June 2023).To access these genomes, filters such as 'Iran' , 'Pakistan, 'India' , 'Turkey' , 'United Arab Emirates' (UAE), 'Russia' , 'Lebanon' and 'Israel' , 'MLST: 147' , 'genome quality: good' were used.The FASTA format of pre-assembled sequences was retrieved to subject Ridom SeqSphere + software to drawing the Minimum Spanning Tree (MST) (described below).Other information, such as the collection year and sample origin for each genome, was collected from PATRIC.
Reference-based analysis was used to draw the phylogenetic tree using reference strain K. pneumoniae strain 4/1-2 (GenBank ID: Cp023839.1).The CSI Phylogeny 1.4 was used to draw the SNP tree [33].The Burrows-Wheeler Aligner (BWA), version 0.7.2 was used to map of paired end reads to the reference genomes and the mpileup module in SAMTool version 0.1.18was used for SNP calling [34].The criteria that were considered for selection of SNP were: (i) presence of minimum 15bp distance between each SNP (pruning), (ii) minimum 10% of average depth, (iii) mapping quality > 30, (iv) quality of SNP > 20, and (v) all indels were excluded.The quality passed SNPs of each genome were concatenated into a single alignment corresponding to the position of the reference strain.The concatenated sequences were subjected to phyML [35] for the construction of a parsimony tree by using the HKY85 substitution model and 1000 bootstrap replicates.The pairwise 23 SNP distances between isolates proposed by Sherry et al. were used for the definition of local transmission [36].The web interface software iTol was used to visualization and labeling of the generated phylogenetic tree.By putting the assembled genomes on Ridom SeqSphere (Ridom GmbH, Munster, Germany) [37], the cgMLST allelic mismatch between strains was used to make the MST.According to the cgMLST SeqSphere + server, closely related genomes are 'lumped' together in complex types (CTs; https:// www.ridom.de/u/ Core_ Genome_ MLST_ Compl ex_ Type.html) and first approximation of close genetic relatedness is defined based on a CT distance of 15 allele differences (https:// www.cgmlst.org/ ncs/ schema/ 21879 31/).This CT allele distance threshold is based on retrospective analysis of well-defined outbreaks and outgroup isolates with the same MLST/MLVA/PFGE profiles, as described elsewhere [38].Moreover, data regarding the virulence factors and resistance genes content of downloaded assemblies was obtained by running the "K.pneumoniae VFDB" and "NCBI AMRFinderPlus" pipelines implemented in Ridom SeqSphere + software.

Transconjugation assay
A filter-mating experiment was performed to evaluate the horizontal gene transfer (HGT) potential of plasmids [39].Three strains (two ST392 and one ST147, all bla OXA-48/NDM-1 +) were considered as potential donors.A colony of K. pneumoniae strains was used to inoculate LB broth (5 ml).In addition, a colony of Escherichia coli DH5α was also used to inoculate LB (5 ml) as recipient.After overnight incubation, the cultures were washed twice with PBS and then mixed at a ratio of 10:1 K. pneumoniae (donor) to E. coli (recipient) in 300 µl PBS.The mixture was plated on a 0.22 µm pore size membrane filter on LB agar and incubated overnight.The cells on the membrane were then re-suspended in 10 ml of PBS and plated on MacConkey agar supplemented with both nalidixic acid (32 µg/ml) and cefotaxime (1 µg/ml).Pink colonies (as recipients) were purified.PCR assays were used to confirm the presence of bla NDM-1 and bla OXA-48 in transconjugants [39].

Statistical methods
Statistical analysis was done using IBM-SPSS Statistics 22. Bivariate analyses of categorical variables were done by Chi-square test.All P values were two-sided.
The majority of patients from whom CC147 strains recovered were admitted to an intensive care unit (internal ICU, surgical ICU), where strains were isolated during their stay.Besides the ICU ward, one of the CC147 infection episodes was recovered from the urine culture of a patient admitted to the CCU (strain no.447, ST392).Temporally, the first NDM positive strain (strain no. 2, ST392) was isolated shortly after starting of sample collection (March 2018), and all except one ST392 strain (strain no.659) were recovered during 2018.In contrast, ST147 appeared in June 2018 and replaced ST392 during the entire sampling period (Fig. 1).In terms of carbapenem MICs, the ST147 clone did better than the ST392/ NDM + strains against this family of antibiotics (Table 1).
Clustering of CC147 genomes based on a minimum spanning tree revealed a high level of proximity among ST147 strains (maximum of 11 allelic distances), while ST392 exhibited shallow branching and one strain (No. 45) (OXA-48 + /NDM − ) had a relatively high allelic  distance (43 allele differences) from the nearest ST392 node (Fig. 2).
On three occasions, it was possible to link replicon type and resistance elements to the same contig: (i) ST147 strains carried bla NDM-1 on an IncFIB (pB171).(ii) ST392 harboured bla NDM-1 on a Col440I, and (iii) bla OXA-48 was embedded in a Tn1999 composite transposon in association with the IncL/M replicon type, the notorious plasmid lineage responsible for the worldwide dissemination of bla OXA-48 .

The bla NDM-1
+ transconjugants was successfully developed By using conjugation experiment, the NDM-1 + plasmids of 3 studied isolates (ST147 and 2 ST392 strains) were successfully transferred to recipient, conferring resistance to carbapenems and cephalosporins in transconjugants.Plasmid gel extraction and the following PCR experiment of the transconjugants revealed that the bla NDM-1 was located on conjugative plasmids.However, in the experiments the OXA-48 + transconjugants were not detected.
The comparable plasmid harboring bla NDM-1 was found in four downloaded genomes clustered with our strains, and IncF pMLST revealed it to be of the K2/K5:A22: B36 replicon type.Considering the ICE and Yersiniabactin types, all United States strains were negative.While 19 ICEKp4 (ybt 1), nine ICEKp12 (ybt 16) and 11 ICEKp3 (ybt 9) positive isolates were identified.Genomic characteristics of ENA downloaded genomes have been shown in Additional file 2.
ST147 K. pneumoniae genomes reported from neighboring countries in our geographic region were included in the cgMLST analysis to identify genetic relatedness.Using a ≤ 15 allele difference threshold in cgMLST analysis, strains were clustered in 13 complex types (CTs) and 18 singletons (Fig. 6).The CTs were formed by genomes from the same countries, except for one cluster (CT1) which was the biggest and included isolates from all countries.Iranian genomes (our 9 isolates and 3 previously reported) were grouped into cluster 3 (11 strains) and one strain remained as a singleton (Ir-2).Considering the clustered strains, the highest diversity was found among Indian strains that were categorized into 5 clusters, followed by genomes from Russia /Turkey (4 clusters) and Pakistan (2 clusters) (Fig. 6).Characteristics of downloaded genomes deposited from neighboring countries are shown in Additional file 3.

Diversity of genetic context involved in spreading of bla NDM-1 , armA and bla CTX-M-15
Analysis of the genetic context of bla NDM-1 in ST147 and ST392 as the positive clones corroborated the previous conserved sequence of this gene, showing that the NDM-1 gene is located downstream of truncated ISAba125 and is followed by ble MBL , Isomerase, protein disulfide reductase dsbD, cutA, and GroES-GroEL genes; however, there were some differences between these two clones based on the arrangement of mobile genetic elements (MGEs) surrounding this segment.Among ST392 strains, only one isoform of MGEs, including ΔISKpn26-NDM core structure-TnAs3-ΔIS3000-Tn5403 was found on plasmid col440I (Fig. 7a).In ST147, the NDM-1 core structure was discovered upstream of the rmtC 16S rRNA methylase gene, flanked by MITESen1 and IS903B in one strain and MITESen1/MITESen1 composite transposon in the other (Fig. 7b, c).In rmtC negative ones, the NDM core structure was bracketed by MITESen1/ISSen4 (Fig. 7d).The plasmid type of NDM in ST147 was IncFIB (PB171).
The bla CTX-M-15 harbored by ST14, ST45, ST48, ST377 and all except one CC147 strain.The core segment was arranged by a truncated and/or intact form of ISEcp1 upstream of CTX-M-15, and the "WbuC family protein fold metalloprotein" gene was located downstream but in a reversed direction along with a copy of Tn2.This core structure was co-localized with (i) bla TEM-1B , tnpA, aph(6)-Id, aph(3)-Ib and sul2 genes, which were bracketed by IS15DIV and IS5075 (IS110 family), and (ii) IS2 and qnrS1.The length of CTX-M-15 carrying contigs varied across STs; however, data suggest a common origin via horizontal transfer across multiple sequence types.(Fig. 7e, f ).From inside to outside of the figure: "deep sky blue "rings"; ST14, "blue" rings; ST40, "purple" rings; ST45, "yellow" rings; ST48, "aquamarine" rings; ST147, "red" rings: ST392, "blue-green [teal]" rings; ST377, "pale green" rings; ST1308.The other undefined colored rings are STs that include one or two strains The other 16S rRNA methylase gene, armA, was detected in seven out of nine ST147 and one ST48 strain (no.639).The core structure which was located on the class 1 integron structure was consisted of armA bracketed by ISEc28 and ISEc29, along with ARR-2, cmlA1, bla OXA-10 , aadA1, qacE, and sul1 in downstream, and msr(E)/mph(E) in upstream.The genetic environment of this structure was conserved in all positive strains and was flanked by two copies of IS15DIV (Fig. 7g).

Discussion
We have identified different lineages of carbapenem non-susceptible K. pneumoniae and notably six carbapenemase (bla OXA-48 , bla NDM-1 , and no bla KPC ) producing clones in our clinical setting, suggesting multiple importation events of this resistant pathogen into the hospital along with their clonal expansion at different levels.While CC147 was the largest clone with the greatest number of resistance markers, its two SLV members (ST147, ST392) varied in their carbapenem MIC values and resistance marker repertoires, with ST147 dominating.To assess the dissemination of bla OXA-48 /bla NDM-1 , we focused on the identification of MGEs, and two types of NDM-1 carrying plasmids ([IncFIB(pB171) exclusively identified in ST147 and Col440I mostly among ST392]) were identified.Furthermore, the IncL/M plasmid was exclusively detected among OXA-48 producers, and we could predict its location on this plasmid type.Different types of NDM-bearing plasmids with distinct dissemination patterns among two members of a clonal complex appear to be largely dependent on the plasmid backbone and the bacterial sequence type harboring them.Our effort in transconjugation assay was successful in transferring of bla NDM-1 carried by two different replicon types, highlighting the important role of these molecular vehicles in NDM circulation.On the other hand, the plasmids harboring bla OXA-48 did not transfer successfully.Maybe it is due to a low efficiency of their conjugation trait during transcojugation assays.However, it seems that the plasmids carrying bla NDM-1 can easily transfer and expand the antimicrobial resistance among inter-and intraspecies of hospital-resident bacteria.In a recent study of carbapenemase producing Enterobacterales collected from a university hospital in Tehran, Iran, the IncFII and IncA/C plasmids were identified as the vehicles for carriage of bla NDM-1 in K. pneumoniae/Enterobacter cloacea and E. coli, respectively [13].Nevertheless, other types of plasmids such as IncL/M, IncFIB(pQil), IncFII (PRSB107) and IncFIB/HI1B hybrid plasmid are also reported for NDM circulation [3,41], indicating that independent acquisition of NDM is more important than its widespread dissemination via major transferable plasmids [42].
The genetic environment the NDM-1 segment (ISAba125-bla NDM-1 -ble MBL -Isomerase-DsbD-cutA-groEL/ES) was different between CC147 members as well.While MGEs arrangement appeared to be conserved in ST392 strains (flanked by ΔISkpn26 and Tn5403), that was completely divergent among ST147, consisting of different patterns of MITESen1 composite transposon, ISSen4, MITESen1, ISEhe3, IS5, and IS1.This rearrangement diversity of MGEs surrounding NDM in a single circulating clone of Klebsiella, with strong evidence of homogeneity based on genomic analysis and the sampled geographic region, was intriguing.This finding indicates that bla NDM-1 is embedded in dynamic genetic platforms.From a clinical point of view, spreading of bla NDM /rmtC/armA coproducing ST147 clone is worrying, as this resistance combination limits the effectiveness of combined carbapenem-aminoglycoside therapy in severe infections.
Genome-wide analysis of study CC147 revealed a tight cluster with < 15 SNPs among ST147, while ST392 strains were more divergent based on genetic context (≈ 50 SNPs difference) and resistance/virulence repertoire.Incorporating the isolation dates of NDM-producers during the sampling period revealed that the NDM gene was first circulated in 2018 by ST392 and gradually replaced by ST147, which was more homogeneous and harbored the most robust resistance repertoire to spread this carbapenemase.It has been shown that K. pneumoniae propensity for successful colonization of hospital environments is strongly influenced by the resistance level, and that carbapenemase producers are prioritized in this regard [42].The probable explanation might be that ecological constraints exerted by antibiotic exposure obstruct the spread of isolates with a lower capacity for expressing resistance [43].Constructing a phylogenetic tree using SNP-based and cgMLST approaches that included our ST147 and publicly available corresponding genomes revealed a monophyletic cluster of our nine strains, indicating a single regional introduction, probably through a locally circulating ST147 strain.Furthermore, the IncF-plasmid replicon found in our ST147 was also detected in four downloaded genomes co-clustered with our strains, suggesting that the IncF plasmid we reported here [IncFIB (pB171)] can be stably associated with a high-risk ST147 clone.
During the search for NDM dissemination in our setting, the ICU was identified as the likely source.There are some points that likely play roles in the spread of NDM-producing CC147.Patients in the ICU are at high risk of acquiring carbapenemase-producing Enterobacteriaceae (CPE), and long-term ICU stays increase the risk of acquiring multidrug-resistant organisms [44].Furthermore, multibed rooms in internal wards connected to ICUs via patient transfer are common, and the facilities themselves lack resources for infection control approaches, both of which could contribute to the intrafacility spread of MDR-high risk clones [45].
Whereas a high diversity of K loci exists, few O loci are described in this organism, with O1, O2 and O3 loci being mostly associated with human disease [46].Two distinct O loci of LPS, O1/O2v1 loci were predominantly found in urine, blood and respiratory origin isolates.O1/ O2v1 loci have been described in association with invasive tissue infection and colonization of internal organs [47].So, these isolation sites and the subtypes of recovered strains should not be undervalued, since they could result from intestinal colonization as an initial step for progression to disease.
Despite the conserved nature of O and K-locus antigens identified within our genomes, the virulence gene content differed even within the study clones.It was highlighted in the ST392 lineage, where half of them were positive for ICEKp3, mrkA-H type 3 "fimberiae synthesis cluster", and a complete fyuA-irp-ybt "yersiniabactin biosynthetic operon".The lowest prevalence of virulence markers was the ferric uptake system (kfuABC) that harbored by 16.9% of isolates, including K1/ST2159 hvKp and ST14 MDR/high-risk clone.Higher rates of siderophore production have been proposed to be associated with hvKp lineages and hypermucoviscosity phenotype.However, this measurement has been shown to be only partially correlated with in vivo virulence differences [48].Conversely, it has been revealed that CC258/ KPC + harboring yersiniabactin shows an enhanced ability to colonize the respiratory tract and cause pneumonia in experimental infection models [49].Overall, it appears that co-carriage of ICEs/ yersiniabactins with carbapenemases/ ESBLs (CC147, ST14, ST45, ST48 and ST377, in our study) is concerning, as this phenomenon not only could be associated with infections but also be a frequent first step in the acquisition of more siderophores (specifically plasmidic siderophores iucABCD/iroBCDN operons considered for hvKp) that enhance gut colonization ability of KpI population and consequently invasive resistant infections with high mortality rates [50].
We acknowledge the limitations of our survey.Firstly, the study was conducted in a single acute-care referral hospital in Iran and the sampling period was short, so rerunning this survey along with bla NDM producing strains collected from other centers will be necessary to confirm current findings.Second, the patient's medical history (specifically antibiotic usage and the hospitalization period before and after of CC147 isolation) was missing, so we couldn't find any link between our phylogenetic data and epidemiological information.Thirdly, the number of deposited public genomes that we analyzed alongside our phylogeny was limited, and we will need to include more ST147 genomes mostly that originated from our geographical region, specifically, Persian Gulf countries, India and Pakistan, to gain a better understanding of how this dominant carbapenemase-producing clone circulates.

Conclusions
The spread of circulating CC147 super-clone with high genomic plasticity in the acquisition of various resistance/virulence markers that propagate along transmission poses a significant threat to public health.The highly convergent nature of CC147 with different subpopulations is linked to specific genomic features and geographic distribution [8].With the help of different arrangements of MGEs, subpopulations of a single clonal complex bring notorious resistance markers such as NDM into circulation together, as we have highlighted.The public health sector should therefore prioritize genomic surveillance of pathogens, detect the introduction and spread of high-risk clones early in an epidemic,  • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year

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Fig. 1 Fig. 2
Fig.1Phylogenetic tree of study 54 K. pneumoniae isolates.Maximum-likelihood phylogenic tree clustered different clones (STs) and CC147 strains (ST147, ST392) were identified as the dominant clone with the highest prevalence of varied resistance elements.The tree was constructed from the whole genome SNPs arising by mutation and labeling and visualization were done by using the iTOL web-interface.The data presented are the date of isolation for CC147, sequence types, capsular types, Integrative and conjugative Elements (ICEs), acquired resistance markers and amikacin/gentamicin susceptibility phenotypes.The hvKp strains have been shown in red

Fig. 3
Fig. 3 Bar-plot the prevalence of different replicon types and their association with STs.The Y-axis represents the number of positive isolates in each STs

Fig. 4
Fig. 4 The virulence genes content of K. pneumoniae.A BLAST Ring Image Generator (BRIG) image shows the presence of virulence factors among different STs.The FFN format file of hvKp (strain No. 290 identified as ST86) was downloaded from VFDB website, and open reading frames (ORFs) are annotated based on the VFDB results and used as reference strains to draw the image.Aligning between the regions of interest and each genome are shown as Colored segments (indicate > 70% similarity), and gray segments (indicate > 50% similarity).From inside to outside of the figure: "deep sky blue "rings"; ST14, "blue" rings; ST40, "purple" rings; ST45, "yellow" rings; ST48, "aquamarine" rings; ST147, "red" rings: ST392, "blue-green [teal]" rings; ST377, "pale green" rings; ST1308.The other undefined colored rings are STs that include one or two strains

Fig. 5
Fig. 5 Maximum-likelihood phylogeny of ST147/NDM + strains based on whole genome SNPs analysis.A phylogenetic reconstruction of 183 strains (including the 174 downloaded genomes and our 9 study strains) with K. pneumoniae strain 4/1-2 (GenBank ID: CP023839.1,NDM + strain), as the reference strain is shown.Colored strips surrounding the phylogram represent the country of origin of each strain, capsular types, ICE types, carriage of NDM/CTX-M-15, OXA-types, and genes involved in gentamicin and amikacin resistance

Fig. 6 Fig. 7
Fig. 6 Minimum Spanning Tree showing distance based on cgMLST of 1826 genes using the parameter 'pairwise ignoring missing values for 130 ST147 genomes.Each circle is named with the geographical origin, including DTU: our ST147 strains, Ir: Iran, Tu: Turkey, In: India, P: Pakistan, Is: Israel, Le: Lebanon, Ru: Russia, UAE: United Arab Emirates.The data of carbapenemase genes is shown by the coloured rings inside each bubble

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Table 1
Demographic characteristics of K. pneumoniae subjected to WGS

Table 1 (continued) Strain No ST a KL, O-type b Ward of isolation Sample c MDR d age Gender e Yersiniabactin; ICE IMI, MEM, ETP f (μg/ml)
Indicates carrying bla OXA-48 , *Indicates carrying bla NDM-1 , a: Sequence type; b: capsular type; c: BC & UC stand for blood and Urine culture, respectively; d: Multidrug resistant; e: Female, Male; f: Minimum Inhibitory Concentrations of imipenem, meropenem and ertapenem are determined for some of the study strains ♦

Table 2
Resistance genotypes in association with phenotypic characteristics Pajand et al.Journal of Biomedical Science (2023) 30:73 • fast, convenient online submission