# Genomic evolution of the globally disseminated multidrug-resistant Klebsiella pneumoniae clonal group 147

Carla Rodrigues, Siddhi Desai, Virginie Passet, Devarshi Gajjar, Sylvain Brisse

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## To cite this version:

Carla Rodrigues, Siddhi Desai, Virginie Passet, Devarshi Gajjar, Sylvain Brisse. Genomic evolution of the globally disseminated multidrug-resistant Klebsiella pneumoniae clonal group 147. Microbial Genomics, 2022, 8 (1), 10.1099/mgen.0.000737. pasteur-03566824

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1

2 **Genomic evolution of the globally disseminated multidrug-resistant**

3 **Klebsiella pneumoniae** clonal group 147

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5 Carla Rodrigues¹, Siddhi Desai², Virginie Passet¹, Devarshi Gajjar² and Sylvain Brisse¹*

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7 ¹Institut Pasteur, Biodiversity and Epidemiology of Bacterial Pathogens, Paris, France.

8 ²Department of Microbiology and Biotechnology Centre, Faculty of Science, The Maharaja

9 Sayajirao University of Baroda, Vadodara, Gujarat, India.

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11 *Corresponding author:

12 Sylvain Brisse

13 Institut Pasteur,

14 Biodiversity and Epidemiology of Bacterial Pathogens,

15 28 rue du Docteur Roux, F-75724 Paris, France.

16 E-mail: sylvain.brisse@pasteur.fr;

17 Phone: +33 1 45 68 83 34

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19 **Keywords:** phylogenomics, pandrug resistance, last-resort antibiotics, high-risk clone,

20 NDM-5, CRISPR/Cas systems, IncF plasmids, genomic epidemiology

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22 ** Repositories:** Reads and genomic sequences of the isolates analysed in this study were

23 deposited at the European Nucleotide Archive (accession number PRJEB41234).
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ABSTRACT

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The rapid emergence of multidrug-resistant *Klebsiella pneumoniae* (Kp) is largely driven by the spread of specific clonal groups (CG). Of these, CG147 includes 7-gene MLST sequence types ST147, ST273 and ST392. CG147 has caused nosocomial outbreaks across the world, but its global population dynamics remain unknown. Here, we report a pandrug-resistant ST147 clinical isolate from India (strain DJ) and define the evolution and global emergence of CG147. Antimicrobial susceptibility testing (EUCAST guidelines) and genome sequencing (Illumina and Oxford Nanopore Technologies, Unicycler assembly) were performed on strain DJ. Additionally, we collated 217 publicly available CG147 genomes (NCBI, May 2019). CG147 evolution was inferred within a temporal phylogenetic framework (BEAST) based on a recombination-free sequence alignment (Roary/Gubbins). Comparative genomic analyses focused on resistance and virulence genes and other genetic elements (BIGSdb, Kleborate, PlasmidFinder, PHASTER, ICEFinder and CRISPRCasFinder).

Strain DJ had a pandrug resistance phenotype. Its genome comprised 7 plasmids and 1 linear phage-plasmid. Four carbapenemase genes were detected: *bla*<sub>NDM-5</sub> and 2 copies of *bla*<sub>OXA-181</sub> in the chromosome, and a second copy of *bla*<sub>NDM-5</sub> on an 84 kb IncFII plasmid. CG147 genomes carried a mean of 13 acquired resistance genes or mutations; 63% carried a carbapenemase gene and 83% harbored *bla*<sub>CTX-M</sub>. All CG147 genomes presented GyrA and ParC mutations and a common subtype IV-E CRISPR-Cas system. ST392 and ST273 emerged in 2005 and 1995, respectively. ST147, the most represented phylogenetic branch, was itself divided into two main clades with distinct capsular loci: KL64 (74%, DJ included, emerged in 1994 and disseminated worldwide, with carbapenemases varying among world regions) and KL10 (20%, 2002, predominantly found in Asian countries, associated with carbapenemases NDM and OXA-48-like). Further, subclades within ST147-
KL64 differed at the yersiniabactin locus, OmpK35/K36 mutations, plasmid replicons and prophages. The absence of IncF plasmids in some subclades was associated with a possible activity of a CRISPR-Cas system.

K. pneumoniae clonal group CG147 comprises pandrug- or extensively-resistant isolates and carries multiple and diverse resistance genes and mobile genetic elements, including chromosomal *bla*<sub>NDM-5</sub>. Its emergence is driven by the spread of several phylogenetic clades marked by their own genomic features and specific temporo-spatial dynamics. These findings highlight the need for precision surveillance strategies to limit the spread of particularly concerning CG147 subsets.

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## 59 Data Summary

Sequence read files and complete genome assembly of DJ have been deposited in European Nucleotide Archive under the BioProject number PRJEB41234.

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62 INTRODUCTION

The increasing number of antimicrobial resistant infections by Klebsiella pneumoniae (Kp), especially by extended-spectrum beta-lactamases (ESBL)- and carbapenemase-producing Kp, led to the declaration of Kp as an ‘urgent threat’ and ‘priority pathogen’ by public health agencies (1,2). Molecular analyses of Kp isolates has evidenced that the rapid emergence of multidrug resistant (MDR) Kp is largely driven by the geographic spread of successful clonal groups (CG; e.g., CG15, CG101, CG147, CG258, CG307) (3), some of them carrying epidemic resistance plasmids (4). In order to treat MDR Kp infections, last-resort drugs such as polymyxins (especially colistin) and tigecycline are used (3,5). Consequently, resistance is also observed to these last resort drugs, especially to colistin, and may culminate in the emergence and spread of pandrug-resistant strains (5,6). Pandrug-resistant Kp strains leave few or no therapeutic options and are associated with high mortality rates (7–11).

The 7-gene MLST sequence type (ST) ST147 has been recognized as a globally distributed antimicrobial resistance clone (12), and is closely related to ST273 and ST392, which themselves comprise MDR isolates. Based on genomic classifications, these three STs are grouped into the CG147 clonal group (13,14). The earliest studies of CG147 date from 2008-2010 in Hungary, and correspond to ciprofloxacin-resistant CTX-M-15-producing ST147 isolates, which had been disseminating in the country since 2005 (15,16). Between 2010 and 2014, CG147 (mainly ST147) was described worldwide in association with several carbapenemases (17). Most reported CG147 isolates are from clinical samples, although some were found in companion animals, river waters, chimpanzees, poultry animals and poultry environment (18–24). The above studies were locally restricted and so far, no study of the global spread and genome dynamics of this clone was performed.
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Here, we report a pandrug-resistant clinical isolate from India belonging to ST147, and investigate the genomic evolution and antimicrobial resistance gene dynamics in the global CG147 population. We also analyze the phylogenetic context of virulence-associated genomic features, CRISPR loci and mobile genetic elements in this successful Kp sublineage.
92 Isolation and phenotypic and genomic characterization of strain DJ

93 Isolate DJ was recovered from the urine of a 45-years old female patient diagnosed
94 with a urinary tract infection (UTI) in Vadodara (Gujarat, India) in October 2016. The isolate
95 was confirmed to be Kp by biochemical tests and 16S rRNA sequencing. Antibiotic
96 susceptibility tests performed using a semi-automated commercial system (Vitek,
97 BioMérieux) revealed resistance to all antibiotics tested. Confirmatory antimicrobial
98 susceptibility tests were carried out for colistin and tigecycline using broth dilution; and for
99 fosfomycin (agar supplemented with 25 mg/L glucose 6-phosphate sodium salt) using agar
100 dilution method. Disk diffusion method was used for the other antimicrobial classes
101 (penicillins, cephalosporins, carbapenems, monobactams, fluoroquinolones, aminoglycosides,
102 macrolides, tetracyclines, phenicols and inhibitors of the folic acid pathway). Results were
103 interpreted using both the Clinical and Laboratory Standards Institute (25) and the European
104 Committee on Antimicrobial Susceptibility Testing (2018) (http://www.eucast.org/)
105 guidelines.

106 DNA extraction was performed using XpressDNA Bacteria kit (MagGenome
107 Technologies Pvt Ltd., India). Whole-genome sequencing data were generated using (i) an
108 Illumina NextSeq-500 platform with a 2 x 150 nt paired-end protocol (Nextera XT library,
109 Illumina, San Diego, CA); and (ii) long-read Oxford Nanopore sequencing using MinION
110 device integrated with a FLO-MIN-106 flow cell and libraries prepared using a 1D ligation
111 sequencing kit (SQK-LSK109) following the protocol for 1D genomic DNA (gDNA) long
112 reads without BluePippin (Oxford Nanopore Technologies, New York, NY, USA). De novo
113 assemblies of the reads were obtained using SPAdes v3.12.0 (26) for Illumina data and using
114 Unicycler v0.4.4 (27) for hybrid assembly. Assembled sequences were annotated using
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Prokka v1.12 (28). Reads and assembly were deposited at the European Nucleotide Archive database (under the BioProject PRJEB41234).

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## Global dataset of publicly-available genomic sequences

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All publicly available CG147 Kp genomes from NCBI RefSeq repository of genome assemblies (May 2019) were downloaded. From the 245 CG147 genomes available, duplicated (n=5) and poor-quality genomes (n=6; genome size and G+C content not matching with Kp and/or more than >1000 contigs), and those without attached isolation year (n=17) were excluded. The final dataset comprised 218 genomes, including strain DJ. Sample information, accession numbers, and biological characteristics of the genomes are given in

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### Table S1.

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## Phylogenetic analyses

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For phylogenetic analyses, a core-genome alignment based on the concatenation of 4,529 core genes was obtained using Roary v3.12 (29) using a blastP identity cut-off of 90% and core genes defined as those being present in more than 90% of the isolates. Recombination events were removed from the core-genome alignment using Gubbins v2.2.0 (30). The final recombination-free alignment comprised 8,450 single-nucleotide variants (SNVs) and was used to construct a maximum-likelihood phylogenetic tree using IQ-TREE v1.6.11 (model GTR+F+ASC+G4). The tree was rooted with a Kp ST258 NJST258_2 (accession number: GCF_000597905.1) and a Kp ST37 INF042 (GCF_002752995.1) (Figure S1).

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To evaluate the strength of the temporal signal of our molecular phylogeny, we first conducted a linear regression analysis of the root-to-tip genetic distances as a function of the sample collection year, using TempEst v1.5.3 (http://tree.bio.ed.ac.uk/software/tempest/)

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(Figure S2). The final recombination-free alignment was then subjected to Bayesian
phylogenetic analysis using BEAST v2.6.1 (run with a Markov chain Monte Carlo length of
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1x 10⁹, sampling every 5x10³ steps) (31). We used model parameters that had the best fit:
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GTR substitution model, lognormal relaxed clock and constant population size. Parameters
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estimates were computed using Tracer v1.7.1, and a maximum clade credibility tree was
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obtained with TreeAnnotator v2.6.0. and visualized in FigTree v1.4.4.
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MLST and genomic analyses of resistance, virulence and other genetic elements

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Multilocus sequence typing (MLST, 7 genes) was performed using the Institut Pasteur
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*Klebsiella* MLST (32) database (https://bigsdb.pasteur.fr/klebsiella/). Kleborate (33) and
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BIGSdb analytical tools (https://bigsdb.pasteur.fr/klebsiella/) (34) were used to define the
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presence of antimicrobial resistance, heavy metal tolerance, virulence genes and to
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characterize the capsular gene cluster . Geneious Prime 2019.1.1 software
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(https://www.geneious.com) was used for further manual curation of antibiotic resistance
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genes, and ISFinder (https://isfinder.biotoul.fr) was used to look for the insertion sequences
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in the resistance genes or in their genetic context. Plasmid replicons were detected using
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PlasmidFinder (https://cge.cbs.dtu.dk/services/PlasmidFinder/) (35), whereas prophages,
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integrative and conjugative elements (ICEs) and CRISPRs were identified using PHASTER
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(https://phaster.ca) (36), ICEfinder (https://db-mml.sjtu.edu.cn/ICEfinder/ICEfinder.html)
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and CRISPRCasFinder (https://crisprcas.i2bc.paris-saclay.fr/CrisprCasFinder/Index) (37),
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respectively. To depict co-resistance genotypes and plasmid networks, we constructed a
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correlation matrix for binary variables (1, presence; 0, absence) using the ‘corr.test’ function
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(Pearson method, which for a pair of binary variables compares to the Phi coefficient) from
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the ‘corrplot’ R package. Significant correlations were visualized with the ‘corrplot’ function
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from the same package. Statistical analyses to check the association of the different

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165 categorical variables within the phylogeny groups were calculated using the χ² test (P values
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# RESULTS

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## Phenotypic and genomic features of pan-drug resistant strain DJ

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Strain DJ was resistant to all tested antimicrobial agents, including last-resort antimicrobials such as carbapenems, colistin, tigecycline and fosfomycin (Table 1), and is therefore pandrug-resistant (6). To define its genetic mechanisms of resistance, a hybrid complete genome assembly was produced (Figure S3a). The 5.7 Mb sequence was 56.9% G+C-rich and made up of one chromosome and 6 circularized plasmids [123kb IncFII(pKPX1); 57 kb IncR; 5.6 kb ColRNAI; 4.7 kb ColRNAI; 2.0 kb ColpVC; and 1.5 kb ColMG828]. In addition, there were two non-circularized contigs: a 84 kb IncFII plasmid and a 57 kb contig corresponding to a N15-like phage-plasmid (P-P) coding for a protelomerase (telN) responsible for the maintenance of its linear genome (38). PHASTER identified four other prophages within the chromosome. Phylogenetic analysis of strain DJ showed it belonged to K. pneumoniae sensu stricto (i.e., phylogroup Kp1) and to the ST147-KL64 lineage previously described as endemic in India (12).

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The long-read sequencing followed by hybrid assembly of strain DJ allowed the identification of multiple copies of unique antibiotic resistance genes in the different genomic elements depicted (chromosomes and plasmids). Strain DJ harbored four carbapenemase genes, corresponding to two copies of each *bla*<sub>NDM-5</sub> and *bla*<sub>OXA-181</sub>. Additionally, three copies of *bla*<sub>CTX-M-15</sub> were detected (Table 1). The above genes were localized as follows (Figure S3, Figure 1): (i) *bla*<sub>NDM-5</sub>: one copy in the chromosome, and the second copy in the 84 kb IncFII plasmid; (ii) *bla*<sub>OXA-181</sub>: two copies in the chromosome; and (iii) *bla*<sub>CTX-M-15</sub>: two copies in the chromosome and one copy in the 57 kb IncR plasmid. In addition, a 61 kb region from the IncFII plasmid, comprising several antimicrobial resistance genes included in a class 1 integron and the replication machinery of the plasmid, was duplicated in the chromosome (Figure S3a,c).
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Additional molecular determinants of antimicrobial resistance were observed in strain DJ. Most notably, the gene *mgrB* was disrupted by an IS5 transposase (1057 bp), consistent with the observed colistin resistance phenotype (MIC=4 µg/ml). This strain also carried *rmtF*, *rmtB*, *strA*, *strB* and *aadA2* genes, associated with resistance to aminoglycosides including amikacin (Table 1). Quinolone resistance determining region (QRDR) mutations were observed, leading to GyrA-S83I and ParC-S80I amino acid alterations. Last, there was a premature stop codon caused by a A580T substitution in RamR, a negative regulator of RamA, itself a transcriptional activator of the *acrAB* genes. Higher production of AcrAB increases the efflux of tigecycline (39), consistent with the resistance phenotype observed for this agent (MIC=16 µg/ml).

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Regarding virulence genes, strain DJ harbored a complete yersiniabactin gene cluster (*ybt10*) located on an ICEKp4 mobile genetic element (Figure S3a). Type 1 (*fimAICDFGH*) and type 3 (*mrkABCD*) fimbriae gene clusters were also observed, but none of the *rmpACD*, aerobactin and salmochelin cluster genes, typically associated with hypervirulence, were present.

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208 **Table 1.** Antimicrobial susceptibility of *Klebsiella pneumoniae* strain DJ, and genes

potentially conferring resistance.

<table><thead><tr><th>Class and antimicrobial agent</th><th>Diameter (mm) or MIC (µg/ml)</th><th>Interpretation<sup>1</sup></th><th>Associated resistance genes (copy number)</th></tr></thead><tbody><tr><td><strong>Beta-lactams</strong></td><td></td><td></td><td>bla<sub>NDM-5</sub> (2), bla<sub>OXA-181</sub> (2), bla<sub>CTX-M-15</sub> (3), bla<sub>TEM-1</sub> (2), bla<sub>SHV-11</sub> (1), disrupted ompK35, mutation in ompK36</td></tr><tr><td>Ampicillin</td><td>9</td><td>R</td><td></td></tr><tr><td>Piperacillin</td><td>13</td><td>R</td><td></td></tr><tr><td>Amoxycillin-clavulanic acid</td><td>11</td><td>R</td><td></td></tr><tr><td>Ticarcillin-clavulanic acid</td><td>14</td><td>R</td><td></td></tr><tr><td>Piperacillin-tazobactam</td><td>14</td><td>R</td><td></td></tr><tr><td>Cefuroxime</td><td>11</td><td>R</td><td></td></tr><tr><td>Cefotaxime</td><td>12</td><td>R</td><td></td></tr><tr><td>Ceftazidime</td><td>15</td><td>R</td><td></td></tr><tr><td>Cefepime</td><td>15</td><td>R</td><td></td></tr><tr><td>Aztreonam</td><td>14</td><td>R</td><td></td></tr><tr><td>Ertapenem</td><td>12</td><td>R</td><td></td></tr><tr><td>Imipenem</td><td>14</td><td>R</td><td></td></tr><tr><td>Meropenem</td><td>15</td><td>R</td><td></td></tr><tr><td><strong>Aminoglycosides</strong></td><td></td><td></td><td>rmtB (1), rmtF (2), aac(6')-Ib (2), aadA2 (3), strAB(2)</td></tr><tr><td>Amikacin</td><td>10</td><td>R</td><td></td></tr><tr><td>Gentamycin</td><td>10</td><td>R</td><td></td></tr><tr><td>Tobramycin</td><td>9</td><td>R</td><td></td></tr><tr><td><strong>Quinolones and Fluoroquinolones</strong></td><td></td><td></td><td>gyrA and parC mutations</td></tr><tr><td>Nalidixic acid</td><td>11</td><td>R</td><td></td></tr><tr><td>Ofloxacin</td><td>11</td><td>R</td><td></td></tr><tr><td><strong>Folate pathway inhibitors</strong></td><td></td><td></td><td>sul1 (3), sul2 (1), dfrA12 (3)</td></tr><tr><td>Trimethoprim/Sulfamethoxazole</td><td>19</td><td>R</td><td></td></tr><tr><td>Trimethoprim</td><td>8</td><td>R</td><td></td></tr><tr><td><strong>Polymyxins</strong></td><td></td><td></td><td>disrupted mgrB</td></tr><tr><td>Colistin</td><td>MIC=4</td><td>R</td><td></td></tr><tr><td><strong>Tetracyclines</strong></td><td></td><td></td><td>ramR mutation<sup>2</sup></td></tr><tr><td>Tigecycline</td><td>MIC=16</td><td>R</td><td></td></tr><tr><td><strong>Phenicol</strong></td><td></td><td></td><td>catA2 (1), catB (2)</td></tr><tr><td>Chloramphenicol</td><td>12</td><td>R</td><td></td></tr><tr><td><strong>Other</strong></td><td></td><td></td><td></td></tr><tr><td>Fosfomycin</td><td>MIC=128</td><td>R</td><td>fosA</td></tr><tr><td>Rifampicin</td><td>NI</td><td>-</td><td>arr-2 (2)</td></tr><tr><td>Macrolides</td><td>NI</td><td>-</td><td>mphA (1), ermB (1)</td></tr></tbody></table>

NI, not included in the antimicrobial susceptibility testing panel; R, resistant; <sup>1</sup>Interpretations were based on CLSI and EUCAST guidelines; <sup>2</sup> Nucleotide mutation resulting in a premature stop codon.

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Time-scaled phylogenetic structure of the global population of sublineage CG147

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We investigated the evolutionary origins of strain DJ within the global diversity of
CG147 using 217 publicly available genomes of isolates collected between 2002 and 2018.

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CG147 genomes were mainly isolated from human samples (90%, 196/218) and hospital
environment (6%, 12/218), mostly in European (40%), Southeastern Asia (17%), Southern

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Asia (11%) and Northern America (10%) countries (Figure 2).

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CG147 was deeply structured into three main branches (Figure S1), each
corresponding to a single MLST sequence type: ST147, ST273 and ST392; the two latter are

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tonB variants of ST147. The number of genome-wide nucleotide substitutions was associated
with isolation dates (root-to-tip regression analysis: $R^2$=0.1123, **Figure S2**), enabling to infer

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a time-scaled phylogeny (Figure 3). The evolutionary rate within CG147 was estimated at
1.45x10⁻⁶ substitutions/site/year (95% HPD, 1.12x10⁻⁶ – 1.78 x10⁻⁶), corresponding to 6.2

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SNPs per genome per year. The last common ancestor of CG147 was estimated around year
1896, with a large uncertainty (95% highest posterior density (HPD): 1817-1962). The ST273

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lineage was the first to diverge, whereas ST147 and ST392 shared a common ancestor,
estimated around 1921 (95% HPD, 1868-1970) (Figure 3).

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ST147 was the most represented (79%, 172/218 genomes) and geographically
widespread lineage (Figure 2), and emerged around 1965 (95% HPD, 1940-1986) (Figure

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3). The phylogenetic structure within ST147 revealed five main clades characterized by
distinct capsular (KL type) and liposaccharide O antigen loci. Clades KL64-O2 (74%;

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128/172) and KL10-O3a (20%, 34/172) emerged in recent years: 1994 [95% HPD, 1990-
1998] and 2002 (95% HPD, 1995-2008), respectively (Figure 3). Whereas KL64-O2

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genomes were predominantly from Europe (54%), KL10-O3a was mainly sampled from Asia
(85%; Figure 2).

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The ST392 and ST273 branches emerged in 2005 (95% HPD, 2001-2009) and 1995

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(95% HPD, 1987-2001), respectively (Figure 3). ST392 (16% of genomes, 34/218) is

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distributed globally and harbors a KL27 capsular gene cluster and a O4 antigen. In contrast,

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ST273 (6%, 12/218) was predominantly found in Asia (64%) and carries KL74 and O3b gene

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clusters (Figure 2, Figure 3). Of note, a group of closely related genomes from the

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Philippines (n=5) lacked a capsular gene cluster, with only *ugd* being detected. This gene had

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100% identity with the *ugd* gene from the KL74 reference strain, suggesting a recent loss of

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the capsular gene cluster.

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## Acquired antimicrobial resistance genes and their evolutionary dynamics within CG147

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All genomes presented QRDR alterations in GyrA and ParC. The topoisomerase ParC

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S80I alteration was fully conserved, whereas the GyrA gyrase subunit S83I amino-acid

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change was observed in all genomes except for clade ST147-KL10, which had S83Y, caused

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by an ATC to TAC codon change. In addition, this clade had an D87A alteration (Figure 3).

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The number of acquired antimicrobial resistance genes or mutations among CG147

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genomes ranged from 2 to 23 (mean: 13; Table S1). Regarding beta-lactam resistance, in

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addition to the conserved chromosomal *bla*<sub>SHV-11</sub> gene, a majority of CG147 isolates carried a

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*bla*<sub>CTX-M</sub> (n=181, 83%), 94% of which was the *bla*<sub>CTX-M-15</sub> variant. In addition, 63% (n=137)

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genomes harbored at least one carbapenemase gene, with 14% (n=19) of these harboring

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more than one copy of the same carbapenemase gene, and/or two or more carbapenemase

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genes from different families (Table S1). Noteworthy, carbapenemase genes were

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significantly more frequent in ST147 (69%) compared to ST392 (35%) and ST273 (58%:

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*p*=0.0005). The two main ST147 clades were similar to this respect (ST147-KL10: 74%;

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ST147-KL64: 71%; *p*=0.78). However, their carbapenemase genes were distinct across world

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regions: there was a predominance of *bla*<sub>NDM</sub> in Southeastern Asia and Northern America,
whereas the combination of *bla*<sub>NDM</sub> and *bla*<sub>OXA-48-like</sub> was almost exclusively detected in Southeastern Asia, as observed in strain DJ. In contrast in Europe, *bla*<sub>OXA-48-like</sub>, *bla*<sub>KPC-2</sub> and *bla*<sub>VIM-1/-27</sub> were the most frequent carbapenemases (**Figure S4; Table S1**).

Different *ybt*/*ICEK*p subtypes (associated with hypervirulence) and OmpK35/K36 mutations (associated with multidrug resistance) were observed within the ST147-KL64 clade (**Figure 3; Table S1**). First, a group of 29 genomes (denominated subclade 1; mean number of SNPs among them: 57), emerged around 2007, was characterized by the presence of *ybt*16/*ICEK*p12 and an altered OmpK35 protein, due to a deletion of 2 nt resulting in a premature stop codon. Within this subclade itself, a subgroup of genomes (n=19/29) harbored *bla*<sub>OXA-48-like</sub> genes and the OmpK36GD mutation observed previously (40). Second, a group of 22 genomes emerged around 2009 (subclade 2; mean of 60 SNPs) was defined by the presence of *ybt*10/*ICEK*p4 and an OmpK35 gene disrupted by *ISEcp1-bla*<sub>CTX-M-15</sub> (**Figure 1; Figure 3, Table S1**). As observed in subclade 1, all subclade 2 isolates carrying *bla*<sub>OXA-181</sub> shared the same OmpK36TD mutation (40). Subclades 1 and 2 also differed in plasmid replicon content: whereas the former were rich in IncR (90%) and IncHIB/IncFIB(Mar) (41%), in contrast IncFII (pKPX1) (82%), IncFII (59%) and IncR (64%) were frequent in the latter (**Table S1**). Differently, the remaining ST147-KL64 genomes (n=77) often carried IncFIB<sub>K</sub> (65%), IncFII<sub>K</sub> (49%; a common pKPN-3-derived plasmid found in Kp harboring *pco* and *sil* clusters; **4,41**) and IncFIA(HI1) (38%).

Strain DJ belonged to subclade 2 and was phylogenetically closely related (<28 SNPs) to five other isolates recovered between 2014 and 2015 in different Asian countries (**Figure S3b; Figure3**) and described as extremely drug resistant or pandrug-resistant (11,42,43), with identical plasmids being observed among them but no chromosomal integration of *bla*<sub>NDM-5</sub> (**Figure S3**).

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286 **Convergence of antimicrobial resistance and virulence**

287 Whereas the yersiniabactin virulence factor gene cluster was rare amongst genomes of
288 ST392 (6%; 2/34) and ST273 (8%; 1/12), it was observed in 53% of ST147 genomes. There
289 were two predominant variants (ybt16/ICEKp12 and ybt10/ICEKp4 associated with subclades
290 1 and 2, respectively), and four minority ones (**Figure 3; Table S1**).

291 Two isolates with hypervirulence genotypes, defined by the presence of *rmpA* and/or
292 *rmpA2* or aerobactin, were observed: KpvST147L (ST147-KL14, 2016, United Kingdom)
293 and B-8658 (ST147-KL10, 2014, Russia). The plasmid from KpvST147L was fully
294 sequenced previously (GenBank accession number NZ_CM007852) and carries *rmpA*,
295 *rmpA2* and aerobactin; it corresponds to a 343 kb IncFIB-IncHIB (pNDM-MAR) plasmid
296 (**Figure S5**) (44). This plasmid was recently described in three ST147 *Kp* isolates recovered
297 in 2018-2019 in the United Kingdom and in other sublineages (45). Here, we found that
298 Russian strain B-8658 (ST147-KL10, 2014) also acquired the pNDM-MAR plasmid, but it
299 lacked the *rmpA* gene (**Figure S5**).

300

301 **Co-occurrence of antimicrobial resistance and heavy metal tolerance genes and**
302 **plasmids**

303 Antimicrobial resistance genes, mutations and plasmids co-occurred in a structured
304 way (Figure S6; Table S1). For example, Col(BS512), ColKp3, IncFIA (HI1), Cml, EreA/B
305 and GyrA S83Y/D87A co-occurred frequently within the ST147-KL10 clade. Associations of
306 (i) VIM, QnrA1, AadB and IncHI2; (ii) OXA-48-like and IncL(pOXA-48) or (iii) KPC and
307 IncFIB(pQIL) were also observed, consistent with previous descriptions of genetic elements
308 co-carrying these genes (46–48). In addition, genes conferring tolerance to copper and silver
309 were associated with IncFII<sub>K</sub>/IncFIB<sub>K</sub> plasmids (*pco* and *sil* operons; *p*<0.00001), whereas
310 genes for mercuric resistance were linked to IncR plasmids (*p*=0.0082). Similarly, a positive
311

association between the tellurite cluster and IncHIB/IncFIB (MAR) was observed (p<0.0001)

312

(4). Last, a negative association between IncR and IncFII<sub>K</sub>/IncFIB<sub>K</sub> was observed.

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314

## CRISPR-Cas systems

315

We investigated whether resistance gene dynamics could be influenced by CRISPR-Cas systems in CG147. There were either one (83%; 180/218) or two (17%; 37/218)

316

317

CRISPR-Cas systems amongst CG147 genomes. A conserved subtype IV-E system was

318

found in all genomes, located in the *iap* – *cysH* region, with a %G+C of 60.6%, and defined

319

arbitrary as CRISPR1. Direct repeat sequences were highly conserved but the number and

320

sequences of spacers varied across CG147 genomes. CRISPR1 variant v0 (43 spacers) was

321

present in 56% of the genomes (122/218) and was used as reference to define CRISPR1

322

variants (Figure S7; TableS2). Four of the CRISPR1 v0 spacers (spacers 1, 28, 29 and 42) matched sequences on MDR IncF plasmids disseminated among different

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*Enterobacteriaceae*, including Kp. Spacer 1 matched a multicopy intergenic region, whereas

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spacers 28 and 29 matched a DUF3560-domain containing protein and spacer 42 a

325

hypothetical protein located upstream of SAM-methyltransferase. Although these spacers

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were highly conserved among CRISPR1 variants, they were also found at high frequency (in

327

28% to 53% of the strains) as protospacers located in plasmid contigs. Noteworthy, in 28%

328

(62/218) of CG147 genomes these plasmid protospacers were not detected, and the majority

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of these strains (65%, 40/62) belonged to subclades 1 and 2, which were also characterized

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by the absence of IncFII(K) and IncFIB(KpQIL) plasmid replicons. The association of the

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lack of IncF plasmids with IncF-targeting spacers in CRISPR1, suggests a possible activity of

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this CRISPR system in subclades 1 and 2. Finally, twelve of the forty-three spacers from

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CRISPR1 v0 targeted prophages; of these, spacers 6 and 25 were found as protospacers in

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some of the CG147 genomes (28% and 18%, respectively).

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336

Six other CRISPR-Cas systems, distinct from CRISPR1, were observed in 37 genomes. CRISPR2 to CRISPR4, of type IV-A3, were strongly associated with IncHIB and/or IncFIB (pNDM-MAR) plasmids (p<0.00001), as previously described (49). Two variants of CRISPR2 (v1; n=12; 17 spacers and v3; n=5, 25 spacers) were prevalent (Figure S7; TableS2). CRISPR2 to CRISPR4 systems shared identical direct repeat sequences but showed a high diversity in the number and sequences of spacers. Their tendency to target IncFII<sub>K</sub>/IncFIB<sub>K</sub> plasmids has led to suggest a role in inter-plasmid competition (49,50). However, here the presence of CRISPR2 to CRISPR4 systems was not uniformly associated with an absence of IncFII<sub>K</sub>/IncFIB<sub>K</sub> plasmids.

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## Prophage elements

347

CG147 genomes harbored 0 to 7 prophages (mean of 4 prophages per genome; considering only the intact ones). Some prophages were frequent, including (i) ST147-VIM1phi7.1-like (GenBank accession no. NC_049451; *Myoviridae* family), which was present in 90% of the genomes (196/218); (ii) *Salmonella* phage 118970 sal3-like (GenBank accession no. NC_031940; *Myoviridae* family; 70% [152/218]); and (iii) *Enterobacteria* phage mEp237-like (GenBank accession no. NC_019704; *Siphoviridae* family; 56% [121/218]) (Figure S7).

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The N15-like phage-plasmid (*Siphoviridae* family) we uncovered in the genomic assembly of strain DJ was present in 37% of CG147 genomes (81/218). It was strongly associated (p<0.00001) with ST147-KL64 subclades 1 and 2 and present in 92% of these genomes (Table S1, Figure S7). These two subclades were also enriched in other prophages (mean number of prophages: 5) compared with the remaining CG147 genomes (mean number of prophages=3).

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357

358

359

360
361 DISCUSSION

362 This study was triggered by the discovery of a pandrug resistance phenotype in strain
363 DJ from India, which prompted us to analyzed its genomic features and understand their
364 dynamics in the context of a large genome dataset of CG147 from multiple world regions.
365 The results revealed its deep phylogenetic structure and a capacity of CG147 members to
366 acquire a wide range of antimicrobial resistance or virulence elements, plasmids and
367 prophages. Of these, Klebsiella capsular locus (KL) switches, which occurred repeatedly,
368 represent prominent phylogenetic markers of CG147 clades. The evolutionary dynamics of
369 these surface structure in CG147 echo those observed in other MDR clonal groups, such as
370 ST258 or ST307 (13,47,51). The multiple clonal expansions of sublineages with distinct KL
371 or O types has clear implications for diagnostic, control or therapeutic strategies such as
372 vaccination and phage therapy. Although its reliance on public genomic sequences may
373 expose this study to a bias towards antibiotic-resistant CG147 isolates, as well as those
374 involved in nosocomial outbreaks, this study provides new insights into the evolutionary
375 history, epidemiology and population dynamics of this important emerging clonal group.

376 The genomic arsenal of antimicrobial resistance features of subclade 2, to which strain
377 DJ belongs, suggest extensively drug and pandrug resistance is not restricted to strain DJ, but
378 rather is a shared characteristic in this particular subclade, already disseminated among Asian
379 countries (11,42,43,52-54). Clonal spread of this subclade between different countries was
380 reported (52). This subclade should be closely monitored and may represent a pioneering
381 situation ushering the worrying prospect of pan-resistance in other CG147 subclades.

382 In the case of strain DJ, we observed the integration of bla<sub>NDM-5</sub> in the chromosome.
383 Previous studies have reported the one or multiple copies of bla<sub>CTX-M-15</sub> and bla<sub>OXA-181</sub>
384 integrated into the chromosome of Kp isolates (55,56), including in the high-risk ST147
385 (52,57). However, the chromosomal integration of bla<sub>NDM</sub> was, to our knowledge, only
reported once in Kp, in two NDM-1 producing ST14 clinical isolates from Thailand (58). In that case, the chromosomal integration was mediated by IS5 and the Tn3 transposase. In strain DJ, the integration of *bla*<sub>NDM-5</sub> may have been mediated by *ISEcp1/IS26* (**Figure 1a**). The genomic rearrangement due to the replicative transposition of IS26 has previously been shown for IncFII-*bla*<sub>NDM-5</sub> bearing plasmids (59). The chromosomal incorporation of carbapenemase and ESBL genes is concerning, as it may stabilize these genes by promoting their vertical dissemination (52).

The convergence of MDR and hypervirulence genotypes is being increasingly observed in Kp (3). Here, this worrisome association was detected in two phylogenetically distinct CG147 isolates from 2014 (Russia) and 2016 (United Kingdom), which shared an MDR-Hv IncHIB/FIB plasmid (44). The recent observation of this plasmid in ST101 and ST147 isolates from the United Kingdom (45), with no epidemiological link with the isolate from 2016, suggests its continuous circulation and further risk of horizontal spread.

The ST147-KL64 lineage is globally disseminated. The evolutionary rate we estimated (1.03x10<sup>-6</sup> substitutions/site/year) is very similar to other MDR global sublineages, such as ST258 (1.03x10<sup>-6</sup> substitutions/site/year) and ST307 (1.18x10<sup>-6</sup> substitutions/site/year) (47,51), and slightly slower than the one estimated for ST101 (2.85x10<sup>-6</sup> substitutions/site/year) (60). It is striking that the emergence of ST147-KL64 lineage occurred approximately at the same time as other MDR sublineages ST258 (year 1995), ST307 (1994) and ST101 (1989) (47,51,60). In addition, the presence of GyrA and ParC QRDR alterations is a common characteristic to these MDR high-risk sublineages [CG258 and ST307: GyrA-S83I and ParC-S80I; ST101: GyrA-S83I, GyrA-D87G/N/A and ParC-S80I] (47,51,60). This phenotypic and temporal conjunction points to common drivers and suggests a role of the usage of fluoroquinolones, introduced into clinical practice in the end of the 1980s, in the emergence of MDR Kp sublineages. This is reminiscent of the
411

scenarios of emergence of *Escherichia coli* ST131 and methicillin-resistant *Staphylococcus aureus* ST22 (61–63).

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413

The drivers of genomic diversification of emerging Kp sublineages may include a combination of ecological opportunities to acquire genetic elements, local selective pressure, and molecular mechanisms that enable or restrict genetic flux. Of these, CRISPR-Cas systems may play a role (64–67). In CG258, an association was suggested between the absence of these systems and the ability to acquire IncF plasmids [such as *bla*<sub>KPC</sub>-IncF(pKpQIL-like plasmids)] (68–70). Here we found a conserved type IV-E CRISPR-Cas system (CRISPR1) within CG147, consistent with early reports (9), with four spacers matching IncF plasmid sequences. The distribution of CRISPR1 in the broader Kp species shows a unique association with CG147, with only two exceptions (ST2746 and ST3700; based on 1001 Kp genomes representing unique STs; selected from a dataset of 4222 genomes from NCBI, November 2018; data not shown). Type IV CRISPR-Cas systems primarily target plasmids (50). However, in the majority of CG147 strains, corresponding protospacers were found in plasmid sequences, advocating that the immunity provided by this CRISPR1 system might not be fully functional. In contrast, ST147-KL64 subclades 1 and 2 were largely devoid of these protospacers and did not carry IncFIA, IncFII<sub>K</sub>, IncFIB<sub>K</sub> and IncFIB (pQil) plasmids (Figure S7). This observation may suggest a possible activity of the CRISPR1 system in these recently emerged subclades. Future experimental studies are needed to explore this hypothesis. Noteworthy, it was also among these subclades that the N15-*like* phage-plasmid was prevalent. Among bacterial phyla, this phage-plasmid family was found almost exclusively in Kp (38), and we found that 58.9% of these belonged to CG147. A possible biological role of plasmid-prophages has yet to be established.

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435

CONCLUSIONS
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443

The presence of pandrug-resistant and extremely-drug resistant isolates in CG147, together with the high genetic plasticity and rapid emergence dynamics of this clone, represents a clear threat to public health. CG147 is globally disseminated but shows a strong phylogenetic structure, with different clades being associated with specific genomic features and geographical distributions. These observations underline how different variants of CG147 contribute to the major public health threat posed by Kp, and call for specific surveillance and directed control strategies of this clone and its particularly concerning clades (12).

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450

A possible link between the absence of IncF plasmids and the activation of CRISPR1 defense system is intriguing. This observation calls for more work on mechanistic drivers of the flux of genetic elements across MDR bacterial lineages. Precise phylogenetic mapping and understanding of the dynamics of antimicrobial resistance features are needed to guide the development of control strategies, especially those that target specific subsets of strains within pathogenic bacterial species, such as CRISPR delivery or toxic conjugation systems (71,72).
451 **DECLARATIONS**

452 **Ethics approval and consent to participate:** To conduct the research, we used bacterial
453 strain DJ, which is not considered a human sample. Accordingly, this research was not
454 considered human research and is out of the scope of the decree n° 2016-1537 of November
455 16, 2016 implementing law n° 2012-300 of March 5, 2012 on research involving human
456 subjects. Therefore, no ethics approval was needed and no informed consent was required.

457 **Consent for publication:** No personal identifying data was used; therefore, no consent was
458 necessary.

459 **Availability of data and materials:** All data generated or analysed during this study are
460 included in this published article and its supplementary information files.

461 **Competing interests:** The authors declare that they have no competing interests.

462 **Funding:** SD availed a UGC-BSR fellowship for research students and was supported
463 financially by EMBO fellowship sponsored by European Molecular Biology Organization
464 (STF_7993) for a visit in the Brisse lab. CR was supported financially by the MedVetKlebs
465 project, a component of European Joint Programme One Health EJP, which has received
466 funding from the European Union's Horizon 2020 research and innovation programme under
467 Grant Agreement No 773830, and by a Roux-Cantarini grant from Institut Pasteur.

468 **Author contributions:** S.D. and D.G. coordinated the microbiological cultures of the isolate
469 DJ and its antimicrobial susceptibility testing and biochemical characterization. V.P., C.R.
470 and S.D. performed the genomic sequencing. C.R. designed and coordinated the comparative
471 genomics study, with input from S.D. C.R. wrote the initial version of the manuscript. All
472 authors provided input to the manuscript and reviewed the final version.
473

**Acknowledgments:** We thank the Plateforme de Microbiologie Mutualisée (P2M) of Institut Pasteur for Illumina sequencing.

474

475

**Authors license statement:** This research was funded, in whole or in part, by Institut Pasteur and by European Union's Horizon 2020 research and innovation programme. For the purpose of open access, the authors have applied a CC-BY public copyright license to any Author Manuscript version arising from this submission.

476

477

478
479 FIGURE LEGENDS

480

481 **Figure 1.** Genetic context of the different copies of *bla*<sub>NDM-5</sub> (**a**), *bla*<sub>OXA-181</sub> (**b**) and *bla*<sub>CTX-M-15</sub> (**c**) genes identified in *K. pneumoniae* strain DJ.

483 Within each panel, the chromosomal and/or plasmid-encoded copies of *bla*<sub>NDM-5</sub>, *bla*<sub>OXA-181</sub> and *bla*<sub>CTX-M-15</sub> detected in DJ are compared between them. Predicted open reading frames (ORFs) are represented on each line by colored arrows, with arrowheads indicating the direction of transcription: antimicrobial resistance genes (red), mobile genetic elements (MGEs) or gene mobilization-related genes (green); RNA-binding proteins (dark yellow); other functions (light gray). Disrupted genes are outlined with dotted lines. Dark gray blocks connecting the distinct gene regions represent homology levels, as indicated in the gradient key. The nucleotide positions of the represented regions are indicated at the left below the copy descriptors. Figures were created using the Easyfig (https://mjsull.github.io/Easyfig/).

492

493 **Figure 2.** Geographic (**main**) and temporal (**inset**) distribution of genomes included in this study.

494

495 The pie charts represent the frequency of each CG147 clade in each country (see size and color keys). **Inset:** Bars represent the number of isolates per year for which genome assemblies were available (NCBI RefSeq) as of May 2019, coloured by clade. Red lines represent (solid line) the number of PubMed-indexed records as of March 2020 (identified using the search criteria '*Klebsiella pneumoniae*' and '*ST147*' or '*ST392*' or '*ST273*' resulting in a total of 99 distinct entries); and (dotted line) the cumulative number of genomes. The scale on the left Y-axis refers to total number whereas the one on the right refers to cumulative numbers.

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499

500

501

502

503
504

**Figure 3.** Time-scaled phylogeny of 218 CG147 genomes and their epidemiological and molecular characteristics.

505

506

The phylogeny was obtained using the BEAST tool. The three main branches correspond to the three main sequence types (ST). Tree tips are colored by world region of isolation (see key). Black dots on main nodes indicate ≥95% posterior probability. The grey boxes delineate subclades 1 and 2, as indicated. Capsular (KL) and O-antigen (O) locus types and the yersiniabactin-carrying ICEK*p* elements are colored according to their variants as shown in the legend. Antimicrobial resistance determinants are indicated by colored rectangles when present. In the antimicrobial resistance determinants columns:¹ dark pink indicates *bla*<sub>KPC-2</sub> and light pink indicates *bla*<sub>KPC-3</sub>;² dark pink indicates *bla*<sub>CTX-M-15</sub> and dark pink indicates other *bla*<sub>CTX-M</sub> variants;³ dark pink indicates *mgrB* mutations and dark pink indicates *pmrB* mutations. AMGs: aminoglycosides; FPIs: folate pathway inhibitors.

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510

511

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514

515
516 Supplementary Data:

517

518 Figure S1. Phylogenetic structure of CG147.

519 The tree was obtained by maximum likelihood analysis (IQ-TREE) based on the final
520 recombination-free alignment of concatenated nucleotide sequence alignments of 4,517 core
521 genes. The tree was rooted with a Kp ST258 NJST258_2 (GCF_000597905.1) and a Kp
522 ST37 INF042 (GCF_002752995.1). Branch lengths represent the number of nucleotide
523 substitutions per site (scale, 0.001 substitution per site). The three main branches are colored
524 by sequence type (ST) and branch tips are colored by world region of isolation (see key).
525 Capsular (KL) and O-antigen (O) locus types and the yersiniabactin-carrying
526 ICEKp elements are colored according to their variants (see key). Subclades 1 and 2 are
527 denoted by rectangle outlines.

528

529 Figure S2. Temporal signal in genomic sequences.

530 The linear correlation between year of isolation and root-to-tip distance from the maximum
531 likelihood phylogeny for CG147 genomes collection was calculated using TempEst.

532

533 Figure S3. Replicons of strain DJ.

534 (a) Visual representation of the hybrid Unicycler assembly graph, obtained using Bandage
535 (Wick et al., 2015). Nine replicons were obtained, including the circularized chromosome, 6
536 circularized plasmids, 1 linear phage-plasmid and 1 non-circularized plasmid contig. The size
537 of replicons is shown.

538 (b) Sequence alignments of the chromosomes of strain DJ and the four closest genomes
539 belonging to subclade 2 (<28 SNPs): MS6671 (GenBank Accession Number:
540 GCA_001455995), DA48896 (GCA_003006175.1); CRKP-1215 (GCA_002786755;
541 =CRKP-2297) and SKGH01 (GCA_001644765.1). Strain DJ was used as reference. The
542 outermost circle is an annotation of the reference strain showing the location of antimicrobial
543 drug resistance genes, ytb/ICEKp, KL64 capsular locus and the IncFII chromosomal
544 integration region. The figure was created using BRIG (Alikhan et al., 2011).

545 (c) Sequence alignments of the non-circular IncFII replicon of strain DJ with the closest
546 plasmids of subclade 2 strains: pCRKP-2297_2 (CP024836.1); pCRKP-1215_2
547 (CP024840.1), MS6671_plasmidE (LN824138.1), SKGH01_p3 (CP015503.1), p48896_1
548 (CP024430.1) and the region integrated in the chromosome of strain DJ. The IncFII non-
549 circularized replicon was used as reference. The outermost circle is an annotation of the
550 reference strain showing the location of antimicrobial drug resistance genes in red. In the
551 table the coverage, identity, size and antimicrobial resistance genes of the plasmids that were
552 used for the comparison. In bold the resistance genes also present in DJ replicon.

553 (d) Sequence alignments of the p48896_1 (CP024430.1) plasmid with the plasmids of the
554 closest strains: pCRKP-2297_2 (CP024836.1); pCRKP-1215_2 (CP024840.1),
555 MS6671_plasmidE (LN824138.1), SKGH01_p3 (CP015503.1), DJ-IncFII non-circularized
556 replicon and DJ-IncR plasmid. p48896_1 was used as reference plasmid. The outermost
557 circle is an annotation of the reference strain showing the location of antimicrobial drug
558 resistance genes in red.

559 (e) Sequence alignments of the 123 kb IncFII(pKPX1) plasmid of strain DJ with the closest
560 plasmids of subclade 2 strains: pCRKP-2297_1 (CP024835.1), pCRKP-1215_1
561 (CP024839.1), MS6671_plasmidB (LN824135.1), SKGH01_p2 (CP015502.1), p48896_2
562 (CP024431.1). The IncFII(pKPX1) plasmid of strain DJ was used as reference. The
563 outermost circle is an annotation of the reference strain showing the location of antimicrobial
564 drug resistance genes in red. In the table the coverage, identity, size and antimicrobial
565

resistance genes of the plasmids that were used for the comparison. In bold the resistance genes also present in DJ replicon.

566

567

568

**Figure S4. Geographic origins of the genomes included in this study and their associated carbapenemase profile.**

569

570

The pie charts represent the frequency of each carbapenemase profile in each country (see key). CPKP-: no carbapenemase.

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572

573

**Figure S5. Sequence alignments of MDR-Hv plasmids identified in CG147 genomes.**

574

Comparison of MDR-Hv plasmids identified in CG147 genomes (KpvST147L_NDM and B-8658) with the reference virulence plasmid pK2044 (AP006726.1) and a similar plasmid recovered in 2019 (pKpvST147B, CP040726.1), which was used as reference. The outermost circle shows annotations of the reference strain.

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578

579

**Figure S6. Correlation between genotypes (Pearson method).**

580

Blank squares represent correlations without statistical significance (p > 0.05). The plot was created with the corrplot R package.

581

582

583

**Figure S7. Time-scaled phylogeny of CG147 genomes and their heavy metal tolerance genes, plasmid replicons, CRIPSR systems, and prophages profile.**

584

585

The phylogeny was obtained using the BEAST tool. The three main branches correspond to the three main sequence types (ST). Tree tips are colored by world region of isolation (see key). Black dots on main nodes indicate ≥95% posterior probability. The grey boxes delineate subclades 1 and 2, as indicated. Capsular (KL) and O-antigen (O) locus types and the yersiniabactin-carrying ICEK*p* elements are colored according to their variants as shown

586

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588

589
590

in the legend. The presence of heavy metal tolerance genes, plasmid replicons, CRISPR-Cas
systems, protospacers and prophages is indicated. In heavy metal tolerance genes columns,
lighter yellow indicates an incomplete operon. In prophages lighter orange indicates an
questionable prophage. HMTG, heavy metal tolerance genes.

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592

593

594

595

**Table S1.** Epidemiological and genomic characteristics of the 218 CG147 *K. pneumoniae* genomes analysed in this study.

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598

**Table S2.** CRISPR/Cas systems and variants identified within CG147 *K. pneumoniae* genomes.

599

600
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