Preferred evolutionary routes of convergence in Klebsiella pneumoniae favor siderophore acquisition over hypervirulence

Preferred evolutionary routes of convergence in Klebsiella pneumoniae favor siderophore acquisition over hypervirulence | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Preferred evolutionary routes of convergence in Klebsiella pneumoniae favor siderophore acquisition over hypervirulence Anjali Sapre, Melissa Martin, Ting Luo, Ulrike Carlino-MacDonald, and 22 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8613390/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The rise of Klebsiella pneumoniae combining antimicrobial resistance and virulence genes poses a major health threat, but the evolutionary routes and phenotypic consequences of this convergence are poorly understood. Here, phylogenetics of 1,468 isolates and population analysis of 7,520 plasmids, from >50 countries through the last two decades, reveal that convergence follows preferred, constrained evolutionary paths. The dominant route involves multidrug-resistant classical K. pneumoniae acquiring conjugative IncFIB(Mar)/IncHI1B plasmids carrying an incomplete set of virulence biomarkers. Across 25 independent convergence events, the acquisition of the aerobactin siderophore locus was the only universal feature. These convergent isolates exhibit enhanced siderophore production but consistently lack the hypervirulent phenotype in vivo. In contrast, genuine hypervirulent strains that gain resistance remain rare. We conclude that enhanced siderophore production, not hypervirulence, is the primary adaptive trait driving the success of globally emerging convergent lineages, representing a distinct evolutionary state optimized for transmission rather than systemic invasion. Biological sciences/Microbiology/Microbial genetics/Bacterial genetics Biological sciences/Microbiology/Antimicrobials/Antimicrobial resistance Biological sciences/Microbiology/Pathogens Biological sciences/Microbiology/Bacteria/Bacterial genomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Klebsiella pneumoniae are ubiquitous bacteria and the causative agent of diverse infections including pneumonia, urinary tract, surgical site, and bloodstream infections ( 1 ). Classical K. pneumoniae (cKp) with acquired resistance determinants are one of the world’s leading causes of healthcare-associated infections ( 2 ). Notably, third-generation cephalosporin-resistant and carbapenem-resistant K. pneumoniae are listed by the World Health Organization as urgently needing novel therapeutics ( 3 ). Antimicrobial resistance (AMR) genes, including extended-spectrum β-lactamases (ESBLs) and carbapenemases, are readily acquired by conjugative plasmids and mobile genetic elements ( 4 – 6 ). Carbapenem-resistant K. pneumoniae were initially associated with globally disseminated clone ST-258, encoding KPC enzymes found on conjugative IncFII(K)/IncFIB(pQil) plasmids ( 4 , 7 , 8 ), however, other lineages ( e.g. ST-307, ST-147, ST-395) have recently emerged internationally carrying bla NDM , bla KPC , and/or bla OXA−48 genes on diverse plasmids ( 9 – 12 ). A distinct pathotype from cKp, hypervirulent K. pneumoniae (hvKp) cause invasive community-acquired infections linked to high mortality, driven by metastatic spread, despite generally remaining amenable to antimicrobial treatment ( 13 ). hvKp infection outcomes are associated with five hvKp-specific virulence plasmid-encoded genes for siderophores aerobactin ( iuc ) and salmochelin ( iro ), mucoid and capsule polysaccharide regulators ( rmpADC and rmpA2 ), and the metabolite transporter ( peg-344 ) ( 14 – 18 ). Genetically distinct from cKp, hvKp clones have lower pan genome diversity, lower capsule type diversity (most commonly KL1 or KL2), and constitute fewer lineages (e.g., ST-23, ST-268, ST-65, ST-420), which are most prevalent in the Asia Pacific Rim ( 5 , 6 , 17 ). Conventionally, hvKp harbor non-conjugative virulence plasmids, mainly the described pLVPK or pK2044 with IncHI1B/IncFIB dual replicon ( 19 , 20 ) or the rarer KP52.145pII plasmid with IncFIB replicon ( 21 ). However, acquired virulence genes are also found on chromosomal integrative conjugative elements (e.g. ICEKp10) and conjugative plasmids ( 6 , 22 ). While historically rare, the overlap of K. pneumoniae strains carrying both hvKp-specific virulence genes and carbapenemase and/or ESBL genes have been increasingly reported within the last decade ( 17 , 23 , 24 ). The genetic characteristics of reported convergent isolates include hvKp lineages (e.g. ST-23-KL1) acquiring conjugative resistance plasmids carrying AMR genes ( 24 ), or MDR-cKp lineages harboring pLVPK-like plasmids ( 25 ). Other evolutionary dynamics that have led to genomic convergence includes hybrid plasmids of the IncFIB(Mar)/IncHI1B dual replicon type carrying resistance ( bla NDM−1 , armA ) and hvKp-specific virulence ( iuc , rmpADC , rmpA2 , and/or peg-344 ) biomarkers that are found in globally epidemic nosocomial MDR lineages such as ST-307 and ST-147 ( 26 , 27 ). Although some confusion remains, because of genetic-only inferences or inadequate virulence models ( 28 ), genomic convergence is not synonymous with phenotypic hypervirulence ( 12 , 15 , 27 , 29 ). In this study we determine that the phenotypic outcomes of convergence in K. pneumoniae are lineage specific and the result of distinct evolutionary routes of plasmids acquisition. Using a unique global dataset of 1,468 multidrug-resistant isolates, and a population plasmidomic analysis of 7,347 complete plasmids (from strains spanning over 47 countries and the last two decades), we describe that convergence follows preferred paths, constrained by the plasmid biology, and that enhanced aerobactin-mediated iron acquisition, not hypervirulence, is the main adaptive trait driving the success of convergent lineages in hospital settings. Results Prevalence of MDR lineages in a global collection of Klebsiella pneumoniae Global surveillance of multi-drug resistant K. pneumoniae (MDR-Kp) resulted in 1,468 clinical isolates recovered from 8 countries on 5 continents, between 2011 and 2021 ( Fig. S1, Table S1 ). The frequency of isolate collection varied by year and country. In silico typing revealed a diverse population comprising 208 sequence types (ST) and 87 capsule types (KL) ( Fig. S1 , Table S1 ). Nevertheless, 8 lineages were represented by > 50 isolates and accounted for 57% (n = 834) of the population. ST-395 and ST-16 were the most prevalent (12% and 11%) and respectively grouped isolates that largely originated from the Republic of Georgia (99%) and Thailand (84%). In contrast, other major lineages (e.g. ST-15 and ST-147) were collected from multiple countries. Within those substantial genetic diversity was observed, with most isolates differing by > 100 allelic differences by core genome MLST ( Fig. S2, Table S2 ). By contrast high genetic relatedness (≤ 10 allelic differences) was observed in geographically clustered subsets, including ST-395 from Georgia, ST-16 from Thailand, and subsets of ST-147 isolates from Thailand and Peru, consistent with local clonal expansion of emerging high-risk lineages. Association of antibiotic resistance and virulence genes with global MDR-Kp lineages For the resistance genes: in this collection, the rate of ESBL carriage remained remarkably stable, between 78 and 82% annually, and gene bla CTX−M−15 (74% of ESBL isolates) was the most represented ( Fig. S1, Table S1) . By contrast, the rate of carbapenemase-producing isolates increased markedly over time, from 6% of MDR-Kp isolates sampled between 2011–2016 to 44% (chi-square 175.5 and p < .01) between 2017–2021. This largely correlated with the sampling of clonal, geographically clustered isolates ( Fig. S1, Table S1 ). Carabapenemase bla NDM genes were the most represented, alone (55%) or in combination with bla OXA−48 (22%). For the virulence genes: 222 isolates (15%) carried at least one virulence biomarker ( iuc, iro , peg-344 , rmpADC , and/or rmpA2 ). By contrast, only 1% of MDR-Kp isolates collected from the U.S. military health system during this same time period carried an acquired virulence gene ( 30 ). Core genome phylogenetic analysis of the global virulence-carrying collection (n = 222) compared to virulence-carrying US isolates (n = 39, including 22 canonical hypervirulent ST-23 isolates) identified 32 distinct virulence gene-carrying lineages including ST-395 (n = 135), ST-23-KL1 (n = 29), ST-15 (n = 13), ST-23-KL57 (n = 13) and ST-2071 (n = 10) (Fig. 1 ). The prevalence of the 5 acquired virulence biomarkers was variable; all 261 isolates carried iuc siderophore loci, represented by four iuc alleles including the hvKp-associated iuc1 (n = 135), followed by iuc3 (n = 107), iuc5 (n = 17), and iuc2 (n = 2) (Fig. 1 , Table S1 ). By contrast, the iro siderophore loci was only found in 23% (n = 60) of isolates, with the hvkp ST-23-KL1 isolates accounting for half. Most isolates carried only 1 biomarker (n = 145), mainly represented by lineage ST-395 from Georgia. Only 49 isolates carried all five biomarkers, predictive of the hvKp phenotype ( 15 ). Within those, ST-23-KL-1 was the most represented (n = 25) (Fig. 1 , Table S1 ). Increased prevalence of convergent isolates in recent years. Convergence, defined by the co-carriage of an ESBL and/or carbapenemase gene in addition to ≥ 1 virulence biomarker ( 15 , 17 , 29 ), was detected for 198 (76%) of the virulence carrying isolates and was significantly enriched (chi-square 27.7 and p < .01) in the last 5 years of sampling ( Table S1 ). Only 7 convergent isolates carried all 5 virulence loci. None of these 7 isolates carried a carbapenemase gene, instead all acquired an ESBL, including ST-420 (n = 2) and ST-268 (n = 1) from Thailand, ST-218 (n = 2) from Kenya, and ST-23-KL-1 (n = 1) from the United States (Fig. 1 , Table S1 ). By contrast, 80% of the isolates with 1–4 virulence loci co-carried a carbapenemase gene. Isolates from Georgia were the most represented (89%) and two lineages were predominant: ST-395 (n = 111) and ST-23-KL57 (n = 13). Within each, differences in patterns of AMR and virulence genes were suggestive of distinct plasmid acquisition/loss events (Fig. 1 ). Population analysis of virulence, resistance, or virulence/resistance carrying plasmids. Complete genomes were obtained for 34 representative convergent isolates and 19 virulence-carrying isolates (Fig. 1 A, blue triangles). Assemblies revealed 173 plasmid sequences, including 172 circular and 1 linear sequence, with between 1 and 7 plasmids identified per isolate ( Table S3) . Replicons were typed and, using a cluster-based approach ( 31 ), all study (MRSN) plasmid sequences were compared to each other and to a publicly available set of 7,347 complete K. pneumoniae plasmids (from genomes in NCBI, representing 306 STs from at least 47 countries throughout the last two decades) ( Fig. S3, Table S4) . The K. pneumoniae plasmidome stratified into 43 discrete plasmid clusters (pC) ( Fig. S.3 ). Within those, the study plasmids (n = 124) clustered in 28 pCs with 4,052 NCBI plasmids sequences ( Fig. 2AB, Table S5 ). The remaining 49 MRSN plasmid sequences were singletons/unassigned. Within the 28 pCs comprising at least one study plasmid, 22 grouped plasmids with an ESBL and/or carbapenemase gene (Fig. 2 C). Cluster pC10, the most numerous, was very homogeneous and 82% of its plasmids carried an OXA-48 carbapenemase ( Table S5 ). Plasmids within pC3, pC38 and pC39 were also frequent carriers of ESBL and/or carbapenemase genes (Fig. 2 C), although a diversity of resistance alleles was noted ( Table S5 ). In alignment with the population-level analysis, ESBL-carrying plasmids (n = 26) from this study were highly diverse and belonged to 11 pCs with pC-38 (n = 6, carrying CTX-M and IncR replicon) and pC-39 [n = 5, carrying CTX-M and IncFII(K)] being the most represented (Fig. 3 , Table S3 ). Carbapenemase-carrying plasmids (n = 20) were also diverse, with 6 pCs represented, though the majority belonged to either pC-10 (n = 9 carrying OXA-48 and IncL/M replicon) or pC-2 (n = 4, carrying NDM and a IncFIB(Mar)/IncHI1B replicon). With a single exception (isolate 582610), conjugative plasmids pC2, -10, -38, and − 39 were exclusively found in MDR-cKp lineages (e.g. ST-395, ST-147 and ST-23-KL57) irrespective of geographical origins (Fig. 3 , Table S3 ). Contrasting with the breadth of diversity of resistance plasmids, only 7 of the 28 pC carried plasmids with virulence biomarkers and over 80% grouped in either pC4 or pC2 (Fig. 2 D). Cluster pC4 was strongly enriched (50% vs 0.001% in other pCs, p < .01) for plasmids carrying all 5 virulence biomarkers (Fig. 2 D), including the canonical, non-conjugative, pLVPK plasmid associated with phenotypic hypervirulence. Unlike plasmids found in pC4, pC2 plasmids rarely carried the complete set of virulence biomarkers (1%) and were more frequently associated with the carriage of only iuc and rmpA2 (23%). In agreement with this population-level analysis, 68% of virulence gene-carrying plasmids characterized in this study belonged pC-4 and pC-2 (Fig. 3 , Table S3, Fig. S4) . Plasmids pC-4, characterized by an IncFIB(K) or dual IncFIB(K)/IncHI1B replicon, were almost exclusively found in hvKp lineages (e.g. ST-23-KL1, ST-592, ST-420 and ST-86) irrespective of geographical origins (Fig. 3 , Table S5 ). A single exception was the detection of pC-4 plasmids in isolates from MDR-cKp ST-23-KL57 from Georgia and Ukraine, which carried an incomplete set of virulence biomarkers (Fig. 3 , Table S3 ). Finally, only 6 (3.5%) plasmids from this study were hybrid and carried both virulence and AMR (ESBL or carbapenemase) determinants. Three belonged to pC-2 followed by pC-33 (n = 2) and pC-4 (n = 1) (Fig. 3 , Table S3 ) and were mainly associated with MDR-cKp lineages (n = 2 ST-395 isolates from Georgia, and one each from ST-14, -37, and − 218 from Jordan, Thailand, and Kenya, respectively). The only exception was the pC-4 plasmid with a complete set of virulence biomarkers and an acquired CTX-M ESBL in the hvKp ST-23-KL1 lineage. Nevertheless, the pC2 cluster was the main source of overlap for virulence and ESBL/carbapenemase resistance genes ( Fig. 2CD ). Convergence is largely driven by the acquisition of conjugative pC2 IncFIB(Mar)/IncHI1B plasmids globally. Besides diversity, to quantify whether convergence resulted from preferred evolutionary routes, the complete set of 1,468 MDR-Kp isolates was analyzed to identify those that originated from shared or independent convergence events. Using maximum parsimony for the presence/absence of virulence and resistance alleles, and the tree topology, 25 independent convergence events were inferred ( Fig. S5 ). These, hereby labeled convergence events A-Y, largely correlated with STs ( i.e. one independent event per lineage) except within MDR-cKp lineages ST-395 and ST-23-KL57 for which convergent isolates were predicted to have emerged from distinct (n = 3 and n = 2, respectively) evolutionary occurrences (Fig. 3 , Table S6 ). At the gene level, carbapenemase and ESBL genes, were equally represented: 12/25 convergent events (48%) acquired carbapenemase genes (NDM-1, NDM-5, or OXA-48) and were non-susceptible to all carbapenems tested while isolates representing the remaining 13 events (52%) all carried an ESBL (CTX-M-15, CTX-M-55, CTX-M-63, SHV-2A) and were non-susceptible to all 3rd and 4th generation cephalosporins. By contrast, the acquisition of an incomplete set of virulence biomarkers was strongly favored (20/25 routes or 80%, p = 0.00012) and only 5/25 convergence events involved acquiring all 5 biomarkers, most predictive of the hvKp phenotype ( 15 ) ( Table S6 ). At the lineage level, am enrichment of convergence events was observed via the acquisition of virulence genes in an MDR-cKp background (events E-Y representing 21/25 routes or 84%). Combined with the plasmid data, the acquisition within a MDR-cKp lineage of a pC2 IncFIB(Mar)/IncHI1B or a pC4 IncFIB(K)/IncHI1B plasmid with an incomplete set of virulence biomarkers were the preferred routes of convergence (8/25 and 2/25, respectively, for a total of 40% of all independent convergent events) in the studied population (Fig. 3 ). By contrast, only 16% of the convergence events (labeled A to D: a ST-23-KL1 from the U.S., and three ST-268, ST-68, ST-420 isolates from Thailand) were due to the acquisition of an ESBL gene (either inserted within the canonical pC-4 virulence plasmid [ Fig. S6 ] or harbored by a standalone plasmid) in isolates from recognized hvKp lineages (Fig. 3 and Table S6 ). Recurring acquisition of genes that encode a functional aerobactin is the common denominator to global and regional emergence of convergent K. pneumoniae . Despite convergence largely (84%) resulting from the acquisition of an incomplete set of virulence biomarkers, it was noted that the aerobactin iuc siderophore loci was acquired in all 25 independent events detected globally (Fig. 3 , Table S6 ). It was most often (11/25) acquired by the gain of a conjugative pC2 IncFIB(Mar)/IncHI1B plasmid but a large diversity of plasmids from other pCs or singletons carried iuc and accounted for the remaining 14 convergence events. This recurring acquisition of iuc -carrying plasmids, albeit observed across MDR-cKp lineages, is best exemplified when reconstructing the evolution of ST-395. Within our sampling of this lineage, 3 independent convergent events (H, I and P) were inferred ( Fig. S5 ) and Bayesian phylogenetics dated their origin within the last decade (Fig. 4 ) . The proposed routes of convergence were reconstructed: routes P and H shared a most recent common ancestor predicted to carry a pC-38 (IncR) plasmid harboring a CTX-M-15 ESBL. From there, the routes diverged and route P involved a stepwise acquisition (between 2016 and 2021) of a 177 kb singleton plasmid carrying the iuc3 aerobactin locus (node 1 ), a pC2 (IncFIB(Mar)/IncHI1B) carrying the NDM-5 carbapenemase (node 2 ), and a transposon harboring the 16S methyltransferase rmtB which inserted into the pC-38 backbone (node 3 ) (Fig. 4 ) . Unlike route P, the stepwise acquisition through route H could not be resolved but this convergent event involved the acquisition of a pC-10 (IncL/M) plasmid co-harboring an OXA-48 carbapenemase and the armA 16S methyltransferase, and a pC-2 (IncFIB(Mar)/IncHI1B) hybrid plasmid carrying 4 virulence biomarkers, including iuc1 , and the NDM-1 carbapenemase (node 4 ). Finally, route I involved the independent acquisition of 3 plasmids (node 5 ): a pC-2 (IncFIB(Mar)/IncHI1B) only carrying two virulence biomarker ( iuc1 and rmpA2 ), a pC-42 (IncFIB(K)/IncFII(K)) harboring a CTX-M-15 ESBL, and a pC-10 (IncL/M) plasmid with OXA-48 and armA like that observed in route H (Fig. 4 ). Phenotypically, representative isolates from each of the ST-395 convergent routes (H, I and P) resulted in extensively drug-resistant isolates with non-susceptibility to all cephalosporins, all carbapenems, and all clinically relevant aminoglycosides ( Table S6 ). Notably, all showed increased siderophore production compared to control isolate cKp1 lacking iuc (p 1x10 7 in the outbred CD1 SQ challenge model [15]), unlike the control hypervirulent isolate kvKp2 (Fig. 5 B). Increased siderophore production, not hypervirulence, is the key adaptive trait driving global convergence in K. pneumoniae . Phenotypic characterization of representative isolates from all 25 independent convergence events showed that, irrespective of lineage, country of origin, or plasmid background, all exhibited increased siderophore production relative to cKp1 control (Fig. 5 A). The only exception was route Y, a ST-25 isolate from Georgia harboring iuc3 on a pC23 plasmid. Across convergence routes, isolates that acquired an incomplete set of virulence genes on a pC2 plasmid (routes G-N) produced siderophore levels comparable to isolates harboring the canonical pC4 virulence plasmid (routes A-F; p > 0.05) (Fig. 5 A C ), a pattern associated with the iuc1 aerobactin allele ( Table S6 ). By contrast, isolates that acquired other virulence plasmid types (routes O-Y), most frequently encoding iuc3 , displayed significantly lower, though still elevated, siderophore production (p < 0.001) (Fig. 5 A C ). Despite this consistent enhancement of iron-scavenging capacity, all convergent MDR-cKp isolates carrying an incomplete set of virulence biomarkers, whether on pC2 or other plasmids (routes G to Y), retained high LD₅₀ values and uniformly lacked the hypervirulent phenotype in the murine subcutaneous infection model ( Fig. 5BD ). In contrast, only hvKp convergent isolates that acquired ESBL or carbapenemase genes, and that maintained the full complement of five virulence biomarkers on a pC4 plasmid (routes A-D), exhibited low LD₅₀ values (LD 50 ≤1x10 7 ) consistent with true hypervirulence. Although rare, only these events result in bona fide MDR-hvKp ( Fig. 5BD ). Discussion The increasing detection of K. pneumoniae isolates that harbor AMR and virulence determinants has raised urgent concerns for human health ( 23 ). Here, we combined evolutionary, functional genomic, and biologic analyses of a global collection of K. pneumoniae to show that plasmid-mediated convergence is frequent but follows preferred evolutionary paths dominated by MDR-cKp lineages acquiring IncFIB(Mar)/IncHI1B (pC2) conjugative plasmids carrying an incomplete set of virulence biomarkers, rather than by hvkp lineages acquiring resistance plasmids. Further, these convergent isolates showed increased siderophore production, due to increased aerobactin production ( 32 ), but lacked in vivo hypervirulence emphasizing a fundamental distinction between genomic and phenotypic convergence. At the lineage level, 25 independent convergent events distributed across 22 sequence types were identified, highlighting the recurring and polyphyletic nature of convergence. The majority resulted from MDR-cKp lineages acquiring virulence genes rather than hvKp acquiring AMR genes. This asymmetry mirrors observations from other studies ( 29 ) and may be explained in that hvKp lineages rarely acquire resistance plasmids due to low conjugation efficiency because of their unique capsule and hypermucoid properties ( 33 ) and perhaps restriction systems ( 34 ). Without these physiological constraints, MDR-cKp are a more permissive recipient, as illustrated by the three genomic convergence events inferred within a single lineage, ST-395, which independently evolved through the last decade alone. These spawned the emergence of NDM and/or OXA-48 producing epidemic subclones which have seemingly outpaced the KPC-producing ST-258 and are now predominant in Georgia and other countries in Europe ( 35 , 36 ). At the plasmid level, our data indicates that convergence is facilitated by a subset of broad-host-range plasmids combining conjugation machinery with partial virulence cargo. Specifically, IncFIB(Mar)/IncHI1B plasmids (plasmid cluster pC2) represented the most common vector for virulence biomarker gene acquisition, albeit an incomplete set, accounting for nearly half of all independent convergence events. Consistent with their non-conjugative nature, the alternative IncFIB(K)/IncHI1B plasmids (pC4), which includes the canonical pLVPK and pK2044 with a complete set of 5 virulence biomarkers, remained primarily confined to hvKp backgrounds ( 5 , 20 ). Further supporting this divide, plasmidome population analysis revealed that resistance and virulence plasmids occupy distinct evolutionary spaces, with limited recombinational events. As such, hybrid plasmids carrying both AMR and virulence loci were rare and largely confined to pC2, confirming previous observations that co-resident plasmids remain the predominant molecular mechanism for genomic convergence ( 29 , 37 ). At the gene level, the aerobactin synthesis locus emerged as the single consistent signature of genomic convergence. All 25 independent events involved the acquisition of iuc and phenotypic assays demonstrated all exhibited increased siderophore production, albeit more pronounced in iuc1 -carrying isolates. The iuc1 variant, typically found on pLVPK-like IncFIB(K)/IncHI1B pC4 plasmids from hvKp strains, was most common, followed by iuc3 more frequently associated with IncFIB(Mar)/IncFII pC2 plasmids, which have been previously associated with strains from animal and environmental sources ( 38 ). The consistent selection of iuc underscores the ecological advantage conferred by enhanced siderophore production ( 32 ). Recent studies have shown that aerobactin provides an ecological advantage and enhances mucosal fitness and epithelial adherence independent of its ability to enhance systemic virulence ( 39 ). Further, increased iron acquisition capacity may improve host colonization as well as persistence in iron-limited environments such as infected tissues and hospital surfaces. These same properties could indirectly promote transmission between patients, suggesting that siderophore-mediated iron acquisition may represent a key adaptive trait underlying the epidemic success of genomic convergent lineages like ST-147 and ST-395 in hospitals worldwide. At the phenotypic level, assays in the murine subcutaneous model reinforce this separation between genomic convergence and pathogenic potential. Adding to our previous studies ( 12 , 27 ) and that of Kochan and colleagues ( 29 ), none of the convergent MDR lineages with an acquired pC2 plasmid harboring an incomplete set of virulence biomarkers displayed systemic lethality characteristic of hvKp, but instead possessed a cKp virulence phenotype. While some confusion originally persisted, it is clear now that genomic convergence resulting in the acquisition of some, but not all, of the virulence biomarkers does not equate to a hypervirulent phenotype but rather defines a new, stable ecological state within the nosocomial setting. Conversely, confirmed hvKp lineages that acquired ESBL genes ( e.g ., ST-23-KL1 with CTX-M-15) retained a pC4 plasmid and full hypervirulence, indicating that convergence in an hvKp background can yield clinically high-risk isolates, albeit less frequently. Although imperfect, for diagnostic purposes, such hypervirulent, drug-resistant isolates are currently best identified by their carriage of a complete set of 5 virulence biomarkers ( 15 ). Unlike the epidemic MDR-cKp convergent lineages, the MDR-hvKp have not spread globally and, so far, have only been sporadically detected in Asia and Europe ( 37 , 40 ), a possible consequence of the metabolic burden of full hypervirulence ( 41 ). Collectively, our results establish a framework for understanding convergence in K. pneumoniae . Genomic convergence is frequent, structured, and dominated by MDR-cKp backgrounds acquiring iuc -carrying plasmids, but it rarely leads to the hypervirulent phenotype. These convergent strains represent an intermediate evolutionary state between cKp and hvKp, optimized for transmission rather than invasion. Understanding the molecular basis and ecological advantages of this intermediate state will be essential for anticipating the future evolution of hospital-adapted Klebsiella and refining surveillance strategies to distinguish true hypervirulence from adaptive convergence. Materials and Methods Bacterial Isolates Klebsiella pneumoniae isolates were collected from Military Health System facilities across the United States and globally in collaboration with the US Department of Defense’s (DoD) Global Emerging Infections Surveillance branch. The 1,468 global isolates were recovered from Thailand (n = 362), Georgia (n = 359), Peru (n = 305), Jordan (n = 215), Kenya (n = 159), Uganda (n = 36), Philippines (n = 25), and Ukraine (n = 7) and were compared to isolates from the United States (n = 39) ( Table S1 ). Whole Genome Sequencing Genomic DNA extraction and Illumina short read WGS were performed as previously described ( 42 ). Libraries were constructed using Kapa HyperPlus Library Preparation kit (Roche Diagnostics) and quantified using the KAPA Library Quantification Kit - Illumina/Bio-Rad iCycler™ (Roche Diagnostics) on a CFX96 real-time cycler (Bio-Rad). Libraries were normalized to 2 nM, pooled, denatured, and diluted to 1 nM. Whole genome sequencing was performed using a MiSeq or NextSeq-500 (Illumina) with MiSeq Reagent Kit v3 (600 cycles; 2 X 300 bp) or NextSeq-500 Reagent kit 500/550 vs (300 cycles, 2 x 150 bp) (Illumina). De novo draft genome assemblies were produced using Newbler v2.7 (Roche Diagnostics). Minimum thresholds for contig size and coverage were set at 200 bp and 49.5+, respectively. Long read sequencing was performed on 53 isolates. 8 isolates were sequenced using single-molecule real-time (SMRT) by Pacific Biosciences RS II Instrument (Pacific Biosciences). Assembly was completed using the Hierarchical Genome Assembly Process (HGAP v3.0). The resulting contigs were imported into Geneious and circularized. Assemblies were polished using Illumina paired-end short reads. 45 isolates were sequenced on a Minion platform using MinION Mk1B (Oxford Nanopore Technologies). Library preparation on genomic DNA was principally performed using EXP-NBD114 and sequenced on a R9.4.1 flowcell. Basecalling was performed using Guppy v6.1.7 using the super accurate basecalling model (r9.4.1_450bps_sup). Prior to assembly, reads were filtered by Filtlong ( https://github.com/rrwick/Filtlong ) to capture the top 95% of reads by quality score. De novo assembly was performed using Trycycler [v0.5.3] ( 43 ). Trycycler outputs a consensus assembly from multiple long-read dedicated assemblers including Flye (v2.9.1), Miniasm (v0.3), and Raven (v1.8.1). Next, the trycylcer consensus assembly was polished with Illumina short reads using Medaka and Illumina short reads using Polypolish ( 44 ). Bioinformatic analysis. Species identification, MLST typing, virulence locus, capsule (K), and lipopolysaccharide (O) loci were identified using Kleborate v3.0.5 ( Table S1 ) ( 17 ). peg-344 was identified using BLASTn search of draft genome assemblies (query sequence pLVPK, accession number AP006726.1). AMRFinderPlus v3.9.8 ( 45 ) and ARIBA v2.14.4 ( 46 ) were used to identify resistance alleles from draft assemblies and processed reads, respectively, followed by deduplication of redundant alleles calls. clast allele assignment and minimum spanning tree generation were performed with SeqSphere ( 47 ). Plasmid replicons were identified using PlasmidFinder v2.1.6 ( 48 ). We created a core gene phylogeny for the 261 isolates harboring ≥ 1 hvKp gene ( Table S1 ). Assemblies were annotated using Prokka v1.14.6 ( 49 ) and were used as input for Roary v3.13.0 ( 50 ) and a SNP-based alignment was generated. Recombination was filtered from the alignments using Gubbins v2.4.1 ( 51 ) and a maximum-likelihood tree was generated with RAxML-NG [v1.1] ( 52 ) using the GTR + G (50 parsimony, 50 random) model 100 random bootstrap replicates. The tree was imported in iTOL [v6.8.1] ( 53 ) for visualization with metadata. Bayesian Evolutionary Phylogenetic Analysis To evaluate the strength of the temporal signal, TempEst v1.5.3 was utilized to visualize the relationship between root-to-tip genetic distances for samples with known collection dates ( 54 ). The bayesian phylogenetic inference was performed using BEAST2 v2.6.5 on a recombination free alignment, removing samples with uncertain collection dates, and accounting for constant sites with beast2_constsites ( https://github.com/andersgs/beast2_constsites ). The HKY substitution model was selected based on evaluation of all possible substitution models in bModelTest v1.2.1 ( 55 ). The random clock model was selected based on support by the marginal likelihood value using the Nested Sampling package v1.1.0 ( 56 ). BEAST2 was run under a coalescent constant population model, with a Markov chain Monte Carlo length of 1 x 10 8 sampling every 5 x 10 3 steps. Analyses were repeated 5 times to confirm consistency between the obtained posterior distributions. Parameter estimates were computed using Tracer v1.7.1. Posterior trees were combined with LogCombiner and summarized in TreeAnnotator after a 50% burn-in. The final MCC target tree was visualized in FigTree v1.4.4 ( https://github.com/rambaut/figtree ) and annotated using iTOL ( 53 ). Plasmidome Analysis mge-cluster plasmid typing [v1.1.0] ( 31 ) was used to investigate plasmid families present in our dataset compared to a global public collection. Mge-cluster is a unitig based classification and pairwise Jaccard distances from unitig presence/absence matrices. All default parameters were used except perplexity, which was set to 100 and unitig filtering set to 30% to deduplicate redundant sequences. First, 7,347 complete plasmid sequences from 2,153 isolates were retrieved on NCBI. Clustering analysis resulted in 43 distinct plasmid clusters that grouped 5,810 plasmid sequences (grey dots, Figure S3). 1,500 NCBI plasmid sequences were unassigned a cluster group and 399 plasmid sequences were excluded from clustering analysis by mge-cluster ( Fig. S3 ). Next, we incorporated our 173 plasmid sequences (recovered from the 53 isolates) into the plasmid cluster network from which 124 plasmids grouped with 28 of the NCBI clusters (44 plasmids were unassigned a cluster group (singletons) and 5 plasmids were excluded from the clustering analysis) ( Fig. 3AB ). The distribution of replicon types, STs, virulence genes, ESBL and/or carbapenemase genes were investigated and visualized for all plasmid sequences located in the 28 discrete clusters (n = 4,052) and the 44 singletons from this study (Fig. 2 , Table S5 ). Quantitative Siderophore Assay Strains were grown overnight at 37°C in iron-chelated M9 minimal medium containing casamino acids (c-M9-CA) ( 18 ) and culture supernatants were assessed using the chromeazurol S dye assay as described ( 32 ). Standards with concentrations of 0, 1.5, 3.1, 6.25, 12.5, 25, 50, and 100 µg/ml enabled quantitation. A minimum of 3 biological assays with 3 technical repeats were performed and the results were reported as the mean ± the SD. Mouse Subcutaneous (SQ) Infection Model Animal studies were reviewed and approved by the Veterans Administration Institutional Animal Care Committee and the University at Buffalo-SUNY and were carried out in strict accordance with the recommendations in the guidelines delineated in the "NIH Guide for the Care and Use of Laboratory Animals"(revised 1985) and the "Ethics of Animal Experimentation Statement" (Canadian Council on Animal Care, July, 1980) as monitored by the Institutional Animal Care and Use Committee. All efforts were made to minimize suffering. Veterinary care for the animals was supplied by the staff of Veterans Administration Animal Facility under the direction of a fully licensed veterinarian. CD1 male mice, 4–6 weeks old, were obtained from Charles River Laboratories, quarantined for 7 days before use, and then challenged via a SQ injection with the isolates of interest (100 µL of bacterial suspension serially diluted to the required titers in 1 x PBS diluted and injected using a 0.5 mL insulin syringe), as previously described ( 57 ). The animals were closely monitored for 14 days after challenge for the development of the study endpoints, survival, or severe illness (in extremis state)/death, which was recorded as a dichotomous variable. Signs that were monitored and which resulted in immediate euthanasia using methods consistent with the recommendations of the American Veterinary Medical Association Guidelines included hunched posture, ruffled fur, labored breathing, reluctance to move, photophobia, and dehydration. Statistical Analyses Desired comparisons between strains for experiments assessing siderophore production were made via ordinary one-way ANOVA, using Šidák’s multiple comparisons test (Prism 10.4.2 for MacIntosh, GraphPad Software Inc.). LD 50 values were estimated using a logistic regression model as described ( 15 ). Pair-wise comparisons of the dose-response curves were used to generate LD 50 values. Desired comparisons between LD 50 values were made by employing a blend of the empirical logit function along with least-squares regression incorporating strain and inoculum factors (CFU/mL) to derive p-values for comparing dose-response curves based on LS-means. Declarations Acknowledgments: The authors are thankful to all the staff of the MRSN and all the members of the Global Emerging Infections Surveillance (GEIS) network. The authors thank MAJ Hunter J. Smith for his leadership of the GEIS AMR portfolio and his assistance in coordinating communications between MRSN and the GEIS overseas partner laboratories. The authors thank Mohammad N. Alhawarat, Wiam Khraisat, and Mohammad J. Gharaibeh from the Ministry of Health of The Hashemite Kingdom of Jordan for their role in coordinating national MDR surveillance efforts. The manuscript has been reviewed by the Walter Reed Army Institute of Research and there is no objection to its presentation. The views expressed herein are those of the author(s) and do not necessarily reflect the official policy or position of the Defense Health Agency, the Department of Defense, nor any agencies under the U.S. Government. Funding: This study was funded by Defense Health Program (DHP) Operation & Maintenance (O&M). Partial funding was provided by the Armed Forces Health Surveillance Division (AFHSD), Global Emerging Infections Surveillance (GEIS) Branch project P0132_23_WR as well as global surveillance projects P0011_AF_25, P0119_18_KY_013, P152_20_KY_06, P0065_21_KY, P0037_22_KY, P0096_23_KY, P0166_22_N3, and P0126_23_N3. This work was also supported by NIH 1R21AI141826-01A1 (Dr. Russo) and the Department of Veterans Affairs VA Merit Review (I01 BX004677-01) (Dr. Russo). Data and materials availability: Individual genomic assemblies accession numbers are listed in Table S1 and genomes of all isolates analysed in this study are publicly available in the NCBI database under the BioProject number PRJNA1354878. Competing interests: The authors declare that they have no competing interests. Ethics approval and consent to participate: For U.S. isolates: the isolates and clinical information were collected as part of the public health surveillance activities of the MRSN, as determined by the WRAIR Commander in accordance with Institutional Review Board (IRB) Policy Memorandum #10; Public Health activity Determination and Oversight Requirements issued June 10th 2019. For Ukrainian samples: hospital permission to conduct the surveillance study was obtained from Hospital Bioethics Committee of MMCC CR, Ministry of Defense, Vinnitsa, Ukraine, protocol reference number 18/2 from 05 May 2014. The patients were included after understanding the study and had signed an informed consent. For Kenya, Uganda and Jordan isolates: the studies were undertaken with ethics approvals from the different country institutional review boards: Kenya, KEMRI SERU#2767/WRAIR #2089/ USAMRMC ORP HRPO) (Log#A-18129); Uganda, MUSPH HDREC #087/UNCST#HS775/WRAIR (#1711); and Jordan, NAMRU3.PJT.2011.0014. 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Supplementary Files TableS3plasmidcontigs.xlsx Table S3 TableS528clustersn4052.xlsx Table S5 TableS4AllNCBIContigsn7347.xlsx Table S4 TableS6ConvergenteventsandPhenotypes.xlsx Table S6 TableS2cgMLSTdistance.xlsx Table S2 TableS1allisolates.xlsx Table S1 SupplementalFiguresandTables.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Research-Africa","correspondingAuthor":false,"prefix":"","firstName":"Lillian","middleName":"","lastName":"Musila","suffix":""},{"id":587139238,"identity":"76674d66-ba49-4207-a019-a45d1d8fefb3","order_by":19,"name":"Paul Rios","email":"","orcid":"","institution":"US Naval Medical Research Unit SOUTH","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Rios","suffix":""},{"id":587139239,"identity":"c14e9864-2064-4ff5-aa32-81471553ecd5","order_by":20,"name":"John Mark Velasco","email":"","orcid":"https://orcid.org/0000-0002-9397-8205","institution":"University of the Philippines Manila","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"Mark","lastName":"Velasco","suffix":""},{"id":587139240,"identity":"b931b4ee-fe3d-40cf-9fe0-0969f36d56d9","order_by":21,"name":"Nattaya Ruamsapp","email":"","orcid":"","institution":"Armed Forces Research Institute of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Nattaya","middleName":"","lastName":"Ruamsapp","suffix":""},{"id":587139241,"identity":"264b382e-2de2-4649-a4ad-c6fcb34092d8","order_by":22,"name":"Jason Bennett","email":"","orcid":"https://orcid.org/0009-0002-8306-3594","institution":"Multidrug-Resistant Organism Repository and Surveillance Network (MRSN), Walter Reed Army Institute of Research, Silver Spring, Maryland, USA.","correspondingAuthor":false,"prefix":"","firstName":"Jason","middleName":"","lastName":"Bennett","suffix":""},{"id":587139242,"identity":"981be1b0-b6ce-4559-9a1c-c94a2ff8c1ff","order_by":23,"name":"Patrick Mc Gann","email":"","orcid":"","institution":"Multidrug-Resistant Organism Repository and Surveillance Network (MRSN), Walter Reed Army Institute of Research, Silver Spring, Maryland, USA.","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"Mc","lastName":"Gann","suffix":""},{"id":587139243,"identity":"5f94b9ea-8098-4cc8-903e-79cab79f6d20","order_by":24,"name":"Thomas Russo","email":"","orcid":"https://orcid.org/0000-0003-4566-7442","institution":"Buffalo","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Russo","suffix":""},{"id":587139218,"identity":"1d9cecdf-064f-4f18-915d-a6cbdfa21fb9","order_by":25,"name":"Francois Lebreton","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-7157-5026","institution":"Walter Reed Army Institute of Research","correspondingAuthor":true,"prefix":"","firstName":"Francois","middleName":"","lastName":"Lebreton","suffix":""}],"badges":[],"createdAt":"2026-01-15 20:05:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8613390/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8613390/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102747936,"identity":"8bdaabcf-f669-4388-899c-858f3aba5070","added_by":"auto","created_at":"2026-02-16 09:05:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2106113,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssociation of antibiotic resistance and virulence genes with global MDR-Kp lineages.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Maximum likelihood phylogenetic tree for 261 isolates carrying at least one acquired virulence gene (\u003cem\u003eiuc, iro, rmpADC, rmpA2, peg-344\u003c/em\u003e). Datasets for ST, capsule KL type, acquired virulence genes (gray), and resistance genes (red) are indicated. Isolates selected for Oxford Nanopore long read sequencing are indicated (blue triangles). (\u003cstrong\u003eB\u003c/strong\u003e) Frequency of isolates stratified by virulence gene carriage (1 to 5) against country of origin, top sequence types, ESBL carriage, and carbapenemase carriage.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8613390/v1/a0ca8b027c5667d1a6cf779f.png"},{"id":102562476,"identity":"e9b40dcd-dfb5-4219-a5bf-9cc264eb3ee4","added_by":"auto","created_at":"2026-02-13 04:41:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2426565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlasmid population cluster analysis\u003c/strong\u003e. \u003cstrong\u003e(A)\u003c/strong\u003e All MRSN plasmids (n=168) (outlined in black) where compared to plasmids from GenBank samples (no fill). pC assignments are indicated and bolded for clusters containing MRSN contigs.\u003cstrong\u003e(B)\u003c/strong\u003e Bar graph depicting the number of MRSN and GenBank plasmids in each pC. \u003cstrong\u003e(C)\u003c/strong\u003e MRSN and GenBank plasmids colored by virulence gene carriage. \u003cstrong\u003e(D)\u003c/strong\u003eMRSN and GenBank plasmids colored by resistance gene carriage.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8613390/v1/3b1ffa3fb74aa092f7dc96bd.png"},{"id":102747680,"identity":"ab8c78ff-5d9e-4c4a-a5fe-933639b8e81e","added_by":"auto","created_at":"2026-02-16 09:05:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":856048,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeatmap of plasmid diversity for convergent isolates. \u003c/strong\u003eConvergent isolates are shown with their corresponding metadata including genotypic classification, country of origin, ST, and replicon fingerprint. Each contig from each sample are categorized by their numerical MGE cluster assignment, or otherwise indicated as chromosomal, and further categorized by virulence gene carriage (blue) or resistance gene carriage (red). KL type (ST-KL), country of origin, virulence and resistance gene cargo, and replicon fingerprint of each closed plasmid from each sample. Numbers denoted in the filled virulence and resistance boxes or replicon fingerprint circles indicate the plasmid type of the harboring plasmid. Plasmids colored in blue represent virulence-carrying plasmids, yellow represent resistance-carrying plasmids, and pink represent both virulence and resistance carrying plasmids (convergent). Chromosomal virulence and resistance gene carriage are designated by blue and yellow circles, respectively.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8613390/v1/73c71161e2823ac233e960e6.png"},{"id":102562484,"identity":"0c063960-1e18-44bc-9a0c-b4df97075881","added_by":"auto","created_at":"2026-02-13 04:41:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2969776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThree proposed routes of convergence in lineage ST-395. \u003c/strong\u003eThree routes (P, H and I) of convergence are inferred based on plasmid content, parsimony, and Bayesian Evolutionary Phylogenetic Analysis (BEAST) of ST-395 \u003cem\u003eK. pneumoniae\u003c/em\u003e sampled from Georgia, between 2017-2021. Key nodes/events are labelled 1-5.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8613390/v1/b3445b90de812328631edf62.png"},{"id":102562481,"identity":"8ecf6485-2962-4209-9ef6-7ad040f70763","added_by":"auto","created_at":"2026-02-13 04:41:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1347859,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic consequences of convergence.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Quantification of siderophore production. Bars represent mean values from ≥3 biological replicates; error bars denote s.d. \u003cstrong\u003e(B)\u003c/strong\u003e LD₅₀ values (log₁₀ CFU) obtained using a murine subcutaneous infection model. See Table S6 for challenge inocula and animal number for each strain. Data for representative isolates for each routes of convergence (labeled A to Y) are indicated and compared to control cKp1 (lacking all virulence biomarkers) and hvKp2 (carrying all 5 biomarkers on a pC4 plasmid) strains. \u003cstrong\u003e(C-D) \u003c/strong\u003eSiderophore production and LD₅₀ values stratified by plasmid cluster carrying the virulence genes (pC4, pC2, or other \u003cem\u003eiuc\u003c/em\u003e-carrying plasmids).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8613390/v1/7cde562dccc969cccea0b82e.png"},{"id":104834924,"identity":"253c3f83-996b-43dd-a38e-84a54f72af7b","added_by":"auto","created_at":"2026-03-17 17:36:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9970294,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8613390/v1/55888304-ce8c-4fdc-b59e-404e06fb518c.pdf"},{"id":102562475,"identity":"49e43dcf-3b6f-42b3-a8c8-4a34f94fe8d1","added_by":"auto","created_at":"2026-02-13 04:41:52","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":36786,"visible":true,"origin":"","legend":"Table S3","description":"","filename":"TableS3plasmidcontigs.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8613390/v1/d82c48cd2b710677350df7d9.xlsx"},{"id":102746899,"identity":"1f818689-838d-4c92-b658-99790e9d0e6e","added_by":"auto","created_at":"2026-02-16 09:02:49","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":894317,"visible":true,"origin":"","legend":"Table S5","description":"","filename":"TableS528clustersn4052.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8613390/v1/fe9dd84e6c0a37b8b0bfcc1b.xlsx"},{"id":102562479,"identity":"25a8cebf-7508-4218-bf01-45a6ea2b5f5c","added_by":"auto","created_at":"2026-02-13 04:41:52","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1578883,"visible":true,"origin":"","legend":"Table S4","description":"","filename":"TableS4AllNCBIContigsn7347.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8613390/v1/ef092ef42b3195c8db0e8ea7.xlsx"},{"id":102747853,"identity":"b7c337ab-9125-465a-8f30-26e9d4eda2dd","added_by":"auto","created_at":"2026-02-16 09:05:30","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":20345,"visible":true,"origin":"","legend":"Table S6","description":"","filename":"TableS6ConvergenteventsandPhenotypes.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8613390/v1/73835f48fabad62cf0cb6494.xlsx"},{"id":102746768,"identity":"4d14e160-4f2e-488a-8194-0f9965b809f1","added_by":"auto","created_at":"2026-02-16 09:01:11","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3821439,"visible":true,"origin":"","legend":"Table S2","description":"","filename":"TableS2cgMLSTdistance.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8613390/v1/f3fc1614ca43d619e43f0091.xlsx"},{"id":102562483,"identity":"94eb2471-b3fe-4d7a-a5d4-e492c12e2b9e","added_by":"auto","created_at":"2026-02-13 04:41:52","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":652780,"visible":true,"origin":"","legend":"Table S1","description":"","filename":"TableS1allisolates.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8613390/v1/105fe3029a70db7d516a9ad1.xlsx"},{"id":102562485,"identity":"abe4cc1b-4daa-4d8e-86ae-6def1c6d688d","added_by":"auto","created_at":"2026-02-13 04:41:52","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":5514619,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFiguresandTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8613390/v1/c49b930db9cf1fa464a58ccb.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Preferred evolutionary routes of convergence in Klebsiella pneumoniae favor siderophore acquisition over hypervirulence","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e are ubiquitous bacteria and the causative agent of diverse infections including pneumonia, urinary tract, surgical site, and bloodstream infections (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Classical \u003cem\u003eK. pneumoniae\u003c/em\u003e (cKp) with acquired resistance determinants are one of the world\u0026rsquo;s leading causes of healthcare-associated infections (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Notably, third-generation cephalosporin-resistant and carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e are listed by the World Health Organization as urgently needing novel therapeutics (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Antimicrobial resistance (AMR) genes, including extended-spectrum β-lactamases (ESBLs) and carbapenemases, are readily acquired by conjugative plasmids and mobile genetic elements (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e were initially associated with globally disseminated clone ST-258, encoding KPC enzymes found on conjugative IncFII(K)/IncFIB(pQil) plasmids (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), however, other lineages (\u003cem\u003ee.g.\u003c/em\u003e ST-307, ST-147, ST-395) have recently emerged internationally carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e, and/or \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;48\u003c/sub\u003e genes on diverse plasmids (\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA distinct pathotype from cKp, hypervirulent \u003cem\u003eK. pneumoniae\u003c/em\u003e (hvKp) cause invasive community-acquired infections linked to high mortality, driven by metastatic spread, despite generally remaining amenable to antimicrobial treatment (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). hvKp infection outcomes are associated with five hvKp-specific virulence plasmid-encoded genes for siderophores aerobactin (\u003cem\u003eiuc\u003c/em\u003e) and salmochelin (\u003cem\u003eiro\u003c/em\u003e), mucoid and capsule polysaccharide regulators (\u003cem\u003ermpADC\u003c/em\u003e and \u003cem\u003ermpA2\u003c/em\u003e), and the metabolite transporter (\u003cem\u003epeg-344\u003c/em\u003e) (\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Genetically distinct from cKp, hvKp clones have lower pan genome diversity, lower capsule type diversity (most commonly KL1 or KL2), and constitute fewer lineages (e.g., ST-23, ST-268, ST-65, ST-420), which are most prevalent in the Asia Pacific Rim (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Conventionally, hvKp harbor non-conjugative virulence plasmids, mainly the described pLVPK or pK2044 with IncHI1B/IncFIB dual replicon (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) or the rarer KP52.145pII plasmid with IncFIB replicon (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). However, acquired virulence genes are also found on chromosomal integrative conjugative elements (e.g. ICEKp10) and conjugative plasmids (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile historically rare, the overlap of \u003cem\u003eK. pneumoniae\u003c/em\u003e strains carrying both hvKp-specific virulence genes and carbapenemase and/or ESBL genes have been increasingly reported within the last decade (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The genetic characteristics of reported convergent isolates include hvKp lineages (e.g. ST-23-KL1) acquiring conjugative resistance plasmids carrying AMR genes (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), or MDR-cKp lineages harboring pLVPK-like plasmids (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Other evolutionary dynamics that have led to genomic convergence includes hybrid plasmids of the IncFIB(Mar)/IncHI1B dual replicon type carrying resistance (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e, \u003cem\u003earmA\u003c/em\u003e) and hvKp-specific virulence (\u003cem\u003eiuc\u003c/em\u003e, \u003cem\u003ermpADC\u003c/em\u003e, \u003cem\u003ermpA2\u003c/em\u003e, and/or \u003cem\u003epeg-344\u003c/em\u003e) biomarkers that are found in globally epidemic nosocomial MDR lineages such as ST-307 and ST-147 (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough some confusion remains, because of genetic-only inferences or inadequate virulence models (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), genomic convergence is not synonymous with phenotypic hypervirulence (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In this study we determine that the phenotypic outcomes of convergence in \u003cem\u003eK. pneumoniae\u003c/em\u003e are lineage specific and the result of distinct evolutionary routes of plasmids acquisition. Using a unique global dataset of 1,468 multidrug-resistant isolates, and a population plasmidomic analysis of 7,347 complete plasmids (from strains spanning over 47 countries and the last two decades), we describe that convergence follows preferred paths, constrained by the plasmid biology, and that enhanced aerobactin-mediated iron acquisition, not hypervirulence, is the main adaptive trait driving the success of convergent lineages in hospital settings.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePrevalence of MDR lineages in a global collection of\u003c/b\u003e \u003cb\u003eKlebsiella pneumoniae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGlobal surveillance of multi-drug resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e (MDR-Kp) resulted in 1,468 clinical isolates recovered from 8 countries on 5 continents, between 2011 and 2021 (\u003cb\u003eFig. S1, Table S1\u003c/b\u003e). The frequency of isolate collection varied by year and country. \u003cem\u003eIn silico\u003c/em\u003e typing revealed a diverse population comprising 208 sequence types (ST) and 87 capsule types (KL) (\u003cb\u003eFig. S1\u003c/b\u003e, \u003cb\u003eTable S1\u003c/b\u003e). Nevertheless, 8 lineages were represented by \u0026gt;\u0026thinsp;50 isolates and accounted for 57% (n\u0026thinsp;=\u0026thinsp;834) of the population. ST-395 and ST-16 were the most prevalent (12% and 11%) and respectively grouped isolates that largely originated from the Republic of Georgia (99%) and Thailand (84%). In contrast, other major lineages (e.g. ST-15 and ST-147) were collected from multiple countries. Within those substantial genetic diversity was observed, with most isolates differing by \u0026gt;\u0026thinsp;100 allelic differences by core genome MLST (\u003cb\u003eFig. S2, Table S2\u003c/b\u003e). By contrast high genetic relatedness (\u0026le;\u0026thinsp;10 allelic differences) was observed in geographically clustered subsets, including ST-395 from Georgia, ST-16 from Thailand, and subsets of ST-147 isolates from Thailand and Peru, consistent with local clonal expansion of emerging high-risk lineages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAssociation of antibiotic resistance and virulence genes with global MDR-Kp lineages\u003c/h2\u003e \u003cp\u003eFor the resistance genes: in this collection, the rate of ESBL carriage remained remarkably stable, between 78 and 82% annually, and gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;15\u003c/sub\u003e (74% of ESBL isolates) was the most represented (\u003cb\u003eFig. S1, Table S1)\u003c/b\u003e. By contrast, the rate of carbapenemase-producing isolates increased markedly over time, from 6% of MDR-Kp isolates sampled between 2011\u0026ndash;2016 to 44% (chi-square 175.5 and p \u0026lt; .01) between 2017\u0026ndash;2021. This largely correlated with the sampling of clonal, geographically clustered isolates (\u003cb\u003eFig. S1, Table S1\u003c/b\u003e). Carabapenemase \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e genes were the most represented, alone (55%) or in combination with \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;48\u003c/sub\u003e (22%).\u003c/p\u003e \u003cp\u003eFor the virulence genes: 222 isolates (15%) carried at least one virulence biomarker (\u003cem\u003eiuc, iro\u003c/em\u003e, \u003cem\u003epeg-344\u003c/em\u003e, \u003cem\u003ermpADC\u003c/em\u003e, and/or \u003cem\u003ermpA2\u003c/em\u003e). By contrast, only 1% of MDR-Kp isolates collected from the U.S. military health system during this same time period carried an acquired virulence gene (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Core genome phylogenetic analysis of the global virulence-carrying collection (n\u0026thinsp;=\u0026thinsp;222) compared to virulence-carrying US isolates (n\u0026thinsp;=\u0026thinsp;39, including 22 canonical hypervirulent ST-23 isolates) identified 32 distinct virulence gene-carrying lineages including ST-395 (n\u0026thinsp;=\u0026thinsp;135), ST-23-KL1 (n\u0026thinsp;=\u0026thinsp;29), ST-15 (n\u0026thinsp;=\u0026thinsp;13), ST-23-KL57 (n\u0026thinsp;=\u0026thinsp;13) and ST-2071 (n\u0026thinsp;=\u0026thinsp;10) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The prevalence of the 5 acquired virulence biomarkers was variable; all 261 isolates carried \u003cem\u003eiuc\u003c/em\u003e siderophore loci, represented by four \u003cem\u003eiuc\u003c/em\u003e alleles including the hvKp-associated \u003cem\u003eiuc1\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;135), followed by \u003cem\u003eiuc3\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;107), \u003cem\u003eiuc5\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;17), and \u003cem\u003eiuc2\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eTable S1\u003c/b\u003e). By contrast, the \u003cem\u003eiro\u003c/em\u003e siderophore loci was only found in 23% (n\u0026thinsp;=\u0026thinsp;60) of isolates, with the hvkp ST-23-KL1 isolates accounting for half. Most isolates carried only 1 biomarker (n\u0026thinsp;=\u0026thinsp;145), mainly represented by lineage ST-395 from Georgia. Only 49 isolates carried all five biomarkers, predictive of the hvKp phenotype (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Within those, ST-23-KL-1 was the most represented (n\u0026thinsp;=\u0026thinsp;25) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eTable S1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIncreased prevalence of convergent isolates in recent years.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eConvergence, defined by the co-carriage of an ESBL and/or carbapenemase gene in addition to \u0026ge;\u0026thinsp;1 virulence biomarker (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), was detected for 198 (76%) of the virulence carrying isolates and was significantly enriched (chi-square 27.7 and p \u0026lt; .01) in the last 5 years of sampling (\u003cb\u003eTable S1\u003c/b\u003e). Only 7 convergent isolates carried all 5 virulence loci. None of these 7 isolates carried a carbapenemase gene, instead all acquired an ESBL, including ST-420 (n\u0026thinsp;=\u0026thinsp;2) and\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eST-268 (n\u0026thinsp;=\u0026thinsp;1) from Thailand, ST-218 (n\u0026thinsp;=\u0026thinsp;2) from Kenya, and ST-23-KL-1 (n\u0026thinsp;=\u0026thinsp;1) from the United States (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eTable S1\u003c/b\u003e). By contrast, 80% of the isolates with 1\u0026ndash;4 virulence loci co-carried a carbapenemase gene. Isolates from Georgia were the most represented (89%) and two lineages were predominant: ST-395 (n\u0026thinsp;=\u0026thinsp;111) and ST-23-KL57 (n\u0026thinsp;=\u0026thinsp;13). Within each, differences in patterns of AMR and virulence genes were suggestive of distinct plasmid acquisition/loss events (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePopulation analysis of virulence, resistance, or virulence/resistance carrying plasmids.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eComplete genomes were obtained for 34 representative convergent isolates and 19 virulence-carrying isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, blue triangles). Assemblies revealed 173 plasmid sequences, including 172 circular and 1 linear sequence, with between 1 and 7 plasmids identified per isolate (\u003cb\u003eTable S3)\u003c/b\u003e. Replicons were typed and, using a cluster-based approach (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), all study (MRSN) plasmid sequences were compared to each other and to a publicly available set of 7,347 complete \u003cem\u003eK. pneumoniae\u003c/em\u003e plasmids (from genomes in NCBI, representing 306 STs from at least 47 countries throughout the last two decades) (\u003cb\u003eFig. S3, Table S4)\u003c/b\u003e. The \u003cem\u003eK. pneumoniae\u003c/em\u003e plasmidome stratified into 43 discrete plasmid clusters (pC) (\u003cb\u003eFig. S.3\u003c/b\u003e). Within those, the study plasmids (n\u0026thinsp;=\u0026thinsp;124) clustered in 28 pCs with 4,052 NCBI plasmids sequences (\u003cb\u003eFig.\u0026nbsp;2AB, Table S5\u003c/b\u003e). The remaining 49 MRSN plasmid sequences were singletons/unassigned.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWithin the 28 pCs comprising at least one study plasmid, 22 grouped plasmids with an ESBL and/or carbapenemase gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Cluster pC10, the most numerous, was very homogeneous and 82% of its plasmids carried an OXA-48 carbapenemase (\u003cb\u003eTable S5\u003c/b\u003e). Plasmids within pC3, pC38 and pC39 were also frequent carriers of ESBL and/or carbapenemase genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), although a diversity of resistance alleles was noted (\u003cb\u003eTable S5\u003c/b\u003e). In alignment with the population-level analysis, ESBL-carrying plasmids (n\u0026thinsp;=\u0026thinsp;26) from this study were highly diverse and belonged to 11 pCs with pC-38 (n\u0026thinsp;=\u0026thinsp;6, carrying CTX-M and IncR replicon) and pC-39 [n\u0026thinsp;=\u0026thinsp;5, carrying CTX-M and IncFII(K)] being the most represented (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003eTable S3\u003c/b\u003e). Carbapenemase-carrying plasmids (n\u0026thinsp;=\u0026thinsp;20) were also diverse, with 6 pCs represented, though the majority belonged to either pC-10 (n\u0026thinsp;=\u0026thinsp;9 carrying OXA-48 and IncL/M replicon) or pC-2 (n\u0026thinsp;=\u0026thinsp;4, carrying NDM and a IncFIB(Mar)/IncHI1B replicon). With a single exception (isolate 582610), conjugative plasmids pC2, -10, -38, and \u0026minus;\u0026thinsp;39 were exclusively found in MDR-cKp lineages (e.g. ST-395, ST-147 and ST-23-KL57) irrespective of geographical origins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003eTable S3\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eContrasting with the breadth of diversity of resistance plasmids, only 7 of the 28 pC carried plasmids with virulence biomarkers and over 80% grouped in either pC4 or pC2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Cluster pC4 was strongly enriched (50% vs 0.001% in other pCs, p \u0026lt; .01) for plasmids carrying all 5 virulence biomarkers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), including the canonical, non-conjugative, pLVPK plasmid associated with phenotypic hypervirulence. Unlike plasmids found in pC4, pC2 plasmids rarely carried the complete set of virulence biomarkers (1%) and were more frequently associated with the carriage of only \u003cem\u003eiuc\u003c/em\u003e and \u003cem\u003ermpA2\u003c/em\u003e (23%). In agreement with this population-level analysis, 68% of virulence gene-carrying plasmids characterized in this study belonged pC-4 and pC-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003eTable S3, Fig. S4)\u003c/b\u003e. Plasmids pC-4, characterized by an IncFIB(K) or dual IncFIB(K)/IncHI1B replicon, were almost exclusively found in hvKp lineages (e.g. ST-23-KL1, ST-592, ST-420 and ST-86) irrespective of geographical origins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003eTable S5\u003c/b\u003e). A single exception was the detection of pC-4 plasmids in isolates from MDR-cKp ST-23-KL57 from Georgia and Ukraine, which carried an incomplete set of virulence biomarkers (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003eTable S3\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, only 6 (3.5%) plasmids from this study were hybrid and carried both virulence and AMR (ESBL or carbapenemase) determinants. Three belonged to pC-2 followed by pC-33 (n\u0026thinsp;=\u0026thinsp;2) and pC-4 (n\u0026thinsp;=\u0026thinsp;1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003eTable S3\u003c/b\u003e) and were mainly associated with MDR-cKp lineages (n\u0026thinsp;=\u0026thinsp;2 ST-395 isolates from Georgia, and one each from ST-14, -37, and \u0026minus;\u0026thinsp;218 from Jordan, Thailand, and Kenya, respectively). The only exception was the pC-4 plasmid with a complete set of virulence biomarkers and an acquired CTX-M ESBL in the hvKp ST-23-KL1 lineage. Nevertheless, the pC2 cluster was the main source of overlap for virulence and ESBL/carbapenemase resistance genes (\u003cb\u003eFig.\u0026nbsp;2CD\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eConvergence is largely driven by the acquisition of conjugative pC2 IncFIB(Mar)/IncHI1B plasmids globally.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBesides diversity, to quantify whether convergence resulted from preferred evolutionary routes, the complete set of 1,468 MDR-Kp isolates was analyzed to identify those that originated from shared or independent convergence events. Using maximum parsimony for the presence/absence of virulence and resistance alleles, and the tree topology, 25 independent convergence events were inferred (\u003cb\u003eFig. S5\u003c/b\u003e). These, hereby labeled convergence events A-Y, largely correlated with STs (\u003cem\u003ei.e.\u003c/em\u003e one independent event per lineage) except within MDR-cKp lineages ST-395 and ST-23-KL57 for which convergent isolates were predicted to have emerged from distinct (n\u0026thinsp;=\u0026thinsp;3 and n\u0026thinsp;=\u0026thinsp;2, respectively) evolutionary occurrences (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003eTable S6\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt the gene level, carbapenemase and ESBL genes, were equally represented: 12/25 convergent events (48%) acquired carbapenemase genes (NDM-1, NDM-5, or OXA-48) and were non-susceptible to all carbapenems tested while isolates representing the remaining 13 events (52%) all carried an ESBL (CTX-M-15, CTX-M-55, CTX-M-63, SHV-2A) and were non-susceptible to all 3rd and 4th generation cephalosporins. By contrast, the acquisition of an incomplete set of virulence biomarkers was strongly favored (20/25 routes or 80%, p\u0026thinsp;=\u0026thinsp;0.00012) and only 5/25 convergence events involved acquiring all 5 biomarkers, most predictive of the hvKp phenotype (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) (\u003cb\u003eTable S6\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eAt the lineage level, am enrichment of convergence events was observed via the acquisition of virulence genes in an MDR-cKp background (events E-Y representing 21/25 routes or 84%). Combined with the plasmid data, the acquisition within a MDR-cKp lineage of a pC2 IncFIB(Mar)/IncHI1B or a pC4 IncFIB(K)/IncHI1B plasmid with an incomplete set of virulence biomarkers were the preferred routes of convergence (8/25 and 2/25, respectively, for a total of 40% of all independent convergent events) in the studied population (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e). By contrast, only 16% of the convergence events (labeled A to D: a ST-23-KL1 from the U.S., and three ST-268, ST-68, ST-420 isolates from Thailand) were due to the acquisition of an ESBL gene (either inserted within the canonical pC-4 virulence plasmid [\u003cb\u003eFig. S6\u003c/b\u003e] or harbored by a standalone plasmid) in isolates from recognized hvKp lineages (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e \u003cb\u003eand Table S6\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRecurring acquisition of genes that encode a functional aerobactin is the common denominator to global and regional emergence of convergent\u003c/b\u003e \u003cb\u003eK. pneumoniae\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eDespite convergence largely (84%) resulting from the acquisition of an incomplete set of virulence biomarkers, it was noted that the aerobactin \u003cem\u003eiuc\u003c/em\u003e siderophore loci was acquired in all 25 independent events detected globally (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003eTable S6\u003c/b\u003e). It was most often (11/25) acquired by the gain of a conjugative pC2 IncFIB(Mar)/IncHI1B plasmid but a large diversity of plasmids from other pCs or singletons carried \u003cem\u003eiuc\u003c/em\u003e and accounted for the remaining 14 convergence events.\u003c/p\u003e \u003cp\u003eThis recurring acquisition of \u003cem\u003eiuc\u003c/em\u003e-carrying plasmids, albeit observed across MDR-cKp lineages, is best exemplified when reconstructing the evolution of ST-395. Within our sampling of this lineage, 3 independent convergent events (H, I and P) were inferred (\u003cb\u003eFig. S5\u003c/b\u003e) and Bayesian phylogenetics dated their origin within the last decade (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. The proposed routes of convergence were reconstructed: routes P and H shared a most recent common ancestor predicted to carry a pC-38 (IncR) plasmid harboring a CTX-M-15 ESBL. From there, the routes diverged and route P involved a stepwise acquisition (between 2016 and 2021) of a 177 kb singleton plasmid carrying the \u003cem\u003eiuc3\u003c/em\u003e aerobactin locus (node \u003cb\u003e1\u003c/b\u003e), a pC2 (IncFIB(Mar)/IncHI1B) carrying the NDM-5 carbapenemase (node \u003cb\u003e2\u003c/b\u003e), and a transposon harboring the 16S methyltransferase \u003cem\u003ermtB\u003c/em\u003e which inserted into the pC-38 backbone (node \u003cb\u003e3\u003c/b\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Unlike route P, the stepwise acquisition through\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eroute H could not be resolved but this convergent event involved the acquisition of a pC-10 (IncL/M) plasmid co-harboring an OXA-48 carbapenemase and the armA 16S methyltransferase, and a pC-2 (IncFIB(Mar)/IncHI1B) hybrid plasmid carrying 4 virulence biomarkers, including \u003cem\u003eiuc1\u003c/em\u003e, and the NDM-1 carbapenemase (node \u003cb\u003e4\u003c/b\u003e). Finally, route I involved the independent acquisition of 3 plasmids (node \u003cb\u003e5\u003c/b\u003e): a pC-2 (IncFIB(Mar)/IncHI1B) only carrying two virulence biomarker (\u003cem\u003eiuc1\u003c/em\u003e and \u003cem\u003ermpA2\u003c/em\u003e), a pC-42 (IncFIB(K)/IncFII(K)) harboring a CTX-M-15 ESBL, and a pC-10 (IncL/M) plasmid with OXA-48 and \u003cem\u003earmA\u003c/em\u003e like that observed in route H (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePhenotypically, representative isolates from each of the ST-395 convergent routes (H, I and P) resulted in extensively drug-resistant isolates with non-susceptibility to all cephalosporins, all carbapenems, and all clinically relevant aminoglycosides (\u003cb\u003eTable S6\u003c/b\u003e). Notably, all showed increased siderophore production compared to control isolate cKp1 lacking \u003cem\u003eiuc\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), but all did not exhibit a hypervirulent phenotype and were categorized as cKp (as defined by LD\u003csub\u003e50\u003c/sub\u003e \u0026gt;1x10\u003csup\u003e7\u003c/sup\u003e in the outbred CD1 SQ challenge model [15]), unlike the control hypervirulent isolate kvKp2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIncreased siderophore production, not hypervirulence, is the key adaptive trait driving global convergence in\u003c/b\u003e \u003cb\u003eK. pneumoniae\u003c/b\u003e.\u003c/p\u003e \u003cp\u003ePhenotypic characterization of representative isolates from all 25 independent convergence events showed that, irrespective of lineage, country of origin, or plasmid background, all exhibited increased siderophore production relative to cKp1 control (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The only exception was route Y, a ST-25 isolate from Georgia harboring \u003cem\u003eiuc3\u003c/em\u003e on a pC23 plasmid. Across convergence routes, isolates that acquired an incomplete set of virulence genes on a pC2 plasmid (routes G-N) produced siderophore levels comparable to isolates harboring the canonical pC4 virulence plasmid (routes A-F; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003eC\u003c/b\u003e), a pattern associated with the \u003cem\u003eiuc1\u003c/em\u003e aerobactin allele (\u003cb\u003eTable S6\u003c/b\u003e). By contrast, isolates that acquired other virulence plasmid types (routes O-Y), most frequently encoding \u003cem\u003eiuc3\u003c/em\u003e, displayed significantly lower, though still elevated, siderophore production (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003eC\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eDespite this consistent enhancement of iron-scavenging capacity, all convergent MDR-cKp isolates carrying an incomplete set of virulence biomarkers, whether on pC2 or other plasmids (routes G to Y), retained high LD₅₀ values and uniformly lacked the hypervirulent phenotype in the murine subcutaneous infection model (\u003cb\u003eFig.\u0026nbsp;5BD\u003c/b\u003e). In contrast, only hvKp convergent isolates that acquired ESBL or carbapenemase genes, and that maintained the full complement of five virulence biomarkers on a pC4 plasmid (routes A-D), exhibited low LD₅₀ values (LD\u003csub\u003e50\u003c/sub\u003e \u0026le;1x10\u003csup\u003e7\u003c/sup\u003e) consistent with true hypervirulence. Although rare, only these events result in \u003cem\u003ebona fide\u003c/em\u003e MDR-hvKp (\u003cb\u003eFig.\u0026nbsp;5BD\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe increasing detection of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates that harbor AMR and virulence determinants has raised urgent concerns for human health (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Here, we combined evolutionary, functional genomic, and biologic analyses of a global collection of \u003cem\u003eK. pneumoniae\u003c/em\u003e to show that plasmid-mediated convergence is frequent but follows preferred evolutionary paths dominated by MDR-cKp lineages acquiring IncFIB(Mar)/IncHI1B (pC2) conjugative plasmids carrying an incomplete set of virulence biomarkers, rather than by hvkp lineages acquiring resistance plasmids. Further, these convergent isolates showed increased siderophore production, due to increased aerobactin production (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), but lacked \u003cem\u003ein vivo\u003c/em\u003e hypervirulence emphasizing a fundamental distinction between genomic and phenotypic convergence.\u003c/p\u003e \u003cp\u003eAt the lineage level, 25 independent convergent events distributed across 22 sequence types were identified, highlighting the recurring and polyphyletic nature of convergence. The majority resulted from MDR-cKp lineages acquiring virulence genes rather than hvKp acquiring AMR genes. This asymmetry mirrors observations from other studies (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) and may be explained in that hvKp lineages rarely acquire resistance plasmids due to low conjugation efficiency because of their unique capsule and hypermucoid properties (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) and perhaps restriction systems (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Without these physiological constraints, MDR-cKp are a more permissive recipient, as illustrated by the three genomic convergence events inferred within a single lineage, ST-395, which independently evolved through the last decade alone. These spawned the emergence of NDM and/or OXA-48 producing epidemic subclones which have seemingly outpaced the KPC-producing ST-258 and are now predominant in Georgia and other countries in Europe (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt the plasmid level, our data indicates that convergence is facilitated by a subset of broad-host-range plasmids combining conjugation machinery with partial virulence cargo. Specifically, IncFIB(Mar)/IncHI1B plasmids (plasmid cluster pC2) represented the most common vector for virulence biomarker gene acquisition, albeit an incomplete set, accounting for nearly half of all independent convergence events. Consistent with their non-conjugative nature, the alternative IncFIB(K)/IncHI1B plasmids (pC4), which includes the canonical pLVPK and pK2044 with a complete set of 5 virulence biomarkers, remained primarily confined to hvKp backgrounds (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Further supporting this divide, plasmidome population analysis revealed that resistance and virulence plasmids occupy distinct evolutionary spaces, with limited recombinational events. As such, hybrid plasmids carrying both AMR and virulence loci were rare and largely confined to pC2, confirming previous observations that co-resident plasmids remain the predominant molecular mechanism for genomic convergence (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt the gene level, the aerobactin synthesis locus emerged as the single consistent signature of genomic convergence. All 25 independent events involved the acquisition of \u003cem\u003eiuc\u003c/em\u003e and phenotypic assays demonstrated all exhibited increased siderophore production, albeit more pronounced in \u003cem\u003eiuc1\u003c/em\u003e-carrying isolates. The \u003cem\u003eiuc1\u003c/em\u003e variant, typically found on pLVPK-like IncFIB(K)/IncHI1B pC4 plasmids from hvKp strains, was most common, followed by \u003cem\u003eiuc3\u003c/em\u003e more frequently associated with IncFIB(Mar)/IncFII pC2 plasmids, which have been previously associated with strains from animal and environmental sources (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). The consistent selection of \u003cem\u003eiuc\u003c/em\u003e underscores the ecological advantage conferred by enhanced siderophore production (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Recent studies have shown that aerobactin provides an ecological advantage and enhances mucosal fitness and epithelial adherence independent of its ability to enhance systemic virulence (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Further, increased iron acquisition capacity may improve host colonization as well as persistence in iron-limited environments such as infected tissues and hospital surfaces. These same properties could indirectly promote transmission between patients, suggesting that siderophore-mediated iron acquisition may represent a key adaptive trait underlying the epidemic success of genomic convergent lineages like ST-147 and ST-395 in hospitals worldwide.\u003c/p\u003e \u003cp\u003eAt the phenotypic level, assays in the murine subcutaneous model reinforce this separation between genomic convergence and pathogenic potential. Adding to our previous studies (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) and that of Kochan and colleagues (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), none of the convergent MDR lineages with an acquired pC2 plasmid harboring an incomplete set of virulence biomarkers displayed systemic lethality characteristic of hvKp, but instead possessed a cKp virulence phenotype. While some confusion originally persisted, it is clear now that genomic convergence resulting in the acquisition of some, but not all, of the virulence biomarkers does not equate to a hypervirulent phenotype but rather defines a new, stable ecological state within the nosocomial setting. Conversely, confirmed hvKp lineages that acquired ESBL genes (\u003cem\u003ee.g\u003c/em\u003e., ST-23-KL1 with CTX-M-15) retained a pC4 plasmid and full hypervirulence, indicating that convergence in an hvKp background can yield clinically high-risk isolates, albeit less frequently. Although imperfect, for diagnostic purposes, such hypervirulent, drug-resistant isolates are currently best identified by their carriage of a complete set of 5 virulence biomarkers (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Unlike the epidemic MDR-cKp convergent lineages, the MDR-hvKp have not spread globally and, so far, have only been sporadically detected in Asia and Europe (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), a possible consequence of the metabolic burden of full hypervirulence (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCollectively, our results establish a framework for understanding convergence in \u003cem\u003eK. pneumoniae\u003c/em\u003e. Genomic convergence is frequent, structured, and dominated by MDR-cKp backgrounds acquiring \u003cem\u003eiuc\u003c/em\u003e-carrying plasmids, but it rarely leads to the hypervirulent phenotype. These convergent strains represent an intermediate evolutionary state between cKp and hvKp, optimized for transmission rather than invasion. Understanding the molecular basis and ecological advantages of this intermediate state will be essential for anticipating the future evolution of hospital-adapted \u003cem\u003eKlebsiella\u003c/em\u003e and refining surveillance strategies to distinguish true hypervirulence from adaptive convergence.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBacterial Isolates\u003c/h2\u003e \u003cp\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e isolates were collected from Military Health System facilities across the United States and globally in collaboration with the US Department of Defense\u0026rsquo;s (DoD) Global Emerging Infections Surveillance branch. The 1,468 global isolates were recovered from Thailand (n\u0026thinsp;=\u0026thinsp;362), Georgia (n\u0026thinsp;=\u0026thinsp;359), Peru (n\u0026thinsp;=\u0026thinsp;305), Jordan (n\u0026thinsp;=\u0026thinsp;215), Kenya (n\u0026thinsp;=\u0026thinsp;159), Uganda (n\u0026thinsp;=\u0026thinsp;36), Philippines (n\u0026thinsp;=\u0026thinsp;25), and Ukraine (n\u0026thinsp;=\u0026thinsp;7) and were compared to isolates from the United States (n\u0026thinsp;=\u0026thinsp;39) (\u003cb\u003eTable S1\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWhole Genome Sequencing\u003c/h3\u003e\n\u003cp\u003eGenomic DNA extraction and Illumina short read WGS were performed as previously described (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Libraries were constructed using Kapa HyperPlus Library Preparation kit (Roche Diagnostics) and quantified using the KAPA Library Quantification Kit - Illumina/Bio-Rad iCycler\u0026trade; (Roche Diagnostics) on a CFX96 real-time cycler (Bio-Rad). Libraries were normalized to 2 nM, pooled, denatured, and diluted to 1 nM. Whole genome sequencing was performed using a MiSeq or NextSeq-500 (Illumina) with MiSeq Reagent Kit v3 (600 cycles; 2 X 300 bp) or NextSeq-500 Reagent kit 500/550 vs (300 cycles, 2 x 150 bp) (Illumina). De novo draft genome assemblies were produced using Newbler v2.7 (Roche Diagnostics). Minimum thresholds for contig size and coverage were set at 200 bp and 49.5+, respectively.\u003c/p\u003e \u003cp\u003eLong read sequencing was performed on 53 isolates. 8 isolates were sequenced using single-molecule real-time (SMRT) by Pacific Biosciences RS II Instrument (Pacific Biosciences). Assembly was completed using the Hierarchical Genome Assembly Process (HGAP v3.0). The resulting contigs were imported into Geneious and circularized. Assemblies were polished using Illumina paired-end short reads. 45 isolates were sequenced on a Minion platform using MinION Mk1B (Oxford Nanopore Technologies). Library preparation on genomic DNA was principally performed using EXP-NBD114 and sequenced on a R9.4.1 flowcell. Basecalling was performed using Guppy v6.1.7 using the super accurate basecalling model (r9.4.1_450bps_sup). Prior to assembly, reads were filtered by Filtlong (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/rrwick/Filtlong\u003c/span\u003e\u003cspan address=\"https://github.com/rrwick/Filtlong\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to capture the top 95% of reads by quality score. De novo assembly was performed using Trycycler [v0.5.3] (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Trycycler outputs a consensus assembly from multiple long-read dedicated assemblers including Flye (v2.9.1), Miniasm (v0.3), and Raven (v1.8.1). Next, the trycylcer consensus assembly was polished with Illumina short reads using Medaka and Illumina short reads using Polypolish (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBioinformatic analysis.\u003c/b\u003e Species identification, MLST typing, virulence locus, capsule (K), and lipopolysaccharide (O) loci were identified using Kleborate v3.0.5 (\u003cb\u003eTable S1\u003c/b\u003e) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). \u003cem\u003epeg-344\u003c/em\u003e was identified using BLASTn search of draft genome assemblies (query sequence pLVPK, accession number AP006726.1). AMRFinderPlus v3.9.8 (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e) and ARIBA v2.14.4 (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) were used to identify resistance alleles from draft assemblies and processed reads, respectively, followed by deduplication of redundant alleles calls. clast allele assignment and minimum spanning tree generation were performed with SeqSphere (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Plasmid replicons were identified using PlasmidFinder v2.1.6 (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe created a core gene phylogeny for the 261 isolates harboring\u0026thinsp;\u003cem\u003e\u0026ge;\u003c/em\u003e\u0026thinsp;1 hvKp gene (\u003cb\u003eTable S1\u003c/b\u003e). Assemblies were annotated using Prokka v1.14.6 (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) and were used as input for Roary v3.13.0 (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e) and a SNP-based alignment was generated. Recombination was filtered from the alignments using Gubbins v2.4.1 (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e) and a maximum-likelihood tree was generated with RAxML-NG [v1.1] (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e) using the GTR\u0026thinsp;+\u0026thinsp;G (50 parsimony, 50 random) model 100 random bootstrap replicates. The tree was imported in iTOL [v6.8.1] (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) for visualization with metadata.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBayesian Evolutionary Phylogenetic Analysis\u003c/h2\u003e \u003cp\u003eTo evaluate the strength of the temporal signal, TempEst v1.5.3 was utilized to visualize the relationship between root-to-tip genetic distances for samples with known collection dates (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). The bayesian phylogenetic inference was performed using BEAST2 v2.6.5 on a recombination free alignment, removing samples with uncertain collection dates, and accounting for constant sites with beast2_constsites (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/andersgs/beast2_constsites\u003c/span\u003e\u003cspan address=\"https://github.com/andersgs/beast2_constsites\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The HKY substitution model was selected based on evaluation of all possible substitution models in bModelTest v1.2.1 (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). The random clock model was selected based on support by the marginal likelihood value using the Nested Sampling package v1.1.0 (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). BEAST2 was run under a coalescent constant population model, with a Markov chain Monte Carlo length of 1 x 10\u003csup\u003e8\u003c/sup\u003e sampling every 5 x 10\u003csup\u003e3\u003c/sup\u003e steps. Analyses were repeated 5 times to confirm consistency between the obtained posterior distributions. Parameter estimates were computed using Tracer v1.7.1. Posterior trees were combined with LogCombiner and summarized in TreeAnnotator after a 50% burn-in. The final MCC target tree was visualized in FigTree v1.4.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/rambaut/figtree\u003c/span\u003e\u003cspan address=\"https://github.com/rambaut/figtree\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and annotated using iTOL (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmidome Analysis\u003c/h3\u003e\n\u003cp\u003emge-cluster plasmid typing [v1.1.0] (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) was used to investigate plasmid families present in our dataset compared to a global public collection. Mge-cluster is a unitig based classification and pairwise Jaccard distances from unitig presence/absence matrices. All default parameters were used except perplexity, which was set to 100 and unitig filtering set to 30% to deduplicate redundant sequences.\u003c/p\u003e \u003cp\u003eFirst, 7,347 complete plasmid sequences from 2,153 isolates were retrieved on NCBI. Clustering analysis resulted in 43 distinct plasmid clusters that grouped 5,810 plasmid sequences (grey dots, Figure S3). 1,500 NCBI plasmid sequences were unassigned a cluster group and 399 plasmid sequences were excluded from clustering analysis by mge-cluster (\u003cb\u003eFig. S3\u003c/b\u003e). Next, we incorporated our 173 plasmid sequences (recovered from the 53 isolates) into the plasmid cluster network from which 124 plasmids grouped with 28 of the NCBI clusters (44 plasmids were unassigned a cluster group (singletons) and 5 plasmids were excluded from the clustering analysis) (\u003cb\u003eFig.\u0026nbsp;3AB\u003c/b\u003e). The distribution of replicon types, STs, virulence genes, ESBL and/or carbapenemase genes were investigated and visualized for all plasmid sequences located in the 28 discrete clusters (n\u0026thinsp;=\u0026thinsp;4,052) and the 44 singletons from this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cb\u003eTable S5\u003c/b\u003e).\u003c/p\u003e\n\u003ch3\u003eQuantitative Siderophore Assay\u003c/h3\u003e\n\u003cp\u003eStrains were grown overnight at 37\u0026deg;C in iron-chelated M9 minimal medium containing casamino acids (c-M9-CA) (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) and culture supernatants were assessed using the chromeazurol S dye assay as described (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Standards with concentrations of 0, 1.5, 3.1, 6.25, 12.5, 25, 50, and 100 \u0026micro;g/ml enabled quantitation. A minimum of 3 biological assays with 3 technical repeats were performed and the results were reported as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;the SD.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMouse Subcutaneous (SQ) Infection Model\u003c/h2\u003e \u003cp\u003e Animal studies were reviewed and approved by the Veterans Administration Institutional Animal Care Committee and the University at Buffalo-SUNY and were carried out in strict accordance with the recommendations in the guidelines delineated in the \"NIH Guide for the Care and Use of Laboratory Animals\"(revised 1985) and the \"Ethics of Animal Experimentation Statement\" (Canadian Council on Animal Care, July, 1980) as monitored by the Institutional Animal Care and Use Committee. All efforts were made to minimize suffering. Veterinary care for the animals was supplied by the staff of Veterans Administration Animal Facility under the direction of a fully licensed veterinarian. CD1 male mice, 4\u0026ndash;6 weeks old, were obtained from Charles River Laboratories, quarantined for 7 days before use, and then challenged via a SQ injection with the isolates of interest (100 \u0026micro;L of bacterial suspension serially diluted to the required titers in 1 x PBS diluted and injected using a 0.5 mL insulin syringe), as previously described (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). The animals were closely monitored for 14 days after challenge for the development of the study endpoints, survival, or severe illness (in extremis state)/death, which was recorded as a dichotomous variable. Signs that were monitored and which resulted in immediate euthanasia using methods consistent with the recommendations of the American Veterinary Medical Association Guidelines included hunched posture, ruffled fur, labored breathing, reluctance to move, photophobia, and dehydration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eDesired comparisons between strains for experiments assessing siderophore production were made via ordinary one-way ANOVA, using Šid\u0026aacute;k\u0026rsquo;s multiple comparisons test (Prism 10.4.2 for MacIntosh, GraphPad Software Inc.).\u003c/p\u003e \u003cp\u003eLD\u003csub\u003e50\u003c/sub\u003e values were estimated using a logistic regression model as described (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Pair-wise comparisons of the dose-response curves were used to generate LD\u003csub\u003e50\u003c/sub\u003e values. Desired comparisons between LD\u003csub\u003e50\u003c/sub\u003e values were made by employing a blend of the empirical logit function along with least-squares regression incorporating strain and inoculum factors (CFU/mL) to derive p-values for comparing dose-response curves based on LS-means.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThe authors are thankful to all the staff of the MRSN and all the members of the Global Emerging Infections Surveillance (GEIS) network. The authors thank MAJ Hunter J. Smith for his leadership of the GEIS AMR portfolio and his assistance in coordinating communications between MRSN and the GEIS overseas partner laboratories. The authors thank Mohammad N. Alhawarat, Wiam Khraisat, and Mohammad J. Gharaibeh from the Ministry of Health of The Hashemite Kingdom of Jordan for their role in coordinating national MDR surveillance efforts. The manuscript has been reviewed by the Walter Reed Army Institute of Research and there is no objection to its presentation. The views expressed herein are those of the author(s) and do not necessarily reflect the official policy or position of the Defense Health Agency, the Department of Defense, nor any agencies under the U.S. Government.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was funded by Defense Health Program (DHP) Operation \u0026amp; Maintenance (O\u0026amp;M). Partial funding was provided by the Armed Forces Health Surveillance Division (AFHSD), Global Emerging Infections Surveillance (GEIS) Branch project P0132_23_WR as well as global surveillance projects P0011_AF_25, P0119_18_KY_013, P152_20_KY_06, P0065_21_KY, P0037_22_KY, P0096_23_KY, P0166_22_N3, and P0126_23_N3. This work was also supported by NIH 1R21AI141826-01A1 (Dr. Russo) and the Department of Veterans Affairs VA Merit Review (I01 BX004677-01) (Dr. Russo).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u0026nbsp;\u003c/strong\u003eIndividual genomic assemblies accession numbers are listed in Table S1 and genomes of all isolates analysed in this study are publicly available in the NCBI database under the BioProject number PRJNA1354878.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003e For U.S. isolates: the isolates and clinical information were collected as part of the public health surveillance activities of the MRSN, as determined by the WRAIR Commander in accordance with Institutional Review Board (IRB) Policy Memorandum #10; Public Health activity Determination and Oversight Requirements issued June 10th 2019. For Ukrainian samples: hospital permission to conduct the surveillance study was obtained from Hospital Bioethics Committee of MMCC CR, Ministry of Defense, Vinnitsa, Ukraine, protocol reference number 18/2 from 05 May 2014. The patients were included after understanding the study and had signed an informed consent. For Kenya, Uganda and Jordan isolates: the studies were undertaken with ethics approvals from the different country institutional review boards: Kenya, KEMRI SERU#2767/WRAIR #2089/ USAMRMC ORP HRPO) (Log#A-18129); Uganda, MUSPH HDREC #087/UNCST#HS775/WRAIR (#1711); and Jordan, NAMRU3.PJT.2011.0014. For Peru: This study was reviewed and approved by the Institutional Review Board at the U.S. Naval Medical Research Unit SOUTH in Peru and was reviewed and approved by the ethics board at each participating institution. For Thailand and Philippines isolates: the isolate collection was approved by the Research Ethic Committee, Naval Medical Department, Royal Thai Navy and Walter Reed Army Institute of Research (WRAIR), Silver Spring, MD, USA. WRAIR Human Subjects Protection Branch determined that this was non-human subjects research (NHSR) and all samples and data associated with clinical isolates was de-identified prior to transfer to AFRIMS. The research conformed to the principles of the Helsinki Declaration.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePodschun R, Ullmann U (1998) Klebsiella spp. as Nosocomial Pathogens: Epidemiology, Taxonomy, Typing Methods, and Pathogenicity Factors. 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Young VB, editor. mSphere. ;6(2):e00045-21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/msphere/6/2/mSph.00045\u0026thinsp;\u0026ndash;\u0026thinsp;21.atom\u003c/span\u003e\u003cspan address=\"http:///msphere/6/2/mSph.00045\u0026thinsp;\u0026ndash;\u0026thinsp;21.atom\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8613390/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8613390/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The rise of Klebsiella pneumoniae combining antimicrobial resistance and virulence genes poses a major health threat, but the evolutionary routes and phenotypic consequences of this convergence are poorly understood. Here, phylogenetics of 1,468 isolates and population analysis of 7,520 plasmids, from \u003e50 countries through the last two decades, reveal that convergence follows preferred, constrained evolutionary paths. The dominant route involves multidrug-resistant classical K. pneumoniae acquiring conjugative IncFIB(Mar)/IncHI1B plasmids carrying an incomplete set of virulence biomarkers. Across 25 independent convergence events, the acquisition of the aerobactin siderophore locus was the only universal feature. These convergent isolates exhibit enhanced siderophore production but consistently lack the hypervirulent phenotype in vivo. In contrast, genuine hypervirulent strains that gain resistance remain rare. We conclude that enhanced siderophore production, not hypervirulence, is the primary adaptive trait driving the success of globally emerging convergent lineages, representing a distinct evolutionary state optimized for transmission rather than systemic invasion.","manuscriptTitle":"Preferred evolutionary routes of convergence in Klebsiella pneumoniae favor siderophore acquisition over hypervirulence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-13 04:41:42","doi":"10.21203/rs.3.rs-8613390/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6d500b30-ee05-4fdb-95b0-046cedf4430e","owner":[],"postedDate":"February 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":62474038,"name":"Biological sciences/Microbiology/Microbial genetics/Bacterial genetics"},{"id":62474039,"name":"Biological sciences/Microbiology/Antimicrobials/Antimicrobial resistance"},{"id":62474040,"name":"Biological sciences/Microbiology/Pathogens"},{"id":62474041,"name":"Biological sciences/Microbiology/Bacteria/Bacterial genomics"}],"tags":[],"updatedAt":"2026-02-13T04:41:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-13 04:41:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8613390","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8613390","identity":"rs-8613390","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}