Klebsiella pneumoniae clinical isolates with features of both multidrug-resistance and hypervirulence have unexpectedly low virulence

Kochan, Travis J.; Nozick, Sophia H.; Valdes, Aliki; Mitra, Sumitra D.; Cheung, Bettina H.; Lebrun-Corbin, Marine; Medernach, Rachel L.; Vessely, Madeleine B.; Mills, Jori O.; Axline, Christopher M. R.; Nelson, Julia A.; VanGosen, Ethan M.; Ward, Timothy J.; Ozer, Egon A.; van Duin, David; Chen, Liang; Kreiswirth, Barry N.; Long, S. Wesley; Musser, James M.; Bulman, Zackery P.; Wunderink, Richard G.; Hauser, Alan R.

doi:10.1038/s41467-023-43802-1

Download PDF

Article
Open access
Published: 02 December 2023

Klebsiella pneumoniae clinical isolates with features of both multidrug-resistance and hypervirulence have unexpectedly low virulence

Nature Communications volume 14, Article number: 7962 (2023) Cite this article

7547 Accesses
2 Citations
61 Altmetric
Metrics details

Subjects

Abstract

Klebsiella pneumoniae has been classified into two types, classical K. pneumoniae (cKP) and hypervirulent K. pneumoniae (hvKP). cKP isolates are highly diverse and important causes of nosocomial infections; they include globally disseminated antibiotic-resistant clones. hvKP isolates are sensitive to most antibiotics but are highly virulent, causing community-acquired infections in healthy individuals. The virulence phenotype of hvKP is associated with pathogenicity loci responsible for siderophore and hypermucoid capsule production. Recently, convergent strains of K. pneumoniae, which possess features of both cKP and hvKP, have emerged and are cause of much concern. Here, we screen the genomes of 2,608 multidrug-resistant K. pneumoniae isolates from the United States and identify 47 convergent isolates. We perform phenotypic and genomic characterization of 12 representative isolates. These 12 convergent isolates contain a variety of antimicrobial resistance plasmids and virulence plasmids. Most convergent isolates contain aerobactin biosynthesis genes and produce more siderophores than cKP isolates but not more capsule. Unexpectedly, only 1 of the 12 tested convergent isolates has a level of virulence consistent with hvKP isolates in a murine pneumonia model. These findings suggest that additional studies should be performed to clarify whether convergent strains are indeed more virulent than cKP in mouse and human infections.

A novel antibiotic class targeting the lipopolysaccharide transporter

Article Open access 03 January 2024

Molecular mechanisms of antibiotic resistance revisited

Article 21 November 2022

Pseudomonas aeruginosa: pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances and emerging therapeutics

Article Open access 25 June 2022

Introduction

Klebsiella pneumoniae is a highly diverse human pathogen and a major cause of antimicrobial resistance-related deaths globally^1,2. Bacteria of this species has been categorized into two broadly defined groups: classical K. pneumoniae (cKP) isolates, which cause nosocomial infections in compromised patients, and hypervirulent K. pneumoniae (hvKP) isolates, which cause community-acquired, disseminated infections in healthy, immunocompetent individuals^3,4,5,6,7,8.

cKP isolates are highly diverse, consisting of hundreds of sequence types (STs) and capsule loci types (KLs)⁹. They are generally of low virulence, which may account for their predominance in hospitalized and immunocompromised patients^10,11. A clinically important subset are multidrug-resistant cKP (MDR-cKP) strains, which are resistant to clinically relevant antibiotics in 3 or more classes. These strains include high-risk clones, which are globally disseminated STs (e.g., ST258 or ST307). MDR-cKP strains frequently produce extended-spectrum beta-lactamases (ESBLs), including CTX-M-15 and SHV-12, which confer resistance to third-generation cephalosporins^9,12,13. Some MDR-cKP isolates are resistant to carbapenems through plasmid-encoded carbapenemases, such as KPC and NDM^13,14.

hvKP isolates are usually susceptible to antibiotics but are exceptionally virulent¹⁵. The hypervirulent phenotype has been attributed to two types of factors: mucoid regulators and siderophores^16,17. Mucoid regulator genes, rmpADC and rmpA2, enhance capsule production and tenacity, resulting in a hypermucoviscosity (hmv) colony morphology^18,19. These capsules allow the bacterium to resist the host’s innate immune response by inhibiting phagocytosis, opsonization, and complement-mediated killing¹⁵. hmv also limits DNA movement into and out of the bacterial cell, restricting recombination and horizontal gene transfer²⁰, potentially explaining the restricted range of hvKP sequence types. Siderophores are molecules secreted by bacteria to scavenge iron. K. pneumoniae isolates may produce up to four siderophores. Biosynthesis genes for enterobactin (ent) are present in nearly all K. pneumoniae strains, whereas biosynthesis genes for aerobactin (iuc) and salmochelin (iro) are frequently present in hvKP strains. Biosynthesis genes for yersiniabactin (ybt) are present in some hvKP and some cKP isolates; they are frequently associated with the genes encoding the genotoxin colibactin (clb) as part of integrated and conjugative elements such as ICEKp10²¹.

Accurately distinguishing between hvKP and cKP strains is challenging and the definitions of these groups remain controversial²². Although hvKP strains are hmv, cKP strains can also have this attribute due to mutations within the capsule locus (wzc)²³. Some studies have defined hvKP as simply the presence of iuc genes^24,25. Russo and colleagues systematically evaluated five genes (rmpA, rmpA2, peg-344, iucA, and iroB) and one phenotype (hmv) for defining hvKP in a sample of 175 hvKP or cKP isolates¹¹. They found that presence of iucA or iroB yielded predictive accuracies of 96% and 97%, respectively, compared to the hmv phenotype, which yielded an accuracy of 90%. However, the clinical implications of these biomarkers across diverse strain backgrounds remain unclear.

Evidence suggests that mouse models of infection, which capture the overall virulence potential of K. pneumoniae isolates, are highly accurate in distinguishing hvKP from cKP¹¹. In a mouse model of pneumonia, hvKP strains cause pre-lethal illness at extremely low doses (some strains <100 CFU), making them easily distinguishable from cKP strains, which usually require doses of 10⁷–10⁹ CFU to cause a similar severity of illness¹¹. For these reasons, mouse models are attractive as a gold standard for identifying hvKP strains^11,26.

Of great concern is the recent emergence of K. pneumoniae strains with features of both hvKP and MDR-cKP. These convergent strains have the potential to cause difficult-to-treat invasive and disseminated infections in healthy individuals. Several reports have documented the existence of typical hvKP strains that have acquired ESBL or carbapenemase genes^27,28,29,30. For example, in 2019 Karlson and colleagues reported an ST23 isolate from the U.S. that carried a bla_KPC-2 plasmid, and in 2016 Cheong and colleagues reported two ST23 isolates in Singapore that carried a bla_CTX-M-15 plasmid^27,30. Others have noted highly antibiotic-resistant cKP strains that have acquired virulence genes usually found in hvKP strains^28,29,31,32. For example, Wyres and colleagues reported that 19 of 22 convergent bloodstream isolates in Southeastern Asia were MDR-cKP isolates that had acquired aerobactin biosynthesis genes. Some reports suggest that convergent strains have enhanced virulence in neutrophil phagocytosis assays or animal models of infection and a few suggest that they cause more severe disease in people^33,34,35,36. For example, Gu and colleagues described five patients infected with an ST11 carbapenem-resistant hvKP strain in China and noted that all five patients died³³. However, data supporting a high degree of virulence associated with convergent strains are limited^31,32,35.

To address this gap in the literature, we screened three large U.S. collections of highly antibiotic-resistant cKP for convergent isolates. We carried out whole-genome sequence analysis and conducted phenotypic tests for established virulence traits of K. pneumoniae.

Results

Detection of convergent isolates among U.S. antibiotic-resistant K. pneumoniae clinical isolates

For the purposes of this study, we refer to convergent K. pneumoniae as strains that both are resistant to third-generation cephalosporins (e.g., ceftriaxone) and contain hvKP virulence genes. We define hvKP virulence genes as iucA, iroB, rmpA, or rmpA2. To identify convergent isolates, we screened the genomes of 2608 isolates from three large collections for at least one of these four hvKP virulence genes.

These collections were as follows: (1) isolates resistant to third-generation cephalosporins from Northwestern Memorial Hospital in Chicago (NMH collection, n = 237), (2) isolates resistant to carbapenems from 49 U.S. hospitals participating in the CRACKLE-2 study (CRACKLE-2 collection, n = 884) and, (3) isolates resistant to third-generation cephalosporins from the Houston-Methodist Hospital System (Houston collection, n = 1486)^10,37,38,39. One additional isolate, DHQP1701672 from the U.S. Center for Disease Control and Prevention, was included because it is one of the few reported carbapenem-resistant hvKP isolates from the U.S.²⁷. Most of the 2608 isolates were from infections of the urinary tract (n = 1266, 48.5%), lung (n = 588, 22.5%), bloodstream (n = 287, 11.0%), and wounds (n = 249, 9.5%) (Table 1).

Table 1 Clinical source of isolate by collection

Full size table

The 2608 K. pneumoniae isolates analyzed in our study exhibited a dominance of two multilocus sequence types (STs) endemic to the U.S., ST258 and ST307 (Figs. 1A and S1A). These STs were primarily capsule loci KL106/KL107 and KL102, respectively. The remaining isolates demonstrated high diversity, comprising 217 STs and 121 KLs, including high-risk clonal groups like ST14, ST15, and ST16 (Figs. 1A, S1A, S1B and Supplementary Data 1). Overall, the most common STs were ST258 (n = 1060, 40.6%), ST307 (n = 576, 22.1%), ST16 (n = 96, 3.7%), ST15 (n = 90, 3.5%), and ST14 (n = 41, 1.6%) (Figs. 1A, S1A and Supplementary Data 1). The most common capsule loci were KL102 (n = 576, 22.1%), KL107 (n = 510, 19.6%), KL106 (n = 391, 15.0%), KL51 (n = 103, 3.9%), and KL24 (n = 71, 2.7%) (Figs. 1A, S1B and Supplementary Data 1).

**Fig. 1: Genomic features of antibiotic-resistant clinical isolates of *K. pneumoniae* from United States hospitals.**

Of the 2608 K. pneumoniae isolates examined, ~90% contained resistance determinants for 3 or more classes of antibiotics (Fig. 1B). While, the majority contained an ESBL gene (n = 2132, 78.6%) or a carbapenemase gene (n = 1165, 43%), some isolates (n = 174, 6.7%) lacked these genes and were presumably resistant to third-generation cephalosporins or carbapenems by other mechanisms (Supplementary Data 1)^40,41.

Further screening identified 47 (1.8%) isolates with at least one hvKP virulence gene (iucA, iroB, rmpA, and rmpA2) and were deemed convergent: 25 (2.8%) of CRACKLE-2, 12 (0.8%) of Houston, and 9 (3.8%) of NMH isolates (Supplementary Data 1). Among these, 45 (95.7%) contained iucA with distinct lineages: 18 (40%) contained iuc5 (a lineage associated with Escherichia coli plasmids), 15 (33.3%) contained iuc1 (a hvKP-associated lineage), and 12 (26.7%) contained novel lineages of iuc (iucOther) (Fig. 1C). None contained iuc2, a second hvKP-associated iuc lineage. Blast analysis of the 12 iucOther lineages revealed they are slight variations of lineages found in E. coli plasmids. Of the 47 convergent isolates, 17 (36.2%) contained iroB and 14 (29.8%) contained either rmpA or rmpA2 (Supplementary Data 1). Multilocus sequence typing of these isolates revealed that 44 (93.6%) were STs associated with cKP lineages, indicating that these highly antibiotic-resistant lineages had acquired virulence genes such as iucA or iroB (Fig. 1D)⁴². The most common of these STs were ST258 (n = 10) and ST16 (n = 6) (Fig. 1D). The remaining three isolates were ST23, a genotype commonly associated with hvKP. These ST23 lineages had acquired the bla_KPC-2 carbapenemase gene (Supplementary Data 1). These findings confirm the presence of rare convergent K. pneumoniae isolates in the U.S.

Phylogenetic analysis of convergent isolates

Twelve of the 47 convergent isolates were chosen for further analysis based on their representation of 9 distinct STs (Table 2 and Supplementary Data 2). Among these isolates, 11 were cKP that acquired hvKP-like virulence factors. The remaining isolate (DHQP1701672) was a typical hvKP sequence type (ST23) that acquired an antibiotic-resistance gene (bla_KPC-2). To further examine how these 12 isolates evolved to become convergent, we compared their whole-genome sequences to other well-characterized isolates from three K. pneumoniae groups: typical hvKP (n = 11), MDR-cKP (n = 11), and relatively antibiotic-susceptible cKP (NON-MDR-cKP) (n = 6) (Fig. 2 and Supplementary Data 2). A core-genome phylogenetic tree was generated to examine the relationships between these isolates (Fig. 2). Convergent isolates clustered together with non-convergent isolates of the same ST or clonal group. For example, the convergent isolate ARLG-3346 clustered with other ST258 isolates, and KPN1409 clustered with other ST15 isolates (Fig. 2). In addition, the convergent isolate K014 is ST268, an ST not traditionally associated with hvKP; however, it did cluster with a confirmed ST268 hvKP isolate from NMH, KPN13 (Fig. 2)⁴². This indicates that the convergent isolates are not phylogenetically distinct from closely related non-convergent isolates and likely have emerged through acquisition of virulence or antimicrobial resistance genes or plasmids (Fig. 2).

Table 2 Characteristics of convergent isolates chosen for further analysis

Full size table

**Fig. 2: Phylogenetic analysis of convergent, MDR-cKP, NON-MDR-cKp, and hvKP isolates.**

Plasmid analysis of convergent isolates

To evaluate plasmid content of the 12 convergent isolates, we performed long-read sequencing. For comparison, complete genomes were obtained, including plasmids, for all 11 of the reference hvKP isolates, all 11 of the MDR-cKP isolates, and all 6 of the NON-MDR-cKP isolates. Plasmid sequence similarity was visualized using a neighbor-joining tree based on Mash distance (Fig. 3). Several observations were apparent: (1) Many of the convergent isolates contained multiple plasmids. For example, KPN1402 and KPN482 each contained five plasmids. (2) Many plasmids had multiple origins of replication, suggesting that they were formed by plasmid fusion. (3) Plasmids containing virulence genes clustered into three major groups. (4) Antibiotic resistance genes, encoding carbapenemases, ESBLs, and aminoglycoside modifying enzymes, were distributed broadly across plasmids, indicating their mobility and ability to integrate into different plasmids. (5) Although most plasmids containing the bla_KPC gene fell into two distinct groups, other examples of plasmids with this gene were also observed (pCRE-265_3, pOC1116_4, pARLG-4365_2, and pCRE-015_3).

**Fig. 3: Plasmid sequence analysis of convergent, MDR-cKP, hvKP, and NON-MDR-cKP isolates.**

Eleven convergent isolates harbored plasmids containing virulence genes (Supplementary Data 3). Six convergent isolates contained plasmids with segments and origins of replication (IncFIB and IncHI1B) highly similar to pK2044, a well-characterized hvKP virulence plasmid (Figs. 3, 4A and Supplementary Data 1). Three (DHQP1701672, ARLG-4365, and K014) contained plasmids with >80% of the sequence content of pK2044. All six contained regions that aligned to both rmpA2 and the iuc locus, but the rmpA2 genes contained frameshift mutations resulting in premature stop codons. Three of these six isolates also contained iro loci and two contained rmpADC (Fig. 4A, Supplementary Data 3). These six plasmids grouped closely with other pK2044-like hvKP plasmids based on genomic similarity (Jaccard Index >0.95) (Fig. 4C). Five other convergent isolates harbored plasmid sequences and replicons (IncFIC, IncFIB, and rep_cluster_2244) similar to portions of pEC244_2, an E. coli virulence plasmid containing iuc and iro loci (Fig. 4B, Supplementary Data 1, and Supplementary Data 3). One plasmid (pARLG-3346_1) contained both iuc and iro loci while four contained only the iuc locus (Fig. 4B and Supplementary Data 1). Based on genomic similarity, these five plasmids grouped together (Jaccard Index >0.95); each carried at least one antimicrobial resistance gene (Fig. 4D). One of these plasmids, pKPN415_1 contained a bla_CTX-M-15 ESBL gene. The twelfth convergent isolate, KPN226, contained a chromosomal copy of ICEKp1, which contained the iro locus and the rmpADC operon (Table 2 and Supplementary Data 1). These findings highlight the diverse plasmids in convergent isolates with various hvKP-associated pathogenicity loci.

**Fig. 4: Virulence plasmids identified in 12 representative convergent isolates.**

All 12 convergent isolates contained antimicrobial resistance plasmids (Fig. 3 and Supplementary Data 3). Six had ESBL genes on plasmids, while 4 others had them in their chromosomes (Fig. 3 and Supplementary Data 3). Carbapenemase genes were found on plasmids from six convergent isolates (Fig. 3 and Supplementary Data 3). Altogether, 10 convergent isolates had plasmids that contained genes encoding ESBLs or carbapenemases. OC1092 carried plasmids that contained both a carbapenemase (bla_KPC-3) and an ESBL (bla_SHV-12) gene. Only ARLG-4803 lacked plasmids containing these genes (Supplementary Data 3). ARLG-4803, was carbapenem-resistant despite lacking a carbapenemase gene (Supplementary Data 3). However, it did contain a bla_CTX-M-15 ESBL gene in its chromosome (Supplementary Data 3).

In vitro phenotypes of convergent isolates

Hypervirulent isolates frequently are resistant to complement-mediated killing in pooled human serum and have mucoid and highly viscous capsules (hmv)¹⁵. We measured these phenotypes in our 12 convergent isolates and compared them to those of typical hvKP (n = 11), MDR-cKP (n = 11), and relatively antibiotic-susceptible cKP (NON-MDR-cKP, n = 6) isolates (Fig. 5 and Supplementary Data 2). The convergent isolates, along with the MDR-cKP isolates, exhibited a large range in serum sensitivity, perhaps due to diversity in capsule and o-antigen types (Fig. 5A and Supplementary Data 2). However, the differences in median serum resistance between convergent isolates and the other groups were not statistically significant (Fig. 5A). Convergent isolates produced slightly more capsule than MDR-cKP and NON-MDR-cKP isolates and slightly less than hvKP isolates, although these differences were not statistically significant (Fig. 5B). As expected, hvKP isolates made more capsule than MDR-cKP and NON-MDR-cKP. To measure the hypermucoviscosity of these capsules, we performed centrifugation assays. As a group, convergent isolates were no more hypermucoviscous than MDR-cKP and NON-MDR-cKP isolates, whereas hvKP isolates were significantly more hypermucoviscous than the other isolates (Fig. 5D). These results were mostly consistent with a string test analysis to measure hmv; only one convergent isolate (K014) had a positive string test, whereas all hvKP isolates did (Fig. S2B and Supplementary Data 2). Interestingly, some string-test-negative K1 isolates were as resistant to sedimentation by centrifugation as string-test-positive isolates with other capsule types (e.g., TK77 and DHQP1701672) (Fig. S2A). These findings show that convergent isolates more closely resemble cKP isolates than hvKP isolates in capsule-related phenotypes.

**Fig. 5: Phenotypic comparison of convergent, MDR-cKP, hvKP, and NON-MDR-cKP isolates.**

Two convergent isolates, KPN226 and DHQP1701672, were hmv-negative despite containing rmpADC loci that usually confer an hmv colony morphology. Upon closer inspection, we found that KPN226 contained an early stop codon mutation in rmpA that likely disrupted its function. DHQP1701672 contained SNVs immediately upstream of rmpA and rmpD that may have altered their expression. Alternatively, DHQP1701672 resisted sedimentation to a degree consistent with other hmv⁺ isolates (Fig. S2A), suggesting that the string test did not adequately measure the hypermucoviscosity of this isolate. One MDR-cKP and three NON-MDR-cKP isolates were also hmv (Fig. S2B), consistent with other reports that cKP isolates may also be hmv²³.

It has been suggested that high levels of siderophore production may account for hypervirulence^43,44. We therefore measured total siderophore production by the four sets of isolates. Convergent isolates secreted significantly more siderophores than MDR-cKP or NON-MDR-cKP but similar amounts as hvKP isolates (Fig. 5C). Among the 12 convergent isolates, 11 contained iuc loci: 6 contained iuc1 (an hvKP-associated lineage) and 5 contained iuc5 (an E. coli-associated lineage) (Supplementary Data 2). Convergent isolates secreted variable amounts of siderophores, and several isolates of both types (iuc1 and iuc5) secreted amounts of siderophores comparable to hvKP isolates (Fig. S3). Overall, 20 (87%) of the 23 iuc⁺ isolates secreted more siderophores than the iuc^- isolates, suggesting that the iuc locus makes a substantial contribution to total siderophore production. iuc1 and iuc2 loci were present in hvKP isolates and were consistently associated with high levels of siderophore production (Fig. S3)¹¹. Overall, having an iuc locus was associated with an increase in siderophore production (two-way ANOVA; p < 0.001), but no statistically significant differences were observed in siderophore production between isolates with different iuc lineages (Tukey’s HSD; p > 0.05). These findings indicate that convergent isolates are capable of secreting large amounts of siderophores, and that this phenotype is not linked to a specific aerobactin lineage.

Virulence of convergent isolates

Although pathogenic genes and in vitro phenotypes are useful markers, the true virulence of a bacterial strain is arguably reflected by its capacity to cause disease in a relevant animal model. For this reason, we quantified the virulence of the 12 convergent isolates using a mouse model of pneumonia by measuring 50% lethal dose (LD₅₀). Convergent isolates had a median LD₅₀ value of ~10⁸ CFU, nearly identical to that of MDR-cKP and only slightly more (i.e., less virulent) than NON-MDR-cKP (Fig. 6). In contrast, hvKP isolates had a median LD₅₀ value of ~10³ CFU, significantly lower (i.e., more virulent) than that of convergent isolates. Only one convergent isolate, K014, had an LD₅₀ value comparable to hvKP isolates (10^4.2 CFU) (Fig. S4A). K014 is an ST268 isolate collected from a patient with meningitis at NMH in 2016. This isolate, which was resistant to third-generation cephalosporins, contained rmpADC, iuc, and iro loci, and is the only convergent isolate that was hmv. Interestingly, the CDC ST23 carbapenem-resistant isolate (DHQP1701672) was not hypermucoviscous (by string test) and had an LD₅₀ value of 10^6.6, over four logs higher (i.e., lower virulence) than that of other ST23 isolates, despite containing a complete pK2044-like plasmid with intact genes in the rmpADC, iro, and iuc loci (Figs. 4A, S4B). Thus, most convergent isolates had relatively low virulence levels despite containing virulence genes that are associated with hypervirulence.

**Fig. 6: Virulence comparison of convergent, MDR-cKP, hvKP, and NON-MDR-cKP isolates.**

Discussion

Historically, K. pneumoniae has existed as two distinct groups: cKP and hvKP. However, recent isolates have emerged with features of both cKP and hvKP, raising the specter of highly invasive strains that are resistant to treatment. Our findings are reassuring and suggest that convergent isolates remain rare in the U.S. Furthermore, although our sample size was small, most of the examined convergent isolates did not have enhanced virulence, as measured by a mouse model of pneumonia. Of note, though, one ESBL-producing isolate did have virulence comparable to typical hvKP isolates, indicating that vigilance for convergent isolates must remain a priority (Fig. 6).

Despite a variety of combinations of virulence genes and strain backgrounds in our 12 convergent isolates, only one had an LD₅₀ value comparable to hvKP strains (Fig. 5D). These observations are consistent with those of Martin and colleagues, who described convergent ST147 and ST307 strains from Italy and Germany, respectively, containing iuc, rmpADC, and rmpA2 loci³². Both strains had unexpectedly attenuated virulence in a mouse subcutaneous infection model, similar to a cKP control strain. However, our collection is small and did not contain representatives of strains recently linked to severe human infections in Asia³³. It remains possible that interrogation of a larger and international collection of convergent isolates would yield a higher proportion of virulent convergent isolates.

There are several potential explanations for the low virulence of our convergent isolates: (1) It is possible that the aerobactin biosynthesis locus may not be sufficient to enhance virulence to the level of hvKP strains. (2) It is conceivable that the E. coli aerobactin lineage (iuc5) confers a lower level of virulence than iuc1 or iuc2, which are usually present in hvKP strains. (3) Convergent isolates exhibit diverse capsule types, some of which may offer lower resistance to phagocytosis or complement compared to hvKP-associated KL1 and KL2 types⁴⁵. (4) The proteins encoded by the rmpADC operons located on plasmids may fail to interact with their cognate chromosomal targets in strains with capsule types or sequence types not typically associated with hvKP. (5) Convergent isolates may have acquired mutations in important pathogenicity genes, as has been reported by others³⁴. In our convergent isolates, we observed mutations in the rmpA and rmpA2 genes. In addition, OC1092 had a frameshift mutation in a gene (wzi) necessary for capsule production. Additional studies and examination of larger numbers of convergent isolates may uncover more reasons for the low virulence of many convergent strains.

Only one (K014) of our 12 convergent isolates was hmv, and this isolate was also the only isolate with high virulence in the mouse model. These findings suggest hmv contributes to the overall virulence of convergent isolates and are consistent with one possible explanation for the attenuation of convergent isolates. Since the evolution of convergent isolates presumably requires acquisition of mobile elements (containing either antibiotic resistance or virulence genes), it is possible that this evolution is restricted by hmv itself. The thick capsules associated with hmv may prevent the transfer of mobile elements, limiting the evolution of convergent isolates to either donor or recipient hvKP strains that have lost the ability to be hmv. In the former case, the defective hvKP donor strain transfers a virulence plasmid containing mutated activators of hmv to an antibiotic-resistant strain, and in the latter case, the defective recipient hvKP strain receives an antimicrobial resistance (AMR) plasmid from an antibiotic-resistant strain. In both cases, the resulting convergent strain is not hmv and presumably not highly virulent. In agreement with this hypothesis, most convergent isolates in our study acquired only siderophore-encoding fragments of virulence plasmids that did not confer the hmv phenotype. Most of those few convergent isolates that acquired AMR plasmids (e.g., DHQP1701672) presumably had mutated to become non-hmv recipients. However, it is important to note that one of our isolates (K014) was indeed hmv, highly virulent, and highly antibiotic resistant, highlighting the fact that if such plasmid transfer barriers do exist, they can be overcome.

Although not an epidemiological study, our results suggest that convergent isolates remain rare in the U.S. Of the 2608 highly antibiotic-resistant K. pneumoniae isolates we screened, only 1.8% contained hvKP-associated genes. This proportion is less than the proportions observed in other studies within the U.S., likely because we focused on collections of antibiotic-resistant isolates rather than bloodstream or liver abscess isolates, which would be expected to be enriched for virulence genes^7,10,46,47. Our prevalence of 1.8% was also lower than the 7.3% prevalence of convergent isolates observed in South and Southeastern Asia bloodstream isolates, perhaps because the higher prevalence of hvKP isolates in Asia facilitates the emergence of convergent strains²⁹.

hvKP strains produce large amounts of siderophores, which may contribute to their ability to thrive in the iron-limited host environment⁴⁸. Here, we confirm that hvKP isolates produce significantly more siderophores than MDR-cKP or NON-MDR-cKP isolates. Interestingly, convergent isolates also produce significantly more siderophores than MDR-cKP or NON-MDR-cKP isolates, and four convergent isolates (ARLG-3346, K014, OC1092, and KPN1402) produced more siderophores than the median amount produced by hvKP isolates. Among the different siderophores, aerobactin is thought to play a major role in hvKP virulence and is responsible for the high amounts of siderophores produced by some hvKP strains^48,49. Although 11 of the 12 convergent isolates we tested had iuc loci (and 6 had the hvKP-associated iuc1 loci), only one (K014) was highly virulent in mice. This suggests that either these iuc loci are non-functional and do not produce substantial amounts of aerobactin or that aerobactin production by itself is not sufficient to confer high levels of virulence in mice. In either case, our findings suggest that the presence of aerobactin biosynthesis genes alone should not be used in future studies to make inferences about the virulence of K. pneumoniae strains. Rather, the total number of virulence factors should be considered when attempting to predict strain virulence. We also emphasize that our study examined associations between pathogenic determinants and virulence; additional genetic studies will be necessary to determine which determinants play a causal role in virulence.

Our study has several limitations. First, we examined isolates from only parts of the U.S., thus our results may not be representative of those occurring in other parts of the world. For example, ST11 and ST23 convergent isolates were rare in our study but are common in parts of Asia²⁹. Second, we screened large collections of antibiotic-resistant cKP for virulence genes; the approach of screening isolate collections from clinically severe or invasive infections for hvKP-associated genes and antibiotic-resistance genes may yield different types of convergent isolates²⁵. Third, we examined only a relatively small number of convergent isolates of limited types for virulence in mice. Nonetheless, our results may serve as a foundation for larger studies in the future. Fourth, we used a mouse model of pneumonia to quantify virulence, and it is possible that other infection models (e.g., intraperitoneal inoculation) may yield different results. However, the pneumonia model has been widely used to quantify the virulence of hvKP strains and nicely distinguishes hvKP from cKP^11,50. Fifth, we cannot exclude the possibility that convergent isolates acquired virulence-attenuating mutations during passaging in our laboratory. However, we minimized the numbers of passages each isolate underwent, and we did not observe attenuation in the 11 similarly passaged hvKP isolates used as comparators. For these reasons, we feel this possibility is less likely. Finally, firm conclusions on the clinical risk associated with convergent isolates requires correlations with clinical outcomes in patients from which these isolates were cultured.

In conclusion, most of the convergent isolates in our study were not as highly virulent as hvKP strains. While containing these additional virulence factors is likely to pose an increased clinical risk in humans, our data suggests that these isolates are unlikely to cause the type of infections typically described for hypervirulent strains³⁶. These findings suggest that additional studies should be performed to clarify whether convergent strains are indeed more virulent than cKP in mouse and human infections. However, strains that are both highly virulent and highly antibiotic resistant do occur, highlighting the need for continued study of convergent isolates.

Methods

Ethical regulations

Experiments using K. pneumoniae isolates were conducted in compliance with the Northwestern Institutional Biosafety Committee. All animal procedures were conducted in accordance with the Northwestern University Animal Care and Use Committee (protocol IS00002172).

Bacterial isolates and growth conditions

This study used three distinct collections of drug-resistant K. pneumoniae isolates collected from U.S. hospitals (Supplementary Data 1). In addition, typical MDR-cKP, NON-MDR-cKP, and hvKP isolates used for comparisons with convergent isolates are listed in Supplementary Data 2. The group of typical hvKP isolates included canonical hypervirulent STs (ST23, ST86, ST66, and ST380) that each contained a large virulence plasmid and was hmv (Supplementary Data 2). In addition, strain KPPR1 was added to the hvKP group because it is commonly used to study hvKP⁵¹. The MDR-cKP group included typical antibiotic-resistant high-risk clones (e.g., ST258, ST11, ST16, ST15, and ST14) that did not contain iucA, iroB, rmpA, or rmpA2 genes. The NON-MDR-cKP group included 5 relatively antibiotic susceptible cKP clinical isolates. In this last group, we included for comparison an hvKP isolate, hvKP5, that had been cured of its virulence plasmid (designated TK77; Supplementary Data 2).

Selected bacterial isolates were recovered from frozen stocks. Bacteria were grown at 37 °C in lysogeny broth (LB) or on LB agar plates.

Genome sequences

Illumina sequencing reads were obtained from NCBI for bioprojects PRJNA658369 (CRACKLE2), PRJNA514245 (Houston-Methodist) and PRJNA788509 and PRJNA395086 (NMH)^{10,37,38,39,52}. Reads were downloaded from NCBI using Sratoolkit v2.11.1. For new strains sequenced as part of this study, a single colony was cultured overnight at 37 °C in LB and used to prepare genomic DNA using the Maxwell 16 system (Promega Corp., Madison, WI, USA). To generate paired-end reads of either 300 bp or 150 bp, Illumina sequencing libraries were created using either the Nextera XT kit (Illumina, Inc., San Diego, CA) or the Seqwell kit (Seqwell, MA, USA). The libraries were then sequenced on an Illumina MiSeq or NextSeq 500 instrument. Reads were trimmed using Trimmomatic (v0.36)^8,53. De novo assembly was performed using SPAdes (v3.9.1)^53,54. Contigs were filtered based on coverage (minimum 5x) in trimmed reads using a custom perl script.

Nanopore sequencing was performed according to previously described protocols^{10,55,56,57,58}. Libraries were prepared using the ligation sequencing kit (SQK-LSK109 or SQK-LSK114, Oxford Nanopore, UK) and sequenced on either a MinION or GridION instrument using either a FLO-MIN106 or FLO-MIN114 flow cell. Sequence reads were demultiplexed and base-called using Guppy (v3.4.5)⁵⁹. Base-called reads were filtered using Filtlong (v0.2.1) removing any reads shorter than 1000 bp. Assemblies were completed using the Trycycler pipeline (v0.5.4)⁶⁰. Filtered reads were subsampled and assembled using Flye (v2.9), Unicycler (v0.5.0), and Raven (v2.3)^61,62,63 Generated contigs were clustered, reconciled, and consensus sequences were generated. Consensus sequences were polished using long-reads (Medaka v1.9.1) and short-reads (Polypolish v0.5.0 and Polca as part of the MaSurCA package v4.1.0) (Supplementary Data 4)^64,65. Finally, annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline⁶⁶.

Sequence analysis

Assembled whole-genome sequences were evaluated for multilocus sequence type, capsule locus type, antimicrobial resistance gene content and the presence of virulence genes using the bioinformatics tools Kleborate v2.1 and Kaptive v0.7.3 with default settings^42,67. BLAST Ring Image generator (BRIG) was used to align plasmids with a sequence similarity threshold of 85%⁶⁸. MASH (v2.3) was utilized to cluster plasmids based on Jaccard index⁶⁹. Cytoscape (v3.8.2) was used to create plasmid networks. MOB-suite (v3.1.7) was used to identify plasmid replicons⁷⁰. Maximum-likelihood phylogenetic trees were constructed using FastTree 2 (v4.0.3) based on the core genome⁷¹. The core genome was defined as sequences present in 95% of isolates⁷¹. Identification of single nucleotide polymorphisms (SNPs) was performed by aligning raw Illumina reads to the genome of the reference strain NTUH-K2044 using bwa-0.7.15. Mashtree (v1.4.3) was used for clustering of individual plasmids^72,73. The phylogenetic trees were visualized and annotated using iTOL (v4)⁷⁴.

Hypermucoviscosity testing

Hypermucoviscosity was measured by string test and by centrifugation. The string test was performed as described previously⁷⁵. Briefly, isolates were grown overnight at 37 °C on LB agar. A colony was lifted with a loop to evaluate the formation of a viscous string between the loop and the colony. A positive string test was defined as a string length ≥5 mm. For centrifugation assays, isolates were cultured overnight on LB agar at 37 °C from frozen stocks. Multiple colonies were picked and cultured overnight in LB at 37 °C with shaking. Cultures were centrifuged at 2000 × g for 5 min, and the optical density at 600 nm (OD₆₀₀) of the supernatant was measured. Data are presented as the OD₆₀₀ post-centrifugation divided by OD₆₀₀ pre-centrifugation.

Serum survival

Pooled, flash-frozen human serum (Innovative Research) was stored at −80 °C. Serum was thawed at room temperature. A portion of the human serum was heat-inactivated by incubation at 55 °C for 1 h. K. pneumoniae were grown overnight in 5 mL of LB and then subcultured in fresh LB to an OD₆₀₀ = 2.5 ± 0.1. Bacteria were then pelleted by centrifugation at 3041 × g for 20 min. The bacterial pellets were resuspended in 1 mL PBS and adjusted to 2 × 10⁶ CFU/mL. Then 100 μL of bacterial suspension was added to 900 μL of active or heat-inactivated human serum and gently vortexed. Experiments were performed in triplicate and bacteria were serial diluted and enumerated following 0 and 180 min of incubation at 37 °C.

Siderophore detection

Siderophores were quantified by the Chrome-azurol S assay⁷⁶. Briefly, 5 mL aliquots of LB medium were inoculated with single colonies of bacteria and grown overnight with aeration at 37 °C. One mL aliquots of overnight cultures were harvested by centrifugation (2 min, 21,130 × g), washed twice with M9 minimal medium supplemented with glucose, and resuspended in 1 mL M9 minimal medium⁷⁶. A total of 50 μL of bacterial resuspension was inoculated into 5 mL of fresh M9 minimal medium and grown for approximately 7 h at 37 °C with aeration to an OD₆₀₀ ∼ 1.3⁷⁶. After centrifugation at 4300 × g for 20 min at 4 °C, supernatant was carefully collected and passed through a 0.22 μm filter. Next, 150 μL of supernatant was mixed with 150 μL of CAS shuttle solution⁷⁶. In addition, serially diluted concentrations of the iron chelator dipyridyl in M9 medium were used to generate a standard curve. The plate was incubated at room temperature for 15 min in the dark, and absorbance was measured at OD₆₃₀ nm using a BioTek plate reader.

Capsule quantification

K. pneumoniae capsule was purified⁵¹. Briefly, bacteria were cultured overnight in LB medium and then subcultured to an OD₆₀₀ = 2.5 ± 0.1 in fresh LB. A total of 500 μL was combined with 100 μL of 1% Zwittergent 3-14 in 100 mM citric acid buffer, pH 2. This mixture was incubated at 50 °C for 30 min with occasional mixing. Bacterial cells were pelleted by centrifugation at 17,000 × g for 2 min, after which 250 μL of supernatant was combined with 1 mL of absolute ethanol (final concentration of 80%) and placed on ice for 30 min. Suspensions were cleared by centrifugation at 17,000 × g for 5 min, and the supernatant was decanted. Pelleted samples were incubated at 37 °C for 30 min or until the precipitates were dry. Then, 200 μL of ddH₂O was added, and the precipitate was allowed to redissolve for 1 h at 37 °C. A total of 1 mL of 12.5 mM sodium tetraborate in concentrated sulfuric acid was added, and the samples were boiled for 5 min at 100 °C and allowed to cool to room temperature. Next, 20 μL of 0.15% 3-hydroxydiphenyl in 0.5% sodium hydroxide was added; 20 μL of 0.5% sodium hydroxide was added to the control set for a background measurement. Absorbance was read at 520 nm.

Mouse studies

Six- to eight-week-old C57BL/6 female mice were purchased from Jackson Labs and infected intranasally¹⁰. Briefly, K. pneumoniae was recovered from frozen stocks and grown overnight on LB agar at 37 °C and then subcultured in LB medium at 37 °C with shaking for 3 hours the day of infection. Bacteria were washed 3 times in PBS, resuspended in PBS, and then diluted with PBS to the indicated doses by measuring OD₆₀₀. Inocula sizes were confirmed by plating bacteria for colony enumeration. Mice were anesthetized by intraperitoneal injection with a mixture of ketamine (100 mg/kg) and xylazine (20 mg/kg). A total of 50 μl of K. pneumoniae suspension was placed on the nares of mice (25 μl per nare) to allow for aspiration into the lungs. Mice were monitored for pre-lethal illness over the subsequent two-week period. Mice were humanely euthanized and scored as dead when they met pre-determined criteria: >20% weight loss, abnormal breathing, or a hunched posture with limited movement. The experimenters were blinded to the strain and dose used. The LD₅₀ was calculated from doses and mouse outcomes (development of pre-lethal illness) using the R package drc⁷⁷. Detailed data, including the strain dose, number of deaths, and total mice infected, are provided in Supplementary Data 5. Sex was not included in the study design as previous experiments found no difference in K. pneumoniae infections among male and female mice. All procedures were conducted in accordance with the Northwestern University Animal Care and Use Committee (protocol IS00002172). Mice were housed at the Northwestern Center for Comparative Medicine at Temperatures of 65–75 °F (~18–23 °C) with 40-60% humidity with a 12:12 light dark cycle.

Statistics and reproducibility

Statistical analyses were done using One-Way ANOVA as part of GraphPad Prism 10. No statistical method was used to predetermine sample size. No data were excluded from the analyses. Groups of mice were determined at random and investigators were blinded to the strains paired with each group of mice.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The whole-genome assemblies of bacteria sequenced for this study have been deposited at GenBank under BioProject number PRJNA788509. Assembly accession numbers are included in Supplementary Data 4. Kleborate data are included in Supplementary Data 1. Phenotypic data for individual strains are found in Supplementary Data 2. There are no restrictions on data availability. Source data are provided with this paper.

Code availability

Code used in this work is available on github.com/tkochan.

References

Antimicrobial Resistance, C. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 399, 629–655 (2022).
Article Google Scholar
Podschun, R. & Ullmann, U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin. Microbiol. Rev. 11, 589–603 (1998).
Article CAS PubMed PubMed Central Google Scholar
Russo, T. A. & Marr, C. M. Hypervirulent Klebsiella pneumoniae. Clin. Microbiol. Rev. 32, e00001–e00019 (2019).
Article PubMed PubMed Central Google Scholar
Marcade, G. et al. The emergence of multidrug-resistant Klebsiella pneumoniae of international clones ST13, ST16, ST35, ST48 and ST101 in a teaching hospital in the Paris region. Epidemiol. Infect. 141, 1705–1712 (2013).
Article CAS PubMed Google Scholar
Meng, X. et al. Assessing molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CR-KP) with MLST and MALDI-TOF in central China. Sci. Rep. 9, 2271 (2019).
Article PubMed PubMed Central Google Scholar
Fang, F. C., Sandler, N. & Libby, S. J. Liver abscess caused by magA+ Klebsiella pneumoniae in North America. J. Clin. Microbiol. 43, 991–992 (2005).
Article PubMed PubMed Central Google Scholar
Pastagia, M. & Arumugam, V. Klebsiella pneumoniae liver abscesses in a public hospital in Queens, New York. Travel Med. Infect. Dis. 6, 228–233 (2008).
Article PubMed Google Scholar
Inniss, N. L. et al. A structural systems biology approach to high-risk CG23 Klebsiella pneumoniae. Microbiol. Resour. Announc. 12, e0101322 (2023).
Article PubMed Google Scholar
Wyres, K. L., Lam, M. M. C. & Holt, K. E. Population genomics of Klebsiella pneumoniae. Nat. Rev. Microbiol. 18, 344–359 (2020).
Article CAS PubMed Google Scholar
Kochan, T. J. et al. Genomic surveillance for multidrug-resistant or hypervirulent Klebsiella pneumoniae among United States bloodstream isolates. BMC Infect. Dis. 22, 603 (2022).
Article CAS PubMed PubMed Central Google Scholar
Russo, T. A. et al. Identification of biomarkers for differentiation of hypervirulent Klebsiella pneumoniae from classical K. pneumoniae. J. Clin. Microbiol. 56, e00776–00718 (2018).
Article CAS PubMed PubMed Central Google Scholar
Iredell, J., Brown, J. & Tagg, K. Antibiotic resistance in Enterobacteriaceae: mechanisms and clinical implications. BMJ 352, h6420 (2016).
Article PubMed Google Scholar
Boyle, D. P. & Zembower, T. R. Epidemiology and management of emerging drug-resistant gram-negative bacteria: extended-spectrum beta-lactamases and beyond. Urol. Clin. North Am. 42, 493–505 (2015).
Article PubMed Google Scholar
Wang, D., Chen, J., Yang, L., Mou, Y. & Yang, Y. Phenotypic and enzymatic comparative analysis of the KPC variants, KPC-2 and its recently discovered variant KPC-15. PLoS ONE 9, e111491 (2014).
Article PubMed PubMed Central ADS Google Scholar
Paczosa, M. K. & Mecsas, J. Klebsiella pneumoniae: going on the offense with a strong defense. Microbiol. Mol. Biol. Rev. 80, 629–661 (2016).
Article CAS PubMed PubMed Central Google Scholar
Patel, P. K., Russo, T. A. & Karchmer, A. W. Hypervirulent Klebsiella pneumoniae. Open Forum Infect. Dis. 1, ofu028 (2014).
Article PubMed PubMed Central Google Scholar
Shon, A. S., Bajwa, R. P. & Russo, T. A. Hypervirulent (hypermucoviscous) Klebsiella pneumoniae: a new and dangerous breed. Virulence 4, 107–118 (2013).
Article PubMed PubMed Central Google Scholar
Lai, Y. C., Peng, H. L. & Chang, H. Y. RmpA2, an activator of capsule biosynthesis in Klebsiella pneumoniae CG43, regulates K2 cps gene expression at the transcriptional level. J. Bacteriol. 185, 788–800 (2003).
Article CAS PubMed PubMed Central Google Scholar
Walker, K. A., Treat, L. P., Sepulveda, V. E. & Miller, V. L. The small protein RmpD drives hypermucoviscosity in Klebsiella pneumoniae. mBio https://doi.org/10.1128/mBio.01750-20 (2020)
Wyres, K. L. et al. Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae. PLoS Genet. 15, e1008114 (2019).
Article CAS PubMed PubMed Central Google Scholar
Lam, M. M. C. et al. Genetic diversity, mobilisation and spread of the yersiniabactin-encoding mobile element ICEKp in Klebsiella pneumoniae populations. Microb. Genom. https://doi.org/10.1099/mgen.0.000196 (2018).
Harada, S. & Doi, Y. Hypervirulent Klebsiella pneumoniae: a call for consensus definition and international collaboration. J. Clin. Microbiol. https://doi.org/10.1128/JCM.00959-18 (2018).
Ernst, C. M. et al. Adaptive evolution of virulence and persistence in carbapenem-resistant Klebsiella pneumoniae. Nat. Med. 26, 705–711 (2020).
Article CAS PubMed PubMed Central Google Scholar
Zhang, Y. et al. High prevalence of hypervirulent Klebsiella pneumoniae infection in China: geographic distribution, clinical characteristics, and antimicrobial resistance. Antimicrob. Agents Chemother. 60, 6115–6120 (2016).
Article CAS PubMed PubMed Central Google Scholar
Liu, C. & Guo, J. Characteristics of ventilator-associated pneumonia due to hypervirulent Klebsiella pneumoniae genotype in genetic background for the elderly in two tertiary hospitals in China. Antimicrob. Resist Infect. Control 7, 95 (2018).
Article PubMed PubMed Central Google Scholar
Li, N. et al. Rapid detection of Klebsiella pneumoniae carrying virulence gene rmpA2 by recombinase polymerase amplification combined with lateral flow strips. Front. Cell Infect. Microbiol. 12, 877649 (2022).
Article CAS PubMed PubMed Central Google Scholar
Karlsson, M. et al. Identification of a carbapenemase-producing hypervirulent Klebsiella pneumoniae Isolate in the United States. Antimicrob. Agents Chemother. https://doi.org/10.1128/AAC.00519-19 (2019).
Turton, J. F. et al. Virulence genes in isolates of Klebsiella pneumoniae from the UK during 2016, including among carbapenemase gene-positive hypervirulent K1-ST23 and ‘non-hypervirulent’ types ST147, ST15 and ST383. J. Med. Microbiol. 67, 118–128 (2018).
Article CAS PubMed Google Scholar
Wyres, K. L. et al. Genomic surveillance for hypervirulence and multi-drug resistance in invasive Klebsiella pneumoniae from South and Southeast Asia. Genome Med. 12, 11 (2020).
Article CAS PubMed PubMed Central Google Scholar
Cheong, H. S. et al. Emergence of an extended-spectrum beta-lactamase-producing serotype K1 Klebsiella pneumoniae ST23 strain from Asian countries. Epidemiol. Infect. 145, 990–994 (2017).
Article CAS PubMed Google Scholar
Huang, Y. H. et al. Emergence of an XDR and carbapenemase-producing hypervirulent Klebsiella pneumoniae strain in Taiwan. J. Antimicrob. Chemother. 73, 2039–2046 (2018).
Article PubMed Google Scholar
Martin, M. J. et al. Anatomy of an extensively drug-resistant Klebsiella pneumoniae outbreak in Tuscany, Italy. Proc. Natl. Acad. Sci. USA https://doi.org/10.1073/pnas.2110227118 (2021).
Gu, D. et al. A fatal outbreak of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae in a Chinese hospital: a molecular epidemiological study. Lancet Infect. Dis. 18, 37–46 (2018).
Article PubMed Google Scholar
Yang, X. et al. Molecular epidemiology of carbapenem-resistant hypervirulent Klebsiella pneumoniae in China. Emerg. Microbes Infect. 11, 841–849 (2022).
Article CAS PubMed PubMed Central Google Scholar
Du, Q. et al. Nosocomial dissemination of hypervirulent Klebsiella pneumoniae with high-risk clones among children in Shanghai. Front. Cell Infect. Microbiol. 12, 984180 (2022).
Article CAS PubMed PubMed Central Google Scholar
Jia, X. et al. Genomic epidemiology study of Klebsiella pneumoniae causing bloodstream infections in China. Clin. Transl. Med. 11, e624 (2021).
Article PubMed PubMed Central Google Scholar
Bulman, Z. P. et al. Genomic features associated with the degree of phenotypic resistance to carbapenems in carbapenem-resistant Klebsiella pneumoniae. mSystems 6, e0019421 (2021).
Article PubMed Google Scholar
van Duin, D. et al. Molecular and clinical epidemiology of carbapenem-resistant Enterobacterales in the USA (CRACKLE-2): a prospective cohort study. Lancet Infect. Dis. 20, 731–741 (2020).
Article PubMed PubMed Central Google Scholar
Long, S. W. et al. Population genomic analysis of 1,777 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae Isolates, Houston, Texas: unexpected abundance of clonal group 307. mBio https://doi.org/10.1128/mBio.00489-17 (2017).
Khalifa, S. M., Abd El-Aziz, A. M., Hassan, R. & Abdelmegeed, E. S. beta-lactam resistance associated with beta-lactamase production and porin alteration in clinical isolates of E. coli and K. pneumoniae. PLoS ONE 16, e0251594 (2021).
Article CAS PubMed PubMed Central Google Scholar
Shropshire, W. C. et al. Systematic analysis of mobile genetic elements mediating beta-lactamase gene amplification in noncarbapenemase-producing carbapenem-resistant enterobacterales bloodstream infections. mSystems 7, e0047622 (2022).
Article CAS PubMed Google Scholar
Lam, M. M. C. et al. A genomic surveillance framework and genotyping tool for Klebsiella pneumoniae and its related species complex. Nat. Commun. 12, 4188 (2021).
Article CAS PubMed PubMed Central ADS Google Scholar
Russo, T. A. et al. Hypervirulent K. pneumoniae secretes more and more active iron-acquisition molecules than “classical” K. pneumoniae thereby enhancing its virulence. PLoS ONE 6, e26734 (2011).
Article CAS PubMed PubMed Central ADS Google Scholar
Russo, T. A. et al. Aerobactin mediates virulence and accounts for increased siderophore production under iron-limiting conditions by hypervirulent (hypermucoviscous) Klebsiella pneumoniae. Infect. Immun. 82, 2356–2367 (2014).
Article PubMed PubMed Central Google Scholar
Lin, J. C. et al. Impaired phagocytosis of capsular serotypes K1 or K2 Klebsiella pneumoniae in type 2 diabetes mellitus patients with poor glycemic control. J. Clin. Endocrinol. Metab. 91, 3084–3087 (2006).
Article CAS PubMed Google Scholar
Parrott, A. M. et al. Detection of multiple hypervirulent Klebsiella pneumoniae strains in a New York City hospital through screening of virulence genes. Clin. Microbiol. Infect. https://doi.org/10.1016/j.cmi.2020.05.012 (2020)
Chou, A. et al. Prevalence of hypervirulent Klebsiella pneumoniae-associated genes rmpA and magA in two tertiary hospitals in Houston, TX, USA. J. Med. Microbiol. 65, 1047–1048 (2016).
Article CAS PubMed PubMed Central ADS Google Scholar
Russo, T. A., Olson, R., MacDonald, U., Beanan, J. & Davidson, B. A. Aerobactin, but not yersiniabactin, salmochelin, or enterobactin, enables the growth/survival of hypervirulent (hypermucoviscous) Klebsiella pneumoniae ex vivo and in vivo. Infect. Immun. 83, 3325–3333 (2015).
Article CAS PubMed PubMed Central Google Scholar
Hsieh, P. F., Lin, T. L., Lee, C. Z., Tsai, S. F. & Wang, J. T. Serum-induced iron-acquisition systems and TonB contribute to virulence in Klebsiella pneumoniae causing primary pyogenic liver abscess. J. Infect. Dis. 197, 1717–1727 (2008).
Article CAS PubMed Google Scholar
Russo, T. A. & MacDonald, U. The Galleria mellonella infection model does not accurately differentiate between hypervirulent and classical Klebsiella pneumoniae. mSphere https://doi.org/10.1128/mSphere.00850-19 (2020).
Mike, L. A. et al. A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness. PLoS Pathog. 17, e1009376 (2021).
Article CAS PubMed PubMed Central Google Scholar
Huang, Y. et al. Aminoglycoside-resistance gene signatures are predictive of aminoglycoside MICs for carbapenem-resistant Klebsiella pneumoniae. J. Antimicrob. Chemother. 77, 356–363 (2022).
Article CAS PubMed Google Scholar
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
Article CAS PubMed PubMed Central Google Scholar
Prjibelski, A., Antipov, D., Meleshko, D., Lapidus, A. & Korobeynikov, A. Using SPAdes de novo assembler. Curr. Protoc. Bioinforma. 70, e102 (2020).
Article CAS Google Scholar
Cherny, K. E., Ozer, E. A., Kochan, T. J., Johnson, S. & Kociolek, L. K. Complete genome sequence of Clostridium innocuum strain LC-LUMC-CI-001, isolated from a patient with recurrent antibiotic-associated diarrhea. Microbiol. Resour. Announc. https://doi.org/10.1128/MRA.00365-20 (2020).
Cherny, K. E., Ozer, E. A., Kochan, T. J. & Kociolek, L. K. Complete genome sequence of Clostridium innocuum strain ATCC 14501. Microbiol. Resour. Announc. https://doi.org/10.1128/MRA.00452-20 (2020).
Cheung, B. H. et al. Genome-wide screens reveal shared and strain-specific genes that facilitate enteric colonization by Klebsiella pneumoniae. mBio https://doi.org/10.1128/mbio.02128-23 (2023)
Turner, T. L. et al. Taxonomic characterization of Pseudomonas hygromyciniae sp. nov., a novel species discovered from a commercially purchased antibiotic. Microbiol. Spectr. 11, e0183821 (2023).
Article PubMed Google Scholar
Wick, R. R., Judd, L. M. & Holt, K. E. Performance of neural network basecalling tools for Oxford Nanopore sequencing. Genome Biol. 20, 129 (2019).
Article PubMed PubMed Central Google Scholar
Wick, R. R. et al. Trycycler: consensus long-read assemblies for bacterial genomes. Genome Biol. 22, 266 (2021).
Article CAS PubMed PubMed Central Google Scholar
Wick, R. R., Judd, L. M., Gorrie, C. L. & Holt, K. E. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput. Biol. 13, e1005595 (2017).
Article PubMed PubMed Central ADS Google Scholar
Vaser, R. & Šikić, M. Time- and memory-efficient genome assembly with Raven. Nat. Comput. Sci. 1, 332–336 (2021).
Article Google Scholar
Kolmogorov, M., Yuan, J., Lin, Y. & Pevzner, P. A. Assembly of long, error-prone reads using repeat graphs. Nat. Biotechnol. 37, 540–546 (2019).
Article CAS PubMed Google Scholar
Wick, R. R. & Holt, K. E. Polypolish: Short-read polishing of long-read bacterial genome assemblies. PLoS Comput. Biol. 18, e1009802 (2022).
Article CAS PubMed PubMed Central ADS Google Scholar
Zimin, A. V. & Salzberg, S. L. The genome polishing tool POLCA makes fast and accurate corrections in genome assemblies. PLoS Comput. Biol. 16, e1007981 (2020).
Article CAS PubMed PubMed Central ADS Google Scholar
Tatusova, T. et al. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 44, 6614–6624 (2016).
Article CAS PubMed PubMed Central Google Scholar
Wick, R. R., Heinz, E., Holt, K. E. & Wyres, K. L. Kaptive web: user-friendly capsule and lipopolysaccharide serotype prediction for Klebsiella genomes. J. Clin. Microbiol. https://doi.org/10.1128/JCM.00197-18 (2018).
Alikhan, N. F., Petty, N. K., Ben Zakour, N. L. & Beatson, S. A. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genom. 12, 402 (2011).
Article CAS Google Scholar
Ondov, B. D. et al. Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol. 17, 132 (2016).
Article PubMed PubMed Central Google Scholar
Robertson, J. & Nash, J. H. E. MOB-suite: software tools for clustering, reconstruction and typing of plasmids from draft assemblies. Microb. Genom. https://doi.org/10.1099/mgen.0.000206 (2018).
Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2-approximately maximum-likelihood trees for large alignments. PLoS ONE 5, e9490 (2010).
Article PubMed PubMed Central ADS Google Scholar
Gardner, S. N., Slezak, T. & Hall, B. G. kSNP3.0: SNP detection and phylogenetic analysis of genomes without genome alignment or reference genome. Bioinformatics 31, 2877–2878 (2015).
Article CAS PubMed Google Scholar
Katz, L. S. et al. Mashtree: a rapid comparison of whole genome sequence files. J. Open Source Softw. https://doi.org/10.21105/joss.01762 (2019).
Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 49, W293–W296 (2021).
Article CAS PubMed PubMed Central Google Scholar
Hadano, Y. String test. BMJ Case Rep. 2013, bcr2012008328 (2013).
Himpsl, S. D. & Mobley, H. L. T. Siderophore detection using chrome azurol S and cross-feeding assays. Methods Mol. Biol. 2021, 97–108 (2019).
Article CAS PubMed Google Scholar
Ritz, C., Baty, F., Streibig, J. C. & Gerhard, D. Dose-response analysis using R. PLoS ONE 10, e0146021 (2015).
Article PubMed PubMed Central Google Scholar

Download references

Acknowledgements

This work was funded by American Heart Association grant 837089 (T.K.), Chicago Biomedical Consortium Catalyst Award (A.H., Z.B.), and National Institute of Health grants T32 AI007476 (T.K.), R01 AI118257 (A.H.), R21 AI153953 (A.H.), K24 AI104831 (A.H.), R21 AI164254 (A.H.), U19 AI35964 (A.H.), T32 AI095207 (R.M., A.H.), R01 AI173064 (Z.P.B., A.H., E.O.) The funding sources had no influence on the design of the study and collection, analysis, interpretation of data, or writing of the manuscript.

Author information

Authors and Affiliations

Laboratory of Respiratory and Special Pathogens, Division of Bacterial, Parasitic, and Allergenic Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA
Travis J. Kochan
Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
Travis J. Kochan, Sophia H. Nozick, Aliki Valdes, Sumitra D. Mitra, Bettina H. Cheung, Marine Lebrun-Corbin, Rachel L. Medernach, Madeleine B. Vessely, Jori O. Mills, Christopher M. R. Axline, Julia A. Nelson, Ethan M. VanGosen, Timothy J. Ward & Alan R. Hauser
Division of Infectious Diseases, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
Rachel L. Medernach, Egon A. Ozer & Alan R. Hauser
Division of Infectious Diseases, University of North Carolina, Chapel Hill, NC, USA
David van Duin
Center for Discovery and Innovation, Hackensack Meridian Health, Nutley, NJ, USA
Liang Chen & Barry N. Kreiswirth
Department of Pathology and Genomic Medicine, Houston Methodist Hospital Research Institute, Houston, TX, USA
S. Wesley Long & James M. Musser
College of Pharmacy, University of Illinois at Chicago, Chicago, IL, USA
Zackery P. Bulman
Division of Pulmonary and Critical Care Medicine, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
Richard G. Wunderink
Simpson Querrey Institute for Epigenetics, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
Richard G. Wunderink

Authors

Travis J. Kochan
View author publications
You can also search for this author in PubMed Google Scholar
Sophia H. Nozick
View author publications
You can also search for this author in PubMed Google Scholar
Aliki Valdes
View author publications
You can also search for this author in PubMed Google Scholar
Sumitra D. Mitra
View author publications
You can also search for this author in PubMed Google Scholar
Bettina H. Cheung
View author publications
You can also search for this author in PubMed Google Scholar
Marine Lebrun-Corbin
View author publications
You can also search for this author in PubMed Google Scholar
Rachel L. Medernach
View author publications
You can also search for this author in PubMed Google Scholar
Madeleine B. Vessely
View author publications
You can also search for this author in PubMed Google Scholar
Jori O. Mills
View author publications
You can also search for this author in PubMed Google Scholar
Christopher M. R. Axline
View author publications
You can also search for this author in PubMed Google Scholar
Julia A. Nelson
View author publications
You can also search for this author in PubMed Google Scholar
Ethan M. VanGosen
View author publications
You can also search for this author in PubMed Google Scholar
Timothy J. Ward
View author publications
You can also search for this author in PubMed Google Scholar
Egon A. Ozer
View author publications
You can also search for this author in PubMed Google Scholar
David van Duin
View author publications
You can also search for this author in PubMed Google Scholar
Liang Chen
View author publications
You can also search for this author in PubMed Google Scholar
Barry N. Kreiswirth
View author publications
You can also search for this author in PubMed Google Scholar
S. Wesley Long
View author publications
You can also search for this author in PubMed Google Scholar
James M. Musser
View author publications
You can also search for this author in PubMed Google Scholar
Zackery P. Bulman
View author publications
You can also search for this author in PubMed Google Scholar
Richard G. Wunderink
View author publications
You can also search for this author in PubMed Google Scholar
Alan R. Hauser
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

T.K. and A.H. designed the study. T.K., S.N., B.C., A.V., M.V., M.L.C., S.M., C.A., T.W., J.N., E.V., and J.O.M. performed the phenotypic experiments. T.K. and E.O. performed the computational analyses. R.M. and B.C. analyzed and interpreted the patient data. T.K., S.N., S.M., E.O., A.V., and A.H. initially analyzed the data. D.D., L.C., B.K., S.W.L., J.M.M., Z.B., and R.W. contributed to interpretation of the data. T.K. and A.H. wrote the paper, and all other authors contributed to the writing. All authors read and approved the manuscript.

Corresponding author

Correspondence to Travis J. Kochan.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks the anonymous, reviewers for their contribution to the peer review of this work. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Peer Review File

Description of Additional Supplementary Files

Supplementary Data 1

Supplementary Data 2

Supplementary Data 3

Supplementary Data 4

Supplementary Data 5

Reporting Summary

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Kochan, T.J., Nozick, S.H., Valdes, A. et al. Klebsiella pneumoniae clinical isolates with features of both multidrug-resistance and hypervirulence have unexpectedly low virulence. Nat Commun 14, 7962 (2023). https://doi.org/10.1038/s41467-023-43802-1

Download citation

Received: 07 July 2023
Accepted: 21 November 2023
Published: 02 December 2023
DOI: https://doi.org/10.1038/s41467-023-43802-1

This article is cited by

Comprehensive study reveals phenotypic heterogeneity in Klebsiella pneumoniae species complex isolates
- Nadia Rodríguez-Medina
- Jonathan Rodríguez-Santiago
- Ulises Garza-Ramos
Scientific Reports (2024)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.