Adaptive evolution of virulence and persistence in carbapenem-resistant Klebsiella pneumoniae

Abstract

Among the most urgent public health threats is the worldwide emergence of carbapenem-resistant Enterobacteriaceae^1-4, which are resistant to the antibiotic class of 'last resort'. In the United States and Europe, carbapenem-resistant strains of the Klebsiella pneumoniae ST258 (ref. ⁵) sequence type are dominant, endemic^6-8 and associated with high mortality^6,9,10. We report the global evolution of pathogenicity in carbapenem-resistant K. pneumoniae, resulting in the repeated convergence of virulence and carbapenem resistance in the United States and Europe, dating back to as early as 2009. We demonstrate that K. pneumoniae can enhance its pathogenicity by adopting two opposing infection programs through easily acquired gain- and loss-of-function mutations. Single-nucleotide polymorphisms in the capsule biosynthesis gene wzc lead to hypercapsule production, which confers phagocytosis resistance, enhanced dissemination and increased mortality in animal models. In contrast, mutations disrupting capsule biosynthesis genes impair capsule production, which enhances epithelial cell invasion, in vitro biofilm formation and persistence in urinary tract infections. These two types of capsule mutants have emerged repeatedly and independently in Europe and the United States, with hypercapsule mutants associated with bloodstream infections and capsule-deficient mutants associated with urinary tract infections. In the latter case, drug-tolerant K. pneumoniae can persist to yield potentially untreatable, persistent infection.

Extended Data Fig. 1 |. Mucoidity phenotypes of clinical ST258 isolates.

a, Representative colony phenotypes of clinical isolates plated on blood agar plates. The colony morphologies of UCI_38 (mucoid), UCI_37 (hypomucoid) and BIDMC_13 (hypermucoid) are shown. b, Hypomucoid clinical isolates display a translucent appearance on LB agar plates. All of the tested colony-purified ST258 isolates displayed the same colony mucoidity phenotype on blood or LB agar plates. 1, UCI_38 (control normal mucoid); 2, BIDMC_18A; 3, BIDMC_14; 4, BIDMC_54; 5, BIDMC_34; 6, UCI_37; 7, BWH_41; 8, UCI_43; 9, MGH_51; 10, MGH_71; 11, BIDMC_68; 12, MGH_73. c, Identification of capsule mutants on LB agar plates. In order to illustrate the feasibility of the identification of capsule mutant subpopulations on LB agar plates, UCI_38 was mixed with UCI_38wzcG565A (left) or UCI_38ΔwbaP (right) to achieve a concentration of mutants of approximately 5%. Hypermucoid colonies could be easily determined by the different colony morphology and bigger size (red arrow, left), while hypomucoid colonies could be easily identified by their translucent appearance and smaller size (red arrow, right). a-c were repeated 3 times independently with similar results.

Extended Data Fig. 2 |. Capsule-deficiency improves biofilm formation and invasion of bladder epithelial cells.

a, wbaP deletion in BWH_36 and BWH_45 abolishes capsule production. Transmission electron microscopy of BWH_36, BWH_45 and corresponding wbaP deletion mutants shown. For every isolate, one representative image from four images obtained from one section is shown. b, Impaired capsule production improves biofilm formation. The isogenic set of ST258 capsule mutants derived from UCI_38 is shown, as well as clinical ST258 capsule mutants and the regular and hypomucoid isolate from the patient specimen UR_5. UCI_37, BIDMC_54 and UR_5_hypo are hypomucoid; BIDMC_13 and BIDMC_16 are hypermucoid. Number of biologically independent experiments indicated. Mean and SEM of at least three independent experiments shown. Significance calculated with an unparalleled two-tailed t-test. c, Capsule production impairs the invasion of bladder epithelial cells. wbaP deletion in UCI_38, BWH_36 and BWH_45 increases the invasion of bladder epithelial cells, while wbaP expression in the isogenic wbaP deletion mutant of UCI_38 impairs bladder epithelial cell invasion. Mean and SEM shown. Significance calculated with an unparalleled two-tailed t-test. n = 3 biologically independent experiments. d, Impaired capsule production increases bladder epithelial cell invasion resulting in a larger intracellular reservoir of K. pneumoniae ST258. UCI_38 and the isogenic wbaP deletion mutant are shown. The net increase in CFUs over 48 h is indicated. Mean and standard deviation of n = 4 biologically independent experiments shown. Significance calculated with an unparalleled two-tailed t-test. e, Antibiotics of last resort are unable to clear intracellular carbapenem-resistant Klebsiella pneumoniae from bladder epithelial cells. Intracellular survival of the clinical capsule-deficient isolate BIDMC_54 shown. Mean and standard deviation of n = 3 biologically independent experiments shown. Significance calculated with an unparalleled two-tailed t-test. f, Intracellular survival of UCI_38_mNeon and UCI_38ΔwbaP_mNeon after 1-minute synchronization of infection (via centrifugation). The centrifugation time was reduced for confocal microscopy in order to more effectively wash off extracellular bacteria from host cells, which decreased invasion efficiency but did not affect the observed net increase in recovered CFUs over 48 h seen in Extended Data Fig. 2d. Mean and standard deviation of n = 4 biologically independent experiments shown. Significance calculated with an unparalleled two-tailed t-test. g, The number of host cells infected with the wild type and mutant is consistent over time, with the mutant displaying a 3fold higher frequency of infection compared to the wildtype. Percentage of infected cells at indicated timepoints, as determined by confocal microscopy shown. n = 2 biologically independent experiments. h, Average number of bacteria per infected cell, as determined by recovered CFUs (Extended Data Fig. 2f) and by the number of infected host cells (Extended Data Fig. 2g) at indicated timepoints. For the 4-hour time point, the number of infected cells determined at 8 hpi was used in order to better distinguish between live and dead bacteria. From 8 to 48 hours post infection, all identified intracellular fluorescent Klebsiella were in LAMP1-positive vacuoles. Mean and SEM shown. n = 2 biologically independent experiments. i, Intracellular Klebsiella grow in Klebsiella-containing LAMP1-positive vacuoles. The fluorescence intensity of mNeon-expressing Klebsiella in LAMP1-positive vacuoles determined via integrated density (IntDen) from confocal microscopy images taken under identical conditions. n = 2 biologically independent experiments. j, Representative image taken with a confocal microscope of bladder epithelial cells infected with mNeon expressing UCI_38ΔwbaP (green, 24 hpi), labeled with anti-LAMP1 antibodies (red) and Hoechst dye (blue). The experiments were repeated 3 times independently with similar results. k, The same image is shown without bacteria to visualize the LAMP1-positive compartments in which Klebsiella pneumoniae persist during infection. l, Heterogeneity of intracellular growth observed in Klebsiella-containing vacuoles. The capsule-deficient mutant displays growth more frequently resulting in 2-fold enhanced net-growth vs the wild type (Extended Data Fig. 2f,h). The integrated density of mNeon-expressing Klebsiella in LAMP1-positive vacuoles is shown (n = 70 UCI_38 vs n = 73 UCI_38ΔwbaP containing compartments, from n = 2 biologically independent experiments shown). Median indicated with a dashed line, quartiles indicated with dashed lines. m, Bacteria recovered from the bladder and the kidney of mice infected transurethrally with UCI_38 or UCI_38ΔwbaP, 3 days post infection. n = 10 mice per group. Significance was calculated with the Mann-Whitney two-tailed U test. P values indicated in the figures. n, The capsule-deficient mutant UCI_38ΔwbaP causes localized infection with no observed immunogenicity at 3 days post infection. Representative histological images of haematoxylin and eosin (H&E) stained liver tissue sections from mouse UTIs, 3 days post infection (n = 3 bladders analyzed). For f-i the mean and SEM is shown. P values are indicated in the figures.

Extended Data Fig. 3 |. SNPs in wzc confer phagocytosis resistance via hypercapsule production.

a, Engineered isogenic strains containing wzc mutations identified in the clinical ST258 isolates produce a hypercapsule. The wzc genes from normal and hypercapsule producing isolates and from the same patient samples (UR_5, UR_11, UR_35) were cloned into the expression plasmid pBAD33 or pBAD33tet (ptet, a tetracycline resistance conferring variant of pBAD33). The ST258 strain UCI_38 and other phylogenetically more distantly related ST258 strains BWH_36 and BWH_45 were transformed with the resulting constructs and were tested for the ability to resist centrifugation as a measure of hypercapsule production. After a brief centrifugation step the optical density of the supernatant was determined. b, Clinical isolates with wzc mutations produce a hypercapsule. Overnight cultures were tested for centrifugation resistance as indicator of excessive capsule production. After a brief centrifugation step the optical density of the supernatant was determined. c, Hypercapsule producing clinical isolates are phagocytosis resistant. UCI_38 and isogenic hypercapsule producing wzc mutants, as well as other clinical isolates with wzc SNPs shown. UR_5 isolates and BIDMC_16 could not be tested for phagocytosis resistance due to extensive drug resistance. d, Mutated wzc induces a hypercapsule in distantly related ST258 strains (for a phylogenetic analysis of these isolates, see Cerqueira et al, 2017). For a-d the mean and SEM of n = 3 biologically independent experiments is shown. Significance calculated with an unparalleled two-tailed t-test. e, Hypercapsule conferring point mutations affect different regions of Wzc. The C-terminal domain of the Wzc protein from ST258 clade 2 and ST512 strains is shown. Wzc controls high level polymerization of capsule in E. coli. Its activity depends on a C-terminal autokinase domain, which consists of Walker A and B boxes that are crucial for the phosphorylation of tyrosine residues located in the tyrosine cluster and which results in the negative regulation capsule polymerization. Hypercapsule production has also been observed in clinical Acinetobacter baumanii isolates with point mutations in wzc and have only in some cases been found to affect autokinase activity. The precise function of Wzc is unknown but it has been hypothesized that it regulates capsule biosynthesis via distinct interactions with other capsule biosynthesis proteins and may have a complex structural role in capsule assembly. Outside of its autokinase activity, Wzc proteins are also known to form oligomers, independent of phosphorylation. A535E is located in a Walker A box, while the G565 mutations are located just outside of the canonical Walker A’ box. However, additional conserved amino acids can be found in Walker A’ boxes of Enterobacteriaceae, potentially extending the Walker A’ box to amino acid 566. L74 is not located in a region of any known activity of Wzc but may affect oligomerization or other complex interactions.

Extended Data Fig. 4 |. Hypercapsule production increases ST258 virulence.

a, Hypercapsule production does not affect the growth rate of UCI_38 (ST258) or SGH_10 (ST23) in axenic culture. UCI_38 and the isogenic hypercapsule mutant UCI_38_wzcG565A, as well as the hypervirulent isolate SGH_10 (ST23) were grown in LB medium or in M9 salts supplemented with 0.2% casamino acids. The experiments were repeated 3 times independently with similar results. b, The hypercapsule producing clinical isolate BIDMC_13 displays increased toxicity in a zebrafish bloodstream infection model compared to normal capsule strain UCI_38. The lethal dose (LD50) required to kill 50% of the zebrafish larvae population is indicated. Meanwhile, a capsule-deficient clinical isolate UCI_37 is more attenuated. Mean and SEM shown. n = 170 UCI_38, n = 111 BIDMC_13, n = 104 UCI_37. P-values indicated relative to the normal capsule UCI_38 strain. c, Survival of zebrafish larvae after bloodstream-infection with UCI_38 (n = 1773 + /-453 CFUs / fish) and the clinical hypercapsule mutant BIDMC_13 (n = 2213 + /−1113 CFUs / fish). P = 0.0024 (log-rank test). d, Hypercapsule facilitates the dissemination of murine UTIs (compare to Fig. 2h). C3H/HeN mice were infected transurethrally with UCI_38 and the isogenic hypercapsule mutant UCI_38_wzcG565A. n = 10 mice were used per strain and timepoint. At day 3 post infection only the hypercapsule mutants were isolated from the bladder and the kidney. Significance was calculated with a two-tailed Mann-Whitney U test. The dashed line indicates the limit of detection. Dpi, days post infection. e, Representative histological images of haematoxylin and eosin (H&E) stained liver tissue sections from mouse UTIs 3 days post infection. The livers of n = 3 infected mice were analyzed per group. f, The hypercapsule mutant disseminates more effectively to the spleen in a mouse model of bloodstream infection. Recovered CFUs from homogenized spleens 3 days post infection shown. n = 5 mice were used per group. Significance was calculated with a two-tailed Mann-Whitney U test. g, Transurethral infection of TLR4-deficient C3H/HeJ mice with the normal capsule strain UCI_38 results in dissemination and persistent infection. Infections were established in 20 mice and CFU counts were determined 7 days post infection. The dashed line indicates the limit of detection. Hypercapsule mutants were isolated. h,i,j, A hypercapsulated ST258 mutant that emerged as a subpopulation in a patient displays enhanced virulence in mouse models of infection. h, Mouse UTI model. Recovered CFUs from the liver of mice infected transurethrally with normal (UC_11) and hyper-capsule producing (UCI_11_wzcL74P) isolates from the urine specimen UR_11 (3 days post infection). The hypercapsule mutant killed 4 of 15 mice, while the parent isolate killed 1 of 15 mice, which is a degree of virulence that is usually not observed in mouse UTIs. Dead mice are indicated by the number of crosses. Livers from deceased mice are not included in the analysis. n = 15 mice per group. Significance calculated with a two-tailed Mann-Whitney U test. i, Pyogenic liver abscess caused by the hypercapsule mutant from UR_11 observed in 4 of 11 mouse UTIs. j, Mouse bloodstream model. The hypercapsule mutant from UR_11 (UCI_11_wzcL74P) displays more rapid lethality in a mouse model of bloodstream infection compared to the normal capsule producing parent at an inoculum of 4×10⁷. n = 5 C3H/HeN mice were infected per group. Mice infected with the normal capsule producing parent were sacrificed due to poor health scores at 16 hpi. Significance calculated with a log-rank test. k, Comparison of capsule production between the representative hypercapsulated ST23 isolate SGH_10 and the hypercapsulated ST258 strain UCI_38_wzcG565A. The capsule was isolated and quantified by determining the amount of uronic acids. Mean and SEM of n = 2 independent experiments shown. l, The representative hypervirulent K. pneumoniae isolate SGH_10 displays rapid lethality in a mouse model of intraperitoneal infection. n = 5 BALB/c mice were infected with an inoculum of 1–2×10⁶ CFUs and mouse survival was followed over time. SGH_10 is a representative Asian ST23 isolate harboring all known hypervirulence-associated genes.

Extended Data Fig. 5 |. identification of isolates with wzc mutations suspected to confer a hypercapsule.

a. ST258 clade 2 reference tree (including single locus ST512 variants), comprised of 117 reference genomes that represent the phylogenetic diversity of the 966 ST258 clade 2 strains identified in the Refseq database (see Methods). b. Phylogenetic distribution of wzc mutants mapped to the closest strain in the reference tree. Mutants harboring mutations close to the hypercapsule-conferring mutations positions that were found to confer a hypercapsule in this study are shown (Extended Data Fig. 3e).

Extended Data Fig. 6 |. Direct isolation of hypercapsule mutants from patients infected with multidrug-resistant K. pneumoniae.

The percentage of hypercapsule and capsule-deficient mutants (Hyper/Deficient) as determined by visual inspection of mucoidity and string-test positivity is shown, for 3 patient urine specimens plated and analyzed directly, as well as the identified wzc mutations (resulting amino acid substitutions shown) and sequence types (ST) for the hypercapsule population isolated from each specimen. The clonality of subpopulations isolated from patients was verified by whole genome sequencing. Point mutations in wzc were identified by comparing the wzc sequence of mutant isolates to regular capsule producing isolates in the same specimen or, in the case of the ST14 isolate, by comparing the sequence to the consensus ST14 sequence in NCBI. The ST307 hypercapsule mutant harbors an additional frameshift mutation (fs) in the tyrosine cluster of wzc (Extended Data Fig. 3e) that affects the last 7 codons (G713-Ter720) and extends the open reading frame by two codons.

Fig. 1 |. Capsule heterogeneity in screened collection of 54 clinical ST258 isolates.

a, Frequency of mucoidity phenotypes of strains isolated from different infection sites. Hypermucoid isolates are associated with bloodstream infections (P = 0.0196) and hypomucoid isolates with UTIs (P = 0.0069). b, Transmission electron microscopy of representative regular, hyper-and hypomucoid clinical isolates and isogenic strains: regular mucoid UCI_38, BIDMC_32 and UCI_38ΔwbaP(pBAD33wbaP); hypermucoid UCI_38_wzcG565A, BIDMC_13 and BIDMC_16; and hypomucoid UCI_38ΔwbaP and UCI_37. Hypermucoid isolates display increased polymerization of the capsule, whereas capsule-deficient isolates do not produce a capsule, compared with regular mucoid UCI_38. One representative image from four images obtained from one section is shown. c, Capsule production measured by uronic acid content of isolates displaying altered mucoidity. Hypermucoid strains produce more capsule, compared with regularly mucoid isolates. The mean and s.e.m. of three independent experiments are shown. P values are displayed above the columns based on the comparison with UCI_38 (unpaired, two-tailed Student’s t-test). d, Mutations found in the capsule biosynthesis genes in the screened collection of ST258 isolates belonging to clade 1 and clade 2, which harbor different capsule operons. The position of the mutations is indicated. Red lines are indicative of deletions (dashed) and ISs. Flags lacking the red pedestal represent non-synonymous SNPs. Linked flags are indicative of multiple mutations found in an isolate. The mucoidity phenotypes associated with IS elements and SNPs are indicated by the color of the flag: white, mucoid; black, hypomucoid; gray, hypermucoid.

Fig. 2 |. Gain-and loss-of-function mutations increase the virulence or the persistence of K. pneumoniae ST258.

a, Capsule production of isogenic mutants of UCI_38 as measured by uronic acid content confirms that disruption of wbaP impairs capsule production, whereas an SNP at position 565 leads to hypercapsule production (n = 4 biologically independent experiments). b, Biofilm formation of isogenic mutants in polystyrene plates. The capsule-deficient mutant forms more robust biofilms (n = 7 biologically independent experiments). c, Impact of capsule production on the invasion of human bladder epithelial cells. The isogenic capsule-deficient mutant displays increased invasion efficiency (n = 3 biologically independent experiments). d, Intracellular K. pneumoniae persist in LAMP1-positive vacuoles: UCI_38ΔwbaP expressing chromosomally integrated mNeon shown 24 h post-infection (see Extended Data Fig. 2f–l for further analysis). The experiments were repeated three times with similar results. e, Persistence of the capsule-deficient isogenic mutant UCI_38ΔwbaP is greater than wild-type UCI_38 in a mouse model of chronic UTI. Bladders were plated 14 d post-infection. The data of two independent experiments are shown. A total of 30 mice were infected per strain. f, Phagocytosis by J774 macrophages. The hypercapsule mutant UCI_38_wzcG565A is more phagocytosis resistant than the wild-type parent UCI_38 and corresponding capsule-deficient mutant (n = 4 biologically independent experiments). g, Bloodstream infection of zebrafish larvae with a non-lethal dose of UCI_38 (inoculum = 1,293 ± 733 c.f.u.; n = 19 infected zebrafish) results in lethality after infection with the isogenic hypercapsule wzc mutant UCI_38_wzcG565A (inoculum 1,556 ± 820 c.f.u.; n = 23 infected zebrafish). h, Dissemination of UCI_38 and UCI_38_wzcG565A from the bladder to the liver in murine UTIs (3 d post-infection; Extended Data Fig. 4d; n = 25 mice (C3H/HeN) infected per group). The hypercapsule mutant displays greater dissemination to the liver than the isogenic, wild-type parent, as demonstrated by bacterial numbers (c.f.u.) recovered in mouse livers (one mouse infected with the hypercapsule mutant died 2 d post-infection). The geometric mean is shown. i, The hypercapsule mutant from UR_11(UCI_11_wzcL74P) is lethal in a mouse model of intraperitoneal infection. Mice were infected with an inoculum of 1–2 × 10⁶ bacteria and mouse survival was followed over time (n = 10 BALB/c mice were infected per group). P values are displayed in the figures (hypercapsulated strains were compared with parent strains). The significance was calculated using an unpaired, two-tailed Student’s t-tests (a–c,f), a two-tailed log-rank test (g) and two-tailed Mann–Whitney U-tests (e,h,i). P values are displayed in the figures and are all based on the comparison with UCI_38 or UR_11. The limit of detection is indicated with a dashed line.

Fig. 3 |. Hypercapsule and capsule-deficient mutants are widespread across the ST258 phylogeny.

a, ST258 clade 2 reference tree, comprising 117 reference genomes that represent the phylogenetic diversity of 966 ST258 clade 2 strains identified in RefSeq (Methods). b, Phylogenetic distribution of isolates in RefSeq carrying wzc mutations at positions 74 and 565, which were found to confer a hypercapsule. The count of strains carrying the designated mutation is mapped on to the phylogenetic reference tree, assigned to the strain in the reference tree that is most similar to the wzc mutation-carrying strain. Different mutations are indicated in different colors. Strains carrying wzc mutations from known hospital outbreaks are indicated: (1) Geneva (Switzerland) and (2) North Carolina (United States). c, Phylogenetic distribution of strains with inactivated wbaP. The count of strains carrying the designated wbaP mutation is mapped on to the phylogenetic reference tree and assigned to the strain in the reference tree that is most similar to the wbaP mutation-carrying strain. Colors indicate the length of the truncated WbaP protein relative to the consensus WbaP sequence of 475 amino acids. Mutations that occurred only once in our dataset are indicated in dark gray (other). Detailed information on the mutants is available in Supplementary Tables 4 and 5.

Fig. 4 |. A model for how capsule remodeling results in different infection phenotypes of K. pneumoniae.

Proposed model for the impact of capsule remodeling in catheter-associated UTIs. Classic K. pneumoniae strains are relatively resistant to phagocytosis during colonization of the bladder, by establishing biofilms on catheters and bladder epithelium and by invading bladder epithelial cells; the capsule, however, limits biofilm formation and invasion of bladder epithelial cells, thereby restricting infection to a relative degree. Increasingly virulent K. pneumoniae, which contain wzc mutations resulting in a phagocytosis resistance-conferring hypercapsule, can disseminate more effectively, but are unable to form a biofilm or invade bladder epithelial cells effectively. Uropersistent K. pneumoniae are impaired in capsule production and therefore are even more effective at forming a biofilm and invading bladder epithelial cells than classic strains. The increased invasion efficiency results in a larger intracellular reservoir, in which K. pneumoniae ST258 can persist in Klebsiella-containing vacuoles in the face of phagocytes, which leads to persistent UTIs and an untreatable intracellular reservoir.