The bloodstream K. pneumoniae population consists of diverse lineagesA total of 136 K. pneumoniae isolates from bloodstream infections in unique pediatric and adult patients at the DHMC were collected between January 2017 and January 2022 (Supplementary Data 1). The maximum likelihood phylogeny built from 188,166 single nucleotide polymorphisms (SNPs) extracted from a 3.378 Mbp sequence alignment of 3,511 core genes revealed a genetically diverse population. We identified 94 known sequence types (ST; Supplementary Data 2). The ST diversity is high (Simpson’s diversity index = 0.983) and not one ST appears to dominate the population (Fig. 1A, B). Only nine STs contained at least three genomes (ST20, six genomes; ST36, ST45 and ST253, five genomes each; ST1380 and ST307, four genomes each; ST111, ST225, ST23 and ST941 with three genomes each). A total of 11 and 73 STs were represented by two and one genome(s), respectively. Thirteen genomes carried novel combinations of the 7-gene multi-locus sequence typing (MLST) loci and were assigned novel ST designations by the Klebsiella-specific database in BIGSdb30: ST6357 (isolate KPB115), ST6358 (KPB126), ST6359 (KPB139), ST6360 (KPB179), ST6361 (KPB28), ST6362 (KPB29), ST6363 (KPB34), ST6364 (KPB41), ST6365 (KPB56), ST6366 (KPB79), ST6367 (KPB89), ST6368 (KPB90), and ST6369 (KPB93).Fig. 1: Genomic features of the 136 K. pneumoniae isolates from bloodstream infection.A A maximum likelihood phylogeny showing the year of isolation and the most frequent sequence types (ST), clonal groups (CG; represented by three or more genomes), and sublineages (SL; represented by four or more genomes). The midpoint-rooted tree is calculated using single nucleotide polymorphic sites from the sequence alignment of 3,511 concatenated core genes. Tree scale represents the number of substitutions per site. The barplots show the proportion of (B) ST (C) CG and (D) SL by sampling year. The numbers above the bars in panel B indicate the number of isolates per year. For visual clarity, only the most frequent ST, CG and SL are shown in colored blocks, while less frequent ones are grouped together as “others” in gray blocks.The 136 genomes can be further classified into 99 known clonal groups (CG; Simpson’s diversity index = 0.986) and 76 known sublineages (SL; Simpson’s diversity index = 0.971) (Fig. 1C,D and Supplementary Data 2). As defined previously31, K. pneumoniae genomes belonging to the same clonal group differ by 43 allelic mismatches, while sublineages differ by 190 allelic mismatches. The most frequently detected clonal groups contained five genomes (CG45), four genomes (CG1380, CG20, CG307, CG36), and three genomes (CG10047, CG111, CG23, CG941). The most frequently detected sublineages were SL17 (consisting of 14 genomes from ST17, ST20, ST422, ST3640, ST5122, ST6364, ST6365), SL3010 (six genomes from ST1, ST5, ST6, ST8, ST9, ST10), SL45 (seven genomes from ST45 and ST987), and SL268 (six genomes from ST36 and ST268). From 2017 to 2021, less common STs, CGs and SLs comprise a large assemblage of the annual population. Overall, these results show that diverse genotypes may similarly cause bloodstream infections, thus emphasizing the opportunistic nature of K. pneumoniae in invasive infections.Antimicrobial resistance is widespread in bloodstream K. pneumoniae
We carried out antimicrobial susceptibility testing of K. pneumoniae isolates against 20 antimicrobial agents from seven classes of antimicrobial drugs (Fig. 2A, Supplementary Data 1). All isolates exhibited resistance to ampicillin, which was not surprising because it is known to be intrinsic in K. pneumoniae32. Resistance was observed against ampicillin-sulbactam (n = 16 isolates, representing 11.76% of the population), cefuroxime (n = 14, 10.29%), sulphamethoxazole (n = 14, 10.29%), and cefazolin (n = 12, 8.82%). A total of 17 (12.5%), 16 (11.76%), seven (5.14%), and two (1.47%) isolates were resistant to at least one antimicrobial agent belonging to cephalosporin, beta-lactam combination agents, quinolone, and aminoglycosides sub/classes, respectively. However, all or nearly all isolates were susceptible to amikacin and meropenem (n = 136, 100%), ertapenem (n = 135, 99.26%), gentamicin (n = 134, 98.52%), cefoxitin (n = 133, 97.79%), amoxicillin-clavulanic acid and piperacillin/tazobactam (n = 132, 97.05%), levofloxacin (n = 131, 96.32). Altogether, we detected 18 unique AMR profiles, each with different combinations of resistance phenotypes (Fig. 2A). A total of 109 isolates were phenotypically resistant to ampicillin only and not to other antimicrobial agents. Isolates KPB140 and KPB97 exhibited resistance to 14 and 13 antimicrobial agents, respectively, while isolates KPB176 and KPB77 were resistant to 12 antimicrobial agents (Supplementary Data 1).Fig. 2: Antimicrobial susceptibility phenotypes and genotypes of the 136 K. pneumoniae isolates from bloodstream infection.A An UpSet plot showing the total number of isolates resistant to antimicrobials tested (left bar plot), the total number of isolates exhibiting a particular antibiogram (top bar plot) and filled dots representing the presence of an antimicrobial resistance (AMR) phenotype. Acronyms: Pip.tazobactam Piperacillin-Tazobactam, SXT Sulphamethoxazole/Trimethoprim, Amp. Sulbactam Ampicillin Sulbactam. B Number of genomes carrying individual AMR genes and gene combinations per antimicrobial class. The AMR genes in the color legend are grouped according to antimicrobial class. C Concordance analysis of resistance phenotypes and predicted genotypes.Using in silico analysis of the genome sequences, we identified the presence of genes that encode AMR determinants (Fig. 2B and Supplementary Fig. S1, Data 3). Across the entire dataset, we detected a total of 64 unique genes encoding resistance to ten antimicrobial drug classes. All 136 genomes harbored at least one AMR gene that confer resistance to beta-lactams. A total of 134 (98.52%) and 123 (90.44% genomes) harbored intrinsic and chromosomally mediated genes encoding resistance to fosfomycin and phenicol/quinolone, respectively. We observed ten combinations of the phenicol/quinolone multi-drug efflux pump alleles occurring in the genomes (Supplementary Data 3). The most frequently detected combinations were the oqxA/B (n = 42, 30.88%), oqxA/B19 (n = 27, 19.85%) and oqxA/B25 (n = 15, 11.02%). Other AMR determinants were also detected but at low frequencies (present in ≤ 21 genomes), including genes that are associated with resistance to aminoglycoside, macrolide, phenicol, quinolone, sulfonamide, tetracycline, and trimethoprim.Among the beta-lactamases, we identified a total of 24 chromosomally encoded blaSHV gene variants (Fig. 2B and Supplementary Data 3). The most frequently occurring variants were blaSHV-11 (n = 37 genomes, representing 27.2% of the population), blaSHV-1 (n = 28 genomes, 20.58%), and blaSHV-27 (n = 11 genomes, 8.08%). The gene blaSHV has been reported to have undergone robust allelic diversification in clinical K. pneumoniae and other Enterobacteriaceae, and our results were consistent with this diversity33. While the presence of the wild type blaSHV is responsible for resistance to ampicillin, amino acid substitutions from allelic diversification may cause them to have expanded functionality such as ESBL activity and/or beta-lactamase inhibitor resistance activity33. The variants blaSHV-38, blaSHV-164, blaSHV-187 that we detected in our dataset are known to be associated with resistance to cephalosporins34. However, we did not observe resistance to cephalosporins when tested in vitro in isolates harboring these genes. Furthermore, isolate KPB57 which carried the gene blaSHV-41 exhibited resistance to first (cefazolin) and second (cefoxitin) generation cephalosporins but not to third generation cephalosporins. Detection of this bla variant has been associated with conflicting ESBL phenotypes35,36.High concordance of AMR phenotype and genotypeWe sought to investigate the level of concordance between the results of the in vitro antimicrobial susceptibility testing and the in silico screening of genetic elements conferring resistance to different antimicrobial agents. We defined the following terms: (1) True positives were isolates with resistant phenotypes harboring a corresponding resistance genetic determinant; (2) True negatives were isolates with susceptible phenotypes and do not carry the corresponding AMR determinant in their genome; (3) False negatives were isolates exhibiting a resistant phenotype but with no corresponding AMR genetic element detected in the genome; and (4) False positives were isolates that exhibit a susceptible phenotype but carry the AMR genetic element in their genome. Overall, we found high concordance between the resistant phenotypes and the presence of the corresponding AMR genes. For the six antimicrobial classes, concordance values range from 90.44% (aminoglycosides) to 100% (carbapenems) (Fig. 2C and Supplementary Data 4).In the phenotype-genotype concordance analysis of carbapenem resistance, the single carbapenem resistant isolate KPB97 (ESBL-producing) lacked a detectable carbapenemase gene. Examination of the gene encoding the outer membrane porin revealed a truncation in the ompK36 gene. We observed ompK36 in the ESBL-producer KPB97, whereas other ompK35 and ompK36 truncations were detected in genomes not containing ESBL (Supplementary Data 3). Previous reports have shown that truncation of ompK36 in the presence of an ESBL is sufficient for non-susceptibility to ertapenem but not to imipenem and meropenem37,38. Hence, we considered this isolate as true positive. We therefore assigned perfect concordance for carbapenem resistance, with specificity of 100% (range: 97.3–100%) and sensitivity of 100% (range: 2.50–100%).Concordance between quinolone resistance phenotype and genotype showed 97% agreement, with specificity of 100% (range: 97.18–100%) and sensitivity of 57.1% (range: 18.4–90.1%). In our study, we associated non-susceptibility to ciprofloxacin and levofloxacin with the presence of either plasmid mediated qnrB1 or the mutations in the quinolone resistance determining regions (gyrA_S83I + parC_S80I)39. A total of four and 129 genomes were true positives and true negatives, respectively. We did not detect mutations in the quinolone resistance determining regions for genomes phenotypically resistant to ciprofloxacin (KPB44 and KPB68) but harbored a qnrB1 gene. We observed no detectable mechanism of resistance in the levofloxacin resistant isolate (KPB87).Phenotype-genotype concordance of sulfonamide resistance was 97.79%, with specificity of 98.36% (range: 94.2–99.8%%) and sensitivity of 92.85% (range: 66.1–99.8%). Cephalosporin phenotype-genotype agreement was 92.64%, with specificity of 99.16% (range: 95.44%–99.97%) and sensitivity of 43.75% (range: 19.75–70.12%). Notably, all ceftriaxone resistant isolates except isolate KPB70 (resistant to all cephalosporins) harbored the ESBL gene blaCTX-M-15. For aminoglycosides, concordance was 90.44%, with specificity of 90.29% (range: 83.98–94.73%) and sensitivity of 100% (range: 15.81–100%).Multidrug resistant and hypervirulent clones are present in bloodstream infectionsMultidrug resistant clones are defined as those encoding acquired resistance determinants to at least three antimicrobial drug classes at high frequencies (≥56%), whereas hypervirulent clones harbored the plasmid-associated virulence genes iuc, iro and/or rmpA/rmpA2 at frequencies between 31–100%40. We used these in silico definitions to determine whether any of our isolates belong to known multidrug resistant or hypervirulent clones. A total of 17 genomes (12.5%) in our dataset matched known multidrug resistant clones (Fig. 3). The multidrug resistant clones included CG14, CG20, CG147, CG711, CG1123, CG10094, CG10156, CG10253, CG10476 (each with one genome), CG10124 and CG10529 with two genomes each, and CG307 with four genomes. When we mapped the multidrug resistant clonal groups against the AMR gene presence and absence results determined using Kleborate41, we found that only members of CG307 harbored resistance genes to multiple antimicrobial classes, including the ESBL gene blaCTX-M-15 (Supplementary Data 4). However, clones that were not designated as multidrug resistant (CG10524, CG429, CG2623, CG540, CG10151, CG12251, CG45, ST6366, and ST3293) as previously reported40 also harbored resistance genes to multiple drug classes in our study (Fig. 3, Supplementary Data 2 and Data 4). Among the unassigned clones, we detected the presence of blaCTX-M-15 in CG10524 (ST1564), CG429 (ST429), and ST3293.Fig. 3: Presence of multidrug resistant and hypervirulent clones.The maximum likelihood phylogeny was built from the single nucleotide polymorphic sites of 3,511 concatenated core genes. The tree is identical to that in Fig. 1A. Only the major CG and ST are shown for visual clarity. The Group category refers to multidrug resistant (MDR; red blocks) and hypervirulent (Hv; blue blocks) K. pneumoniae clones defined in references40,47 and implemented on Kleborate41. The matrix shows the presence (purple blocks) or absence (white) of at least one gene conferring resistance to each antimicrobial class. AGLY Acronyms, AGLY aminoglycoside, FLQ fluoroquinolones, PHE Phenicol, SUL sulfonamides, TET tetracycline, TMT trimethoprim, BLA beta-lactamase. The presence of chromosomally encoded blaSHV is indicated by the light blue blocks.Experimental evidence from previous studies has identified the presence of key markers for hypervirulence that includes the siderophores yersiniabactin, aerobactin and salmochelin as well as hypermucoidy via capsule overproduction28,40. In our study, we identified only one known hypervirulent clone based on the previous classification40, and this clone included three genomes belonging to CG23/ST23. We did not find convergent clones in our dataset, i.e., those that are both hypervirulent and multi-drug resistant42.Diversity and transmission of plasmid-encoded ESBL gene bla
CTX-M-15
Plasmids encoding the ESBL gene blaCTX-M-15 often harbor resistance determinants to additional drug classes26. We sought to understand the epidemiology of plasmids carrying the blaCTX-M-15 by sequencing plasmid genomes. We identified blaCTX-M-15 in seven (5.14%) bloodstream isolates. Because multidrug resistant isolates from non-blood samples (e.g., urine) are also routinely archived by DHMC as part of patient care, we also identified another isolate (KPB102) from a urine sample that carried blaCTX-M-15. From the long-read sequencing data of the eight isolates, we obtained 12 circular plasmid genomes (Fig. 4A and Supplementary Data 5). Of these, four isolates carried two plasmids (KBP68, KPB77, KBP97, KBP176), while the other four isolates carried only one plasmid (KBP44, KBP140, KBP166, KBP102). The eight isolates came from four STs representing four CGs, of which five isolates are members of ST307 (CG307). The gene blaCTX-M-15 was present in either of the two replicon types IncFIB(K)/IncFII(K) (n = 6 plasmid genomes) or IncFIB(K) (n = 2 plasmid genomes). The sizes of these plasmids ranged from 131,110 bp (pKPB68_2) to 246,740 bp (pKPB176_2). Four plasmid genomes with an unknown replicon type were also detected and classified as untyped. The sizes of the untyped plasmids ranged from 3559 bp (pKPB77) to 367,083 bp (pKPB97).Fig. 4: Plasmid types and transmission of plasmid carrying blaCTX-M-15.A Bar plot showing the number of plasmid genomes and replicon types detected in the eight isolates harboring the gene blaCTX-M-15. B Timeline of transmission of plasmids encoding blaCTX-M-15 between isolates. Horizontal lines connecting circles show the mash distances between plasmid genomes in each linked group. In group 1, both isolates are from ST307/CG307. In group 2, the black asterisk (*) on pKBP97_3 indicates that the plasmid came from an ST429/CG429, while the other two isolates are members of ST307/CG307. For both panels A and B, circles are colored by clinical source (red for blood, yellow for urine). C Structural comparison of plasmid genomes encoding blaCTX-M-15 and belonging to group 1 and group 2 in panel 2B. Gray areas between plasmids represent regions with 80–100% sequence identity. Genes are represented by arrows and are color-coded according to general function: magenta—antimicrobial resistance, brown—heavy metal resistance, green—conjugation transfer, yellow—mobile elements such as insertion sequences, transposons and integrases.Two groups of plasmid sequences sampled from different patients exhibited high sequence identity (Fig. 4B and Supplementary Fig. S2A, B). The first group consisted of plasmids pKPB102_2 and pKPB166_2, which are both of the IncFIB(K)/IncFII(K) replicon type. The two plasmid sequences are of the same size (231,545 bp) and have 100% sequence identity and sequence coverage, as well as a low pairwise mash distance of 7.14e-6 (Fig. 4B). Each plasmid was retrieved from two different isolates (KPB102 and KPB166) that both belonged to ST307, and which differed by 13 SNPs in their core genome sequence alignment. One isolate was collected from a urine sample in October 2019 and the second isolate from a blood sample in September 2021 from different patients. The high identity between the two plasmid sequences and their common strain background exemplifies a transmission pattern within the same clonal lineage (ST307).The second group of highly similar plasmid sequences consisted of three IncFIB(K)/IncFII(K) plasmids (pKPB77_2, pKPB97_3, pKPB176_2) (Fig. 4B). The three isolates that carried these plasmids were sampled from three different patients. Plasmids pKPB77_2 (245434 bp) and pKPB97_3 (243759 bp) shared 100% sequence identity, 99% sequence coverage, and a mash distance of 3.1e-5. These two plasmids were retrieved from two distinct lineages—isolate KPB77 is a member of ST307/CG307 and KPB97 is a member of ST429/CG429. Both isolates were from blood samples collected in March 2019 and August 2019, respectively. The two isolates differed by 19,080 SNPs in their core genome alignment. ST307 and ST427 are 4-locus variants in the MLST scheme (gapA, pgi, phoE, tonB). A third isolate in this group is KPB176 (pKPB176_2), which was collected from a blood sample in November 2021. This plasmid (pKPB176_2) exhibited high identity with pKPB77_2 (99% sequence identity, 99.99% sequence coverage, mash distance of 3.1e-4). The isolates from which plasmids pKPB77_2 and pKPB176_2 were derived from both belonged to ST307 and differed by 63 SNPs in their core genome sequence alignment. Such variation in their core SNPs appear to have accumulated over 2 years between March 2019 and November 2021. These results show that this plasmid type was mobilized through both horizontal transmission between phylogenetically distinct lineages (in the case of pKPB77_2 and pKPB97_3) as well as transmission within the same clonal lineage (in the case of pKPB77_2 and pKPB176_2 in ST307). The notable difference between the two groups of plasmids (i.e., group 1 and group 2 in Fig. 4B) is that group 2 contains a ~13 kb cluster of genes that includes IS6 family transposases, Tn5403 family transposase, and AMR genes catB3, blaOXA-1, aac(6’)-Ib-cr5, and tetA (Fig. 4C).Lastly, we observed the presence of multiple resistance genes carried on different plasmids within a single genome. Multidrug resistance in isolate KPB68 (ST3293/CG12467 sampled in 2018) was mediated by resistance determinants present in two plasmids. The plasmid pKPB68_2 has a genome size of 131,110 bp, replicon type IncFIB(K), and carries blaCTX-M-15, aph(3”)-Ib, aph(6)-Id, and sul2, blaTEM-1. The plasmid pKPB68_3 has a genome size of 32,881 bp with an untyped replicon and carries aac(6’)-Ib-cr, qnrB1, catB3, blaOXA-1, tetA, and dfrA14 (Supplementary Fig. S2A, B).Antigenic diversity and hypervirulence markersUsing in silico screening of the genomes, we inspected our isolates for the presence and diversity of the surface polysaccharide capsule (KL) and lipopolysaccharide (O) that determine the antigenic serotypes. These two structures activate the human immune system during infection and are also widely used in strain typing43,44. We identified 53 known capsular KL locus types and four genomes with unknown KL types (Simpson’s diversity index = 0.971) (Fig. 5A and Supplementary Fig. S3A, Supplementary Data 3). The most frequent KL types in our dataset were KL28 (present in nine genomes), KL24 (eight genomes), and KL102 (seven genomes). The KL1 type which is a genetically homogenous capsular type commonly associated with hypervirulent K. pneumoniae clones45 was detected in three genomes belonging to ST23. We also identified 11 different O types in our dataset (Simpson’s diversity index = 0.731). Three O types (O1/O2v2 = 54 genomes, O1/O2v1 = 37 genomes, O3b = 25 genomes) accounted for >85% of our dataset (Supplementary Fig. S3B). Two unknown O types were detected in ST2004 (KPB152) and ST6366 (KPB79) genomes.Fig. 5: Diversity and distribution of surface polysaccharides and hypervirulence markers.A Different combinations of the surface polysaccharides capsule (KL) and lipopolysaccharide (O) loci are shown. The colors and sizes of the bubbles correspond to the number of genomes that carry unique KL and O combinations. B The maximum likelihood core genome phylogeny showing the phylogenetic distribution of genes encoding yersiniabactin (ybt), colibactin (clb2), aerobactin (iuc1), salmochelin (iro1), and rmpA/2. The tree is identical to that in Fig. 1A. Only the major CGs and STs are shown for visual clarity.We searched our short-read sequence dataset for the presence of Klebsiella-specific virulence factors. First, we screened the genomes for yersiniabactin, a siderophore system for sequestering iron that enhances bacteria survival and replication within the host46. This genetic marker is encoded by the ybt gene and is transferred by mobile genetic elements, in particular the integrative conjugative element ICEkp46. In our study, we detected five known ybt variants (ybt 1, 4, 9, 10 and 14) in 24 genomes representing a total of 17.64% of the population (Fig. 5B). The five ybt variants were widely distributed across the core genome phylogeny. The most common ybt variants were ybt 10, ybt 1, ybt 4, ybt 14, and ybt 9. Variant ybt 10 was present in five genomes from ST45/CG45 and one genome each belonging to ST36/CG36, ST14/CG10156 and ST1564/CG10524. ybt 1 was present in three genomes from ST23/CG23 and one genome from ST260/CG10671. ybt 4 was present in one genome each from ST37/CG10094, ST2217/CG2217, ST2.248/CG10668 and ST6360/CG12462. ybt 14 was present in one genome each from ST20/CG20, ST111/CG111, ST942/CG942. ybt 9 was present in one genome each from ST35/CG35, ST307/CG307 and ST393/CG12458. Two genomes possessed unknown ybt variants (KPB59 from ST36 and KPB141 from ST1554).Using the short- and long-read hybrid assemblies, we screened for the presence of other key virulence determinants such as clb2 (colibactin), iuc1 (aerobactin), iro1 (salmochelin), and rmpA/A2 (activator for capsule biosynthesis) in plasmid genomes. These genes have been previously identified as genetic markers of hypervirulence in K. pneumoniae47. Yersiniabactin, aerobactin, and salmochelin are siderophores that promote bacterial survival in nutrient-poor environments by chelating iron48 and is therefore particularly important in the growth, replication, and metabolism of bacteria in the blood. The RmpA/2 activator of capsule biosynthesis leads to hypermucoidy49. We detected the presence of these four virulence markers in only four genomes (Fig. 5B and Supplementary Data 3). Three of these four genomes are known hypervirulent clones (ST23/CG23), whereas the fourth genome belongs to clonal group ST260/CG10671, which is a double-locus variant of ST23 (in genes pgi and phoE). These four genomes clustered together in the core genome phylogeny and they all possessed the ybt 1 variant, KL1 capsular type, and two O types (O1/O2v1 in KPB51 and O1/O2v2 in the other three).Analysis of long read sequencing data on these four isolates showed that the hypervirulence genes are borne on plasmids ranging in size from 178,418 bp (pKPB165_2 in isolate KPB165) to 228,574 bp (pKPB147_2 in isolate KPB147) (Supplementary Data 6). The sequences of the hypervirulence-carrying plasmids were characterized by rearrangement, loss, and/or truncation of genomic regions (Supplementary Fig. S4). For instance, a ~50 kb region present in all other hypervirulence plasmids was missing in pKPB165. This region consists of several genes including those encoding tellurium resistance (terABCDEXZ) and the transposase Tn3. Although the hypervirulence plasmids were frequently flanked by transposases and insertion sequences, they lack a conjugative apparatus, except for the presence of the gene encoding the TraI protein that functions in site and strand nicking50,51. We did not identify an origin of transfer (oriT) site in any of the four hypervirulence plasmids, suggesting that they are not themselves able to initiate their transfer via conjugation.
Clonal background and routes of plasmid transmission underlie antimicrobial resistance features of bloodstream Klebsiella pneumoniae
