Genomic and functional analysis of rmp locus variants in Klebsiella pneumoniae
Department of Infectious Diseases, School of Translational Medicine, Monash University, Melbourne, Australia
Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC, USA
Institut Pasteur, Université Paris Cité, Biodiversity and Epidemiology of Bacterial Pathogens, Paris, France
Department of Genetics, University of North Carolina School of Medicine, Chapel Hill, NC, USA
Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, UK
*Corresponding author: margaret.lam@monash.eduAbstract
Background
Klebsiella pneumoniae is an opportunistic pathogen and a leading cause of healthcare-associated infections in hospitals, which are frequently antimicrobial resistant (AMR). Exacerbating the public health threat posed by K. pneumoniae, some strains also harbor additional hypervirulence determinants typically acquired via mobile genetic elements such as the well-characterised large virulence plasmid KpVP-1. The rmpADC locus is considered a key virulence feature of K. pneumoniae and is associated with upregulated capsule expression and the hypermucoid phenotype, which can enhance virulence by contributing to serum resistance. Typically such strains have been susceptible to all antimicrobials besides ampicillin, however the recent emergence of AMR hypermucoid strains is concerning.
Methods
Here, we investigate the genetic diversity, evolution, mobilisation and prevalence of rmpADC, in a dataset of 14000 genomes from isolates of the Klebsiella pneumoniae species complex, and describe the RmST virulence typing scheme for tracking rmpADC variants for the purposes of genomic surveillance. Additionally, we examine the functionality of representatives for variants of rmpADC introduced into a mutant strain lacking its native rmpADC locus.
Results
The rmpADC locus was detected in 7% of the dataset, mostly from genomes of K. pneumoniae and a very small number of K. variicola and K. quasipneumoniae. Sequence variants of rmpADC grouped into five distinct lineages (rmp1, rmp2, rmp2A, rmp3 and rmp4) that corresponded to unique mobile elements, and were differentially distributed across different populations (i.e. clonal groups) of K. pneumoniae. All variants were demonstrated to produce enhanced capsule production and hypermucoviscosity.
Conclusion
These results provide an overview of the diversity and evolution of a prominent K. pneumoniae virulence factor and support the idea that screening for rmpADC in K. pneumoniae isolates and genomes is valuable to monitor the emergence and spread of hypermucoid K. pneumoniae, including AMR strains.
Article notes
Competing Interest Statement
The authors have declared no competing interest.
Background
A distinct pathotype of Klebsiella pneumoniae, often referred to as hypervirulent K. pneumoniae (hvKp), poses a significant public health challenge outside of clinical settings where it causes severe and sometimes life-threatening infections1–3. These are regarded as being distinct from ‘classical Kp’ strains, which typically cause opportunistic infections mostly within healthcare settings, and are often multidrug-resistant (MDR). Community-acquired infections can arise in otherwise healthy and immunocompetent individuals, although there are reportedly associations with comorbidities such as diabetes4. Common examples of infections include pyogenic liver abscess, endophthalmitis, pneumonia and meningitis, but they can also present as metastatic, multisite infections. Earlier reports of hypervirulent, community-acquired infections were largely confined to countries in Eastern Asia. Cases are now being more widely reported in other regions including Europe, North America and Australia, although often associated with individuals of East Asian descent5.
The overwhelming majority of hvKp infections are associated with strains from distinct genetic backgrounds or lineages; these include clonal groups CG23, CG25, CG65, CG66, CG86 and CG3806,7. Several features are considered hallmark characteristics of these hvKp strains7,8. Most produce a K1 or K2 capsule, encoded by the cps (K) loci KL1 and KL2, respectively, and O1 lipopolysaccharide (OL1 locus). Many hvKp strains also exhibit hypermucoviscosity (HMV), which is defined by a positive string test and/or low OD600 measurements in a sedimentation assay, and is associated with presence of the rmpADC locus and/or rmpA2 gene. Lastly, most hvKp also synthesise the siderophores aerobactin (iuc locus) and salmochelin (iro locus), in addition to the intrinsic siderophore enterobactin (ent locus)7,8. The acquired siderophore loci, rmpADC and rmpA2 (which also appears to be part of a locus including homologs of rmpD and rmpC), are typically mobilised by the large K. pneumoniae virulence plasmids (KpVP) that have been stably maintained for over 100 years in some HvKp clones including CG23 and CG86, although iro and rmpADC can also be mobilised via the chromosomal integrative conjugative element ICEKp19–11.
In K. pneumoniae, the hypermucoid or HMV phenotype has long been associated with the gene rmpA although the exact mechanisms leading to the phenotype were unknown12–14. Based on observations and experimental evidence from earlier studies using knockout mutants, it was proposed that the expression of rmpA upregulated expression of cps thereby increasing capsule production and subsequently resulting in HMV. Subsequent work has confirmed that the rmpA LuxR-like transcriptional regulator is part of a larger operon (herein called rmp) together with the rmpD and rmpC genes located downstream15,16. This work has further clarified that enhanced capsule expression and HMV are two discrete features requiring rmpC (regulator of cps) and rmpD (encoding a small protein required for HMV), respectively. While HMV can be attained in the absence of elevated cps expression (i.e. in a rmpC mutant), it was not observed in capsule-defect mutants, suggesting that HMV does rely on the presence of some capsular components but these need not be hyperexpressed. Accordingly, it was recently shown that HMV is driven through elongation of the capsule polysaccharide chain, driven by a direct interaction between RmpD and Wzc (transmembrane protein), and presumptive indirect interaction with Wzy (capsule repeat unit polymerase), both of which are components of the core capsule synthesis and export machinery17,18. The rmpA and rmpA2 genes appear to be frequently subjected to insertions or deletions (indels) within a poly(G) tract that consequently encode a truncated and presumably non-functional product, and this has been suggested as a mechanism by which differential expression of the two genes is achieved14.
The presence or absence of rmpA in clinical isolates has been investigated in dozens of studies focused on hypervirulent infections, and rmpA has been identified as one of several biomarkers for distinguishing hvKp strains from non-hvKp19. However, their detection shows a variable degree of correlation with HMV as measured by string test (51-98%). The functional impact of allelic variation in the rmp locus genes has not yet been explored, although this is likely to be important for explaining or predicting HMV based on rmp locus sequences. Here we investigate the genetic diversity and distribution of the rmp locus in the K. pneumoniae species complex (KpSC), identify key variant lineages of rmp and their associated mobile genetic elements (MGEs), and demonstrate that representatives of each of the lineages are able to induce HMV and elevated capsule production when introduced into an hvKp isolate lacking its native rmp locus (KPPR1S rmp).
Methods
Genome sequences and genotyping
The initial screening for rmpADC was conducted on the same 2733 K. pneumoniae species complex (KpSC) genomes included in a 2018 study10 examining the aerobactin- and salmochelin-encoding loci iuc and iro, which are often co-localised with rmp on the same mobile genetic elements. The SRST2-table-from-assemblies.py Python script (github.com/rrwick/SRST2-table-from-assemblies) was used to screen these assemblies for the presence of existing rmpA alleles from the virulence database on BIGSdb-Kp (37 alleles as of October 2020; http://bigsdb.pasteur.fr/klebsiella/klebsiella.html) along with the reference rmpD and rmpC sequences from Walker et al.15,16 with BLAST+ v2.2.21, and novel alleles extracted with the --report_new_consensus flag. Unique alleles from 160/2733 genomes with an intact rmp locus (defined as those in which all genes in the locus could be assigned an allele) were assigned allele numbers, and unique allele combinations were used to define unique ‘rmp sequence types (RmSTs)’ using a multi-locus sequence typing (MLST) approach. These rmpADC alleles and RmST profiles were then incorporated along with rmpA2 alleles into the genotyping pipeline Kleborate v2.0.0, and applied to screen 13,156 publicly available Klebsiella genomes20. From this dataset of 13,156 genomes, 944 genomes positive for rmp and/or rmpA2 (including genomes with an incomplete rmp locus) were included for analysis in this study (see Additional File 1 for genome accessions, isolate metadata and genotyping information). We also included rmp-positive genomes from three additional datasets: 4/208 isolates collected at an Australian hospital in 200221, in addition to 36/392 isolates from the same hospital in 2020, and 8/276 isolates from the Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) network22. Novel rmpADC alleles and RmST profiles were added to the BIGSdb-Kp database. Clonal groups, and designations of clones as hypervirulent or MDR, were done using previously defined ST-CG assignments23.
Phylogenetic analyses
For each unique RmST, an alignment of the concatenated rmpA, rmpD and rmpC sequences was generated with MUSCLE v3.8.31, and used as an input for maximum likelihood phylogenetic inference with RAxML v8.2.924 run five times with the generalised time-reversible GTR+gamma model. Similarly, phylogenies were also generated for rmpA, rmpD and rmpC genes individually using the same approach described for RmST. Rmp lineages were defined based on monophyletic groups of RmSTs mobilised by a unique mobile element (see below).
Phylogenetic trees for genomes belonging to hypervirulent clonal groups CG23, CG65 and CG86 were generated using cgMLST implemented in Pathogenwatch (pathogen.watch)25.
Comparison of rmp genetic contexts
Contigs containing rmp were manually inspected in Bandage v0.8.126 to determine whether the locus was located on the chromosome or on a previously described virulence plasmid (KpVP-1 reference pSGH10 with accession CP025081.1, or KpVP-2 reference Kp52.145 plasmid II with accession FO834905.1). For any assemblies where the rmp-containing contig did not match to any of the contexts described above, the contig sequence was screened against the NCBI non-redundant nucleotide database and top hits noted. A single annotated representative (complete sequence where possible) was selected for each rmp lineage to compare the overall genetic structures of the neighbouring regions of rmp (i.e. up to 15 kbp upstream and downstream of rmp). Annotations were performed with Prokka v1.14.627 and comparisons between the annotated genes were visualised with clinker v0.0.21 (github.com/gamcil/clinker)28.
Bacterial strains, plasmids and growth conditions
The K. pneumoniae strains and plasmids used in this study are listed in Table 1. The strains from which representative rmp was amplified were as follows: SGH10 for rmp1, 52.145 for rmp2, NCTC 13669 for rmp2A, KPPR1S for rmp3, and NCTC 1936 for rmp4. Strains were grown at 37°C in LB medium (10 g tryptone, 5 g yeast extract, 10 g NaCl). Saturated overnight cultures were diluted to an OD600 of 0.2 and grown for 5.5 hours. Antibiotics were used where appropriate: kanamycin (Kan), 50 μg/ml; rifampin (Rif), 30 μg/ml; spectinomycin (Sp), 50 μg/ml. All plasmids were introduced into K. pneumoniae by electroporation as previously described15. For expression of genes cloned into the pMWO-078 vector, 100 ng/ml anhydrous tetracycline (aTc) was added to the medium at the time of subculture.
Primers used for the construction of expression vectors are listed in Additional File 2. All rmp loci were amplified from genomic DNA by PCR and then cloned into pMWO-07829 by Gibson assembly (NEB). The rmp expression vectors were then transformed into KPPR1S Δrmp. KPPR1S (ST493; K2/O1) is a well-characterised streptomycin and rifampicin-resistant derivative of ATCC 43816, and has been used in previous studies to characterise rmpD, rmpC, and HMV15–17. Wildtype KPPR1S, ΔwcaJ, and Δrmp strains transformed with pMWO-078 were also included as control strains for measuring HMV. The wcaJ mutant does not produce capsule and is HMV negative, while the rmp mutant produces capsule but is HMV negative as it does not produce RmpD.
Assessment of hypermucoviscosity
Saturated overnight cultures of strains containing rmp plasmids were subcultured in fresh LB for 5.5 hours at 37°C with 100 ng/ml anhydrotetracycline (ATc) to induce rmp expression. Cultures were normalised to 1 OD600/ml and centrifuged at 1000 x g for 5 minutes. Mucoviscosity of cultures was determined by normalising OD600 of the culture supernatant to the starting culture as previously described31.
Uronic acid measurement
Uronic acid (UA) was measured following an established protocol32 from cultures grown as described for assessing HMV. Briefly, UA was extracted from 500 μl of culture with zwittergent, precipitated with ethanol, and resuspended in tetraborate/sulfuric acid. Phenylphenol was added and absorbance at 520 nm was measured. UA amounts were determined from a standard curve generated with glucuronolactone.
Results
Prevalence of rmp in the K. pneumoniae species complex
Screening for the rmp locus in publicly available KpSC genomes identified its presence in 992/13993 genomes across three KpSC species; K. pneumoniae (980/11967, 8.2%), K. quasipneumoniae subsp. similipneumoniae (Kqs; 5/522, 1.0%) and K. variicola subsp. variicola (Kv; 7/626, 1.1%) (see Table 2). The majority of these included complete rmp loci with intact coding sequences for rmpA, rmpC and rmpD (i.e. functional variants, detected in 73% of rmp+ genomes), however some loci were incomplete (i.e. deletion variants missing at least one gene within the locus or arising from assembly fragmentation) or had mutations resulting in premature stop codons truncating one or more of the encoded proteins (i.e. truncation variants) (see Table 2). Fifteen genomes were identified with multiple rmp loci (two rmp each); the majority of these carried at least one functional rmp locus (73%; eight genomes with two functional rmp loci and three genomes with one functional rmp) while the remaining four genomes each had two non-functional rmp loci arising from deletion and/or truncation variants. BLASTn search of all non-redundant bacteria in NCBI with default parameters identified complete rmp loci in four non-Klebsiella genomes, in all cases rmp was present in plasmids sequenced from Escherichia coli transconjugants that had been mated with a K. pneumoniae strain carrying KpVP-1-like virulence plasmids (GenBank accessions MN200130.1, MN182750.1, MZ475697.1, CP068571.1). Incomplete rmp loci (44% coverage) were detected in an additional five plasmids that were also sequenced from E. coli transconjugants.
Genetic diversity of the rmp locus
Each of the genes comprising the rmp locus showed some degree of genetic diversity, with 85 rmpA alleles, 77 rmpD alleles and 42 rmpC alleles observed across rmp+ genomes. For the 825 genomes carrying an intact ‘typeable’ rmp locus (838 loci total accounting for genomes with two rmp loci), allelic variants could be assigned to all three genes, and resulted in 170 unique combinations which were each assigned a unique RmST (rmp sequence type). The rmp loci from the remaining 167 genomes were designated ‘non-typeable’ due to the locus being incomplete (i.e. missing at least one gene or comprising a fragmented gene to which an allele could not be designated).
Maximum likelihood phylogenetic analysis of the concatenated rmpA, rmpD and rmpC sequences belonging to each unique RmST revealed the grouping of sequences into five distinct lineages, which we labelled rmp1, rmp2, rmp2A, rmp3 and rmp4 (Figure 1). These lineages were labelled as such to match with their associated iro and/or iuc lineages. The nucleotide divergence between lineages ranged from 0.7-11% (mean 4.7%), decreasing to 0-5.9% within lineages (mean 0.4%). No rmp gene alleles were shared between lineages (see individual gene phylogenies in Additional File 3), suggesting an absence of recombination between the lineages. The rmp3 lineage was notably divergent from the other rmp lineages, with a mean divergence of 8.4% compared to 1.8% between all other lineages (Table 3). Further, the rmpD alleles of the rmp3 lineage were longer than those of other lineages, measuring 176-177 bp in length compared to the 151-162 bp alleles observed in rmpD from the other lineages.
Diversity of rmp-associated mobile genetic elements
Each of the rmp lineages were associated with unique plasmids or chromosomal contexts (see Figure 1), many of which have been previously characterised in a study examining the aerobactin- and salmochelin- encoding loci iuc and iro10, which are typically co-localised with rmpA2 and rmp respectively on the same mobile elements. The dominant K. pneumoniae virulence plasmid, KpVP-1 (associated with iuc1 and iro1 loci) was associated with the rmp1 lineage and accounted for the majority of rmp carriage (785 genomes, 79.1%). Next most common were the iuc2A virulence plasmids (associated with iuc2A)10, carrying rmp2A lineage (77 genomes, 7.8%); followed by ICEKp1 carrying rmp3 (58 genomes, 5.8%); and KpVP-2 virulence plasmids with rmp2 (associated with iuc2 and iro2, 45 genomes, 4.6%). Two additional genetic contexts were observed in this study: (i) the rmp4 lineage was chromosomally-encoded and associated exclusively with K. pneumoniae CG67 genomes (six genomes, identified as ST67 or single-locus variants thereof, also known as K. pneumoniae subspecies rhinoscleromatis33); and (ii) transposition of rmp3 into the yersiniabactin-encoding ybt4-type plasmids (three genomes; note partial deletion of rmp locus in two of these genomes). For the remaining genomes, one was associated with a KpVP-2/iuc2A hybrid plasmid as previously described10, nine carried multiple rmp loci (due to presence of both KpVP-1 and ICEKp1), and 17 were unresolved due to assembly issues. KpVP-1 was detected in 785 genomes, where it typically carried both rmp and rmpA2 (669/785). However, deletion variants of KpVP-1 were common (i.e. coverage of KpVP-1 reference ranged from 26.7% coverage in rmp1+ genomes): we observed 116/785 genomes with rmp only without rmpA2 (20 and 32 of these lacking an intact iro1 and iuc1, respectively).
The genetic context of rmp in representatives of each of the key rmp lineages is shown in Figure 2. The rmp locus was located adjacent to the iro locus and peg-344 gene (which has been mis-labelled as pagO in some studies) in most cases, with the exception of the ybt4 plasmids (which lack iro and peg-344 genes) and iuc2A plasmids (which carry iroB only). The iro locus is typically intact in KpVP-1, KpVP-2 and ICEKp1, but is partially disrupted by insertion sequences (IS) in the ST67 (rhinoscleromatis) chromosome and iuc2A plasmids. IS flank the rmp/iro region in all contexts, however the specific IS vary and it is not clear which have played a role in mobilising rmp and/or iro. Notably, all contexts include IS3 on one or both ends of the rmp/iro region. The closely related rmp1, rmp2, rmp2A and rmp4 lineages all have IS3 in a conserved position adjacent to rmpA; this also appears to be conserved in the divergent lineage rmp3, suggesting it was likely present in the common ancestor of all rmp lineages and may have played a role historically in the mobilisation of rmp/iro between genetic backgrounds (see Figure 2). However all rmp/iro regions have additional IS near or within the locus, some of which may have contributed to further mobilisation and/or degradation of the distinct lineages over time. While the assembly of the rmp region in the ybt4 plasmid was incomplete, an IS3 fragment was detected next to rmp.
Hypermucoviscosity of rmp variants in a KPPR1S background
To determine if there were functional differences between the level of HMV conferred by the rmp lineages, we ectopically expressed variants of the rmp locus cloned into pMWO-078 in an HMV-negative rmp deletion mutant KPPR1S Δrmp. Wildtype KPPR1S is a ST493 K2/O1 isolate containing rmp3 (ICEKp1), which is the most divergent rmp lineage (Figure 1); KPPR1S Δrmp therefore provides a good background to assess potential functional differences between rmp variants. HMV was assessed using a sedimentation assay. The ΔwcaJ and Δrmp vector controls fully sedimented, whereas the KPPR1S vector control did not sediment well (Figure 3A). Ectopical expression of the native locus, rmp3, conferred elevated HMV in Δrmp compared to the KPPR1S vector control. This elevated phenotype is likely due to the overexpression of rmp, and this complemented strain therefore serves as a reference to compare the effects of expressing the other rmp loci. Expression of the rmp genes from rmp1, rmp2 and rmp2A also conferred elevated HMV levels in Δrmp (Figure 3A), and there were no statistically significant differences in sedimentation resistance between these strains versus that expressing rmp3. However, expression of rmp4 conferred a significantly lower level of HMV compared to the other lineages.
Capsule production of rmp variants in a KPPR1S background
Elevated capsule production is an rmpC-dependent phenotype in KPPR1S, independent of HMV, whereby RmpC upregulates capsule expression via the promoters upstream of the galF and manC genes in the K locus, and KPPR1S Δrmp mutants display decreased capsule production15,16. To further investigate any functional differences between rmp variants, we assayed the same transformants described above for uronic acid (UA) production, which is used as an indicator of the amount of capsule32. KPPR1S, ΔwcaJ (i.e. capsule-negative), and Δrmp strains were included as controls. Expression of rmp from each of the lineages increased capsule production (Figure 3B). Expression of rmp1, rmp2A and rmp4 in the Δrmp mutant increased capsule production to the same level observed when expressing the native rmp3 locus (Figure 3B). Expression of rmp2 produced slightly but significantly higher uronic acid levels compared to rmp3 (p < 0.05). Collectively, these data suggest that there are subtle but significant functional differences between the rmp lineages.
Distribution of rmp in the K. pneumoniae species complex
The rmp locus was detected in 129 unique K. pneumoniae STs, 3 Kqs STs and 5 Kv STs, representing 143 unique combinations of STs and rmp lineages. As expected, the prevalence of rmp is typically quite high within known hvKp clones (≥80% prevalence within any given ST assigned to a hypervirulent clonal group with the exception of CG25, which has recently been shown to comprise of two distinct lineages; mean 83.6%) (Figure 4). Rmp was also common in CG67 (rhinoscleromatis, 100% rmp4; 25% truncated) and CG91 (ozaenae, 79% rmp2A incomplete or truncated); both lineages have previously been defined as subspecies due to their distinct pathotypes33. However, relatively high rmp prevalence was also observed in some ‘generalist’ (i.e. non-HvKp and non-MDR) K. pneumoniae clones (1.6-100% rmp prevalence, mean rmp prevalence=75.0%). Conversely, the MDR clones together with CG36 and CG45, which are also clones that are commonly detected in healthcare settings, had relatively lower frequencies of rmp (mean prevalence=11.4%).
KpVP-1-rmp1 was the most widely disseminated, and was not only detected in the hypervirulent clones from which they were initially characterised but also in MDR clones. Numbers of KpVP-1-rmp1 were notably high in ST11 (211 genomes) and ST15 (50 genomes), reflecting recently reported MDR-hypervirulent convergent variants of these well-known MDR clones34. KpVP-1 also accounted for the majority of rmp acquisitions in the other KpSC species where the genetic context could be resolved. In comparison, the other mobile elements appeared in relatively fewer STs, but also appeared to be fixed in most of these clones that likely serve as native hosts to the respective mobile elements (see Figure 4).
Amongst rmp+ genomes with a confident K locus call (917/992), 38 different K loci were detected (Figure 5). Half (i.e. 19) were detected in two or fewer genomes. The most common K loci were KL1 (319 genomes), KL2 (203), KL64 (140), which collectively account for 72.2% of genomes with a confident K locus, followed by KL57 (45) and KL20 (40). The dominant K loci are largely driven by their associations with over-represented STs (Figure 5), many of which are known hvKp lineages such as KL1 in CG23 (which harbours KpVP-1), and KL2 in CG380 (KpVP2), CG65 and CG86 (KpVP-1). The next most common K locus, KL64, is associated with a Chinese sublineage of MDR clone ST11, known to have acquired variants of KpVP-134.
Given previous reports and observations highlighting the common occurrence of truncations in rmpA due to insertions and deletions (indels) within a poly-G tract14, we next examined the rmp locus for the occurrence of truncations. Truncations were detected in 30/85 rmpA, 9/77 rmpD and 16/42 rmpC alleles across 63, 16 and 55 genomes respectively (Additional File 3). The majority of truncations (45/55 truncated alleles) were caused by indels within homopolymer tracts resulting in premature stop codons arising from frameshift mutations (Additional File 4). Further, some homopolymer regions had a higher frequency of mutations compared to others, including the previously reported poly-G tract in rmpA spanning nucleotide position 276 to 285 whereby indels were detected in 16 rmpA variants across 46 genomes. The frequency of truncated rmp loci compared to intact loci was lower within the hypervirulent clones and appeared to be more common outside of these clones (Figures 4 and 5). Closer inspection of the phylogenies for hypervirulent clones CG23, CG65 and CG86 (Additional Files 5 - 7) revealed a random distribution of truncated rmp alleles throughout these populations.
Discussion
We previously reported briefly on the sequence variation of the rmp locus, which clustered into four lineages and were each associated with distinct MGEs20. In this study we provide detailed insights into the phylogenetic relationships, genetic contexts, distribution and phenotypic functionality of rmp variants. Building on the initial rmp scheme, we characterised an additional rmp4 lineage observed only in genomes belonging to a sublineage of K. pneumoniae (CG67), updating the number of rmp lineages to five.
The 1:1 correlation observed between rmp lineages and the MGEs that mobilise them also extends to iro, shown here to be typically located adjacent to rmp (Figure 2), and to iuc, which is often located elsewhere on the same plasmids10. This association highlights the co-evolution of these virulence loci, and presumably other common genes shared between the MGEs. While specific details or the order of events in the evolutionary history of these MGEs cannot be determined, similarities in the genetic contexts surrounding rmp do suggest a shared ancestry and the role of IS, particularly IS3, in the mobilisation of rmp/iro between different MGEs. Interestingly, the presence of IS3 upstream of rmpA2 has been flagged as being necessary for complete activation of the promoter for rmpA2, and may likewise serve a similar purpose for rmpA35. We also observed three instances of a novel genetic context for rmp, where a variant of rmp3 (ICEKp1) had been introduced into a yersiniabactin plasmid (i.e. another virulence MGE)36 likely via IS3-mediated transposition (Figure 2). Outside of K. pneumoniae, rmp (KpVP-1 and ICEKp1) was only detected in two other KpSC species at rare frequencies, and did not appear to have been acquired naturally in any other species. K. pneumoniae is therefore likely to be the original host for rmp, although the same may not hold true for the other virulence loci given that iro and iuc variants have been detected in other non-Klebsiella species including Enterobacter spp. and Escherichia coli10.
Even with the larger genome dataset screened in this present study, the prevalence of rmp overall (7.5%) and each of the MGEs was very similar to that reported in our earlier study characterising iuc (8.7%) and iro (7.2%) (i.e. 13000 versus 2700 genomes). The rmp1 lineage (KpVP-1) was by far the most dominant accounting for 80% of the rmp burden, in part driven by their association and maintenance via clonal expansion within the key hypervirulent clones such as CG2311, but also due to the more recent transmission and spread within MDR clones such as ST11 and ST15 (Figures 1 and 4). Based on growing reports of ‘hypervirulent ST11’ from China, it is possible that KpVP-1 is also being stably maintained within this clone following its acquisition. With the exception of KpVP-1-rmp1 and ICEKp1-rmp3, which were detected in multiple STs/CGs, the remaining MGEs also appeared to be stably conserved within a small number of clones in which they were detected (93.9-100% prevalence; Figure 4).
Each of the rmp lineages was shown to be functional, resulting in HMV and elevated capsule production when a representative of each was introduced into a single strain background, KPPR1S rmp. Notably, this ST493 strain has a K2 capsule, which is one of two dominant capsule types among known hvKp clones such as CG86 (Figure 5). We also observed variability in the extent of HMV and capsule production for different rmp, which further reiterates that HMV and capsule production are two separable traits15,16; for example, the expression of rmp4 does not restore HMV to the same level as KPPR1S rmp3 but does restore capsule production (Figure 3). It is unclear if expressing these rmp (or other representatives of the same lineages) in other KpSC strains with different serotypes will have the same impact, especially given the extensive diversity of Wzc in different K loci and its interaction with RmpD. These insights will be particularly useful for predicting the impacts of rmp acquisition in other clones, including those considered to be MDR, and is the subject of ongoing work by our team.
Loss of function mutations of rmpA arising from indels within the poly(G) tract has been well documented in many studies14, and while this site (i.e. bases 267 to 285 of the rmpA_2 reference) does account for the majority of rmpA truncations in this dataset, indels within additional homopolymer tracts in rmpA, rmpD and rmpC were also observed (Additional File 4). Loss of function mutations in the rmp genes were observed in at least 145 genomes (14.6% of rmp+ genomes, including three with multiple rmp), most often in the non-hypervirulent clones. Further, we observed parallel evolution of the same loss of function mutations on multiple occasions within different hypervirulent clones (e.g. allele rmpA_4 in CG23, CG65 and CG86; Additional Files 5-7). Taken together, these findings highlight the potential reversibility of homopolymer tract mutations, which may take place to help alleviate the negative selection pressure following acquisition of KpVP-1 or other MGEs. Importantly, these loss of function mutations also need to be carefully considered when interpreting data based solely on PCR detection of rmpA/A2, and may partly explain the discrepancies in the literature reporting on the association between rmp presence and HMV. Although, other reasons also include inconsistencies and the unreliability of string testing, which is heavily influenced by temperature dependencies and strain genetic background37.
Conclusions
Our findings reveal that, similar to the other key virulence loci co-localised on the same MGEs, genetic variation within the rmp locus is highly structured and this information can be harnessed to track novel rmp acquisitions. To this end, detection and genotyping of the rmp genes (i.e. RmST typing), alongside the reporting of locus disruptions, has already been implemented in our genotyping tool for KpSC genomes, Kleborate (github.com/klebgenomics/Kleborate). The tool also outputs a virulence score that is currently calculated from the presence of various siderophores (ybt and iuc) and colibactin (clb), and does not take into account rmp (or iro). Given the apparent role of rmp in hypervirulence, the virulence score in addition to rmp presence should both be considered when assessing the virulence of a given strain or genome. Ongoing research investigating the expression of rmp variants in different strain and capsule backgrounds will yield additional important insights into the function of rmp, particularly for novel strains such as those from the MDR clones that acquire them, and can be used to further improve the genotyping output for rmp.
Supporting information
Declarations
Ethics approval and consent to participate
Not applicable
Consent for publication
Not applicable
Availability of data and materials
All whole-genome sequences analysed in this study are publicly available on NCBI, and the accession numbers are listed in Additional File 1. The RmST scheme is available in the K. pneumoniae BIGSdb database (https://bigsdb.pasteur.fr/klebsiella/) and in the Kleborate distribution (https://github.com/klebgenomics/Kleborate)
Competing interests
Not applicable
Funding
This work was supported, in whole or in part, by the Bill & Melinda Gates Foundation [OPP025280]. Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. MMCL is supported by an Australian National Health and Medical Research Council Investigator Grant [APP2009163].
Acknowledgements
We thank the Institut Pasteur curation team of BIGSdb-Pasteur for importing novel alleles, profiles and/or isolates at https://bigsdb.pasteur.fr/.
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Additional file 1
Strain information and Kleborate genotyping output for genomes included in this study (XLS)
Additional file 2
Primers used in this study (DOC)
Additional file 3
Phylogenetic relationships of the A. rmpA, B. rmpD and C. rmpC genes. (DOC)
Additional file 4
Description of truncated allelic variants of rmpA, rmpD and rmpC (XLS)
Additional file 5
Distribution of rmpADC allelic variants and KpVP-1 associated virulence loci rmpA2, iuc and iro in Klebsiella pneumoniae clonal group 23 (DOC)
Additional file 6
Distribution of rmpADC allelic variants and KpVP-1 associated virulence loci rmpA2, iuc and iro in Klebsiella pneumoniae clonal group 65 (DOC)
Additional file 7
Distribution of rmpADC allelic variants and KpVP-1 associated virulence loci rmpA2, iuc and iro in Klebsiella pneumoniae clonal group 86 (DOC)