Clonal relatedness of strains harboring HVPs

Seventy-nine non-redundant HVPs were found in GenBank. Their corresponding hvKp strains were belonged to clonal complex (CC)23 (including ST23, ST485, ST1660, ST1265, ST1946, ST268, ST36, ST65 and ST375) as well as single clonal lineages ST7, ST11, ST15, ST29, ST34, ST147, ST86, ST101, ST90, ST374, ST383, ST3685, ST83 and ST3913. See Fig. 1. Six plasmids were found in strains with undetermined ST. See Additional file 1: Table S1.

Fig. 1

The minimum spanning tree of STs carrying the Klebsiella pneumoniae hypervirulent plasmids with similarity cut off of > 4 allelic types (green: STs carrying all 14 hypervirulent genes, purple: STs not carrying all hypervirulent genes). Clonal complex 23 (CC23) including ST23, ST1941, ST1660, ST485, ST268, ST36, ST1265, ST65, and ST375 harbored complete hypervirulence pattern. While, non-CC23 including ST11, ST3813, ST86, and ST29 lack several hypervirulence genes

Genetic plasticity and phylogenic analysis and of HVPs

High heterogeneity was observed among different HVPs. However, they have similarity in some regions regardless of their strains STs. See Fig. 2A.

Fig. 2

A The multiple circular alignment of 15 K. pneumoniae hypervirulent plasmids. High heterogenicity was detected among plasmids. However, they were similar in some regions. B The phylogenetic NJ dendrogram of 79 K. pneumoniae hypervirulent plasmids and distribution of drug-resistant hybrids. The subclade distribution of plasmids within the leaves was not exactly associated with their strains STs. Conjugative antimicrobial-resistant hybrids were mainly distributed in clade II and III. Besides, almost all MDR-HVPs were located in clade III. While, clade I mainly contained non-drug-resistant HVPs

A number of 2952 of genes with > 10% occurrence rate were identified for plasmids by BacCompare. The results showed that the HVPs had 1589 unique genes. The heatmap of 79 plasmids showed that the plasmids have different allele numbers in 185 loci. See Additional file 3: Fig. S1. The phylogenetic dendrogram based on 2952 gene classified the HVPs in three main clades (I, II and III). See Fig. 2B.

Genetic characteristics of HVPs

The size of plasmids ranged from 96,085 to 479,335 bp. Twenty-one plasmids were potentially conjugative carrying all four conjugal constituents including oriT, relaxase, type IV coupling protein (T4CP) and type IV secretion system (T4SS). Three plasmids including CP034421.1, CP045692.1 and CP032834.1 were mobilizable (only lacked oriT). All plasmids were positive for T4CP and T4SS.

A number of 56 plasmids had IncFIB and IncHI1B. Thirteen plasmids had only IncFIB. A number of seven plasmids had the IncFIB and IncFII replicon types. CP025640.1 and CP029227.1 had only IncHI1B. MK181633.1, CP035384.1 and MF398271.1 had three replicon types (IncFIB, IncFIB and IncHI1B). IncFIB and IncR were detected in CP045692.1. CP040994.1 had IncFIA and IncFIB. See Additional file 1: Table S1 and Fig. 3A.

Fig. 3

A The prevalence of different Inc groups among 79 K. pneumoniae hypervirulent plasmids. IncFIB was the most abundant (97%) replicon type among HVPs which co-existed with IncHI1B in 70% of the plasmids. B The prevalence of ARGs K. pneumoniae hypervirulent plasmids against different drug classes. The plasmids were mainly resistant to beta-lactamand aminoglycosides modifying enzymes. C The frequency of major ESBLs and carbapenemases. blaTEM, blaCTX-M and blaSHV were the most abundant ESBL genes among hypervirulent plasmids

Hypervirulence profile

A number of 32 plasmids carried all 14 hypervirulence related genes including iroB, iroC, iroD, iroN, iucA, iucB, iucC, iucD, iutA, terB, rmpA, rmpA2, peg-589 and peg-344. Out of 79 plasmids, 78 were positive for iutA and iucA. The iucC, iucB and iucD were found in 77 plasmids. The rmpA2, peg-589 and terB genes were detected in 73, 70 and 66 HVPs, respectively. peg-344 and iroN were identified in 51 and 49 plasmids, respectively. A number of 41 plasmids were positive for iroD, iroC and iroB genes. The fewest number of hypervirulence genes was for CP040994.1 (peg-344, iroB and rmpA). The schematic comparison of plasmids hypervirulence profile has been presented in Fig. 4.

Fig. 4

The hypervirulence profile (iroB, iroC, iroD, iroN, iucA, iucB, iucC, iucD, iutA, terB, rmpA, rmpA2, peg-589 and peg-344) of 79 K. pneumoniae hypervirulent plasmids. The iucA, iutA, iucD, iucB, iucC, rmpA2, peg-589 and terB genes were highly prevalent among plasmids

Antimicrobial resistance associated genetic elements in HVPsARGs

A number of 24 plasmids carried different ARGs against extended-spectrum beta-lactams (blaTEM-1, blaDHA-1, blaCTX-M-15, blaCTX-M-24, blaSHV-11, blaSHV-64, blaSHV-5 and blaCMY-6), carbapenems (blaOXA-1, blaKPC-2 and blaNDM-1), quinolones (qnrB4, qnrB17 and qnrS1), aminoglycosides (armA, aph(3'')-Ib, aph(3')-Ia, aac(6')-Ib-cr6, aac(6′)-lb-cr5, aac(6')-Ib10, aac(6′)-lb, aac(3)-lld, aac(3)-IIe, rmtC and ant(3'')-IIa), chloramphenicol (catI, catII, cmlA1, and catB3), tetracycline (tetA, tetB, tetD, and tetR) and macrolide (mphE, msrE, and mphA). See Additional file 1: Table S1.

Quinolone resistance genes were identified in four plasmids including MK649823.1, MK191024.1, MK649829.1, and MK181633.1. Macrolide, tetracycline and chloramphenicol resistance genes were detected in 12, six, and five plasmids, respectively. A number of 25 and 23 aminoglycosidase and beta-lactamase encoding genes were found, respectively. See Fig. 3B. blaCTX-M, blaTEM and blaSHV were carried by six, five and four plasmids, respectively. CP028791.1 (blaOXA-1), MK181633.1 (blaOXA-1), MK649825.1 (blaOXA-1), MF398271.1 (blaKPC-2) and CP034201.1 (blaNDM-1) were carbapenemase producers. See Fig. 3C. Six plasmids were MDR-HVPs. See Additional file 1: Table S1.

Mobile genetic elements associated with AMRProphages

A number of 13 HVPs had prophage structures with Salmon_SJ46 and Escher_RCS47 being the most prevalent. The blaCTX-M and blaTEM-1 genes were major ESBLs carried by prophage structures. blaOXA-1 of CP028791.1 was detected in Escher_RCS47. Genes encoding aminoglycosides modifying enzymes in MK181633.1, CP034046.1, CP034054.1, CP040595.1 and CP028791.1 carried by different incomplete prophages (including Bacill_Shanette, Salmon_SJ46, Escher_RCS47 and Escher_Av). Some ARGs against other antimicrobial drug classes including quinolones (qnrS1), tetracycline (tetA) and chloramphenicol (catB3) were also phage-mediated. Prophages in five plasmids did not carry any ARGs. See Additional file 2: Table S2.

Integrons

Class 1 integron (intI1) was found in eight plasmids including CP034421.1, CP040595.1, CP040726.1, MK649825.1, CP030270.1, CP028791.1, CP034046.1, and MK181633.1. See Additional file 3: Fig. S2. ARGs which were found around class 1 integron were as follows: aac(3)-lld, aac(6’)-lb and aac(6’)-lb-cr5 on plasmids CP034421.1, CP040595.1 and CP028791.1, respectively; dfrA5 on CP040726.1, MK649825.1 and MK181633.1; cmlA and catB3 on CP030270.1 and CP028791.1, respectively and arr3 on CP028791.1. Class 2 integron (sat2) was detected only on CP034046.1

Insertion sequences and transposons

The IS26 (IS6-like), ISVsa5 (IS4-like), IS4321 (IS110-like), ISCfr1 (IS1182-like), ISEc9 (IS1380-like), ISKpn11(IS3-like), IS1326 (IS2-like), IS110, IS1 elements as well as TnAs3 (Tn3-like) and Tn3 family transposases were truncated by intI1 and multiple ARGs. See Additional file 3: Fig. S2. IS6, IS110 and IS1380 were frequently found on prophages surrounding beta-lactamase and other ARGs. See Additional file 3: Table S2.