Open Access

Comparative genomic analysis of Klebsiella pneumoniae subsp. pneumoniae KP617 and PittNDM01, NUHL24835, and ATCC BAA-2146 reveals unique evolutionary history of this strain

  • Taesoo Kwon1,
  • Young-Hee Jung2,
  • Sanghyun Lee3,
  • Mi-ran Yun3,
  • Won Kim1 and
  • Dae-Won Kim3Email author
Contributed equally
Gut Pathogens20168:34

https://doi.org/10.1186/s13099-016-0117-1

Received: 17 April 2016

Accepted: 16 June 2016

Published: 11 July 2016

Abstract

Background

Klebsiella pneumoniae subsp. pneumoniae KP617 is a pathogenic strain that coproduces OXA-232 and NDM-1 carbapenemases. We sequenced the genome of KP617, which was isolated from the wound of a Korean burn patient, and performed a comparative genomic analysis with three additional strains: PittNDM01, NUHL24835 and ATCC BAA-2146.

Results

The complete genome of KP617 was obtained via multi-platform whole-genome sequencing. Phylogenetic analysis along with whole genome and multi-locus sequence typing of genes of the Klebsiella pneumoniae species showed that KP617 belongs to the WGLW2 group, which includes PittNDM01 and NUHL24835. Comparison of annotated genes showed that KP617 shares 98.3 % of its genes with PittNDM01. Nineteen antibiotic resistance genes were identified in the KP617 genome: bla OXA-1 and bla SHV-28 in the chromosome, bla NDM-1 in plasmid 1, and bla OXA-232 in plasmid 2 conferred resistance to beta-lactams; however, colistin- and tetracycline-resistance genes were not found. We identified 117 virulence factors in the KP617 genome, and discovered that the genes encoding these factors were also harbored by the reference strains; eight genes were lipopolysaccharide-related and four were capsular polysaccharide-related. A comparative analysis of phage-associated regions indicated that two phage regions are specific to the KP617 genome and that prophages did not act as a vehicle for transfer of antimicrobial resistance genes in this strain.

Conclusions

Whole-genome sequencing and bioinformatics analysis revealed similarity in the genome sequences and content, and differences in phage-related genes, plasmids and antimicrobial resistance genes between KP617 and the references. In order to elucidate the precise role of these factors in the pathogenicity of KP617, further studies are required.

Keywords

Klebsiella pneumoniae OXA-232 NDM-1 Carbapenemases

Background

Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated, facultative anaerobic bacterium, which belongs to the family Enterobacteriaceae. K. pneumoniae is found in the normal flora of the mouth, skin, and intestines; however, this bacterium may act as an opportunistic pathogen, causing severe nosocomial infections such as septicemia, pneumonia, and urinary tract infections in hospitalized and immune-comprised patients with chronic ailments [1, 2].

Beta-lactam antibiotics, used as therapeutic agents against a broad range of bacteria, bind to the penicillin-binding protein and inhibit biosynthesis of the bacterial cell membrane. However, the extended spectrum β-lactamases (ESBLs) and carbapenemases confer resistance to penicillin, cephalosporins, or carbapenem [3, 4]. The β-lactamases are divided into four classes on the basis of the Ambler scheme: class A (Klebsiella pneumoniae carbapenemase, KPC; imipenem-hydrolyzing β-lactamase, IMI; Serratia marcescens enzyme, SME; Serratia fonticola carbapenemase, SFC), class B (Verona integron-encoded metallo-β-lactamase, VIM; imipenem-resistant Pseudomonas, IMP; New Delhi metallo-β-lactamase, NDM), class C (AmpC-type β-lactamase, ACT; cephamycin-hydrolyzing β-lactamase, CMY), and class D (oxacillinase, OXA) [5] are composed of transposon, cassettes, and integrons and transferred within and between species by HGT (horizontal gene transfer). Numerous carbapenemase-producing bacteria similarly harbor drug resistance genes that are transferred to other strains by horizontal gene transfer [6, 7]; infections caused by such multi-drug-resistant bacteria are difficult to treat [8]. The emergence of the novel carbapenemase NDM-1 (the New Delhi metallo-β-lactamase) is of great concern, as no therapeutic agents are available to treat infections caused by NDM-1-producing bacterial strains [9]. NDM-1-producing K. pneumoniae strains were first isolated from a Swedish patient who had travelled to India in 2009 [10]. Since then, NDM-1 has been reported to be produced by various species of Enterobacteriaceae, such as K. pneumoniae, Escherichia coli, Enterobacter spp. and Acinetobacter spp., in numerous countries [11].

The carbapenem-hydrolyzing β-lactamase OXA-232, which was first reported in E. coli and two K. pneumoniae strains [12], belongs to the OXA-48-like family. Carbapenemase-producing Gram-negative bacteria are often multi-drug resistant [13]. K. pneumoniae isolates that coproduce OXA-48-like β-lactamase and NDM-1 have been isolated in numerous countries [1416]. Recently, K. pneumoniae isolates coproducing two carbapenemases, bla NDM-1 and bla OXA-232 , have been identified in several countries; of these, two isolates originating in India were recovered in the USA and Korea, in January 2013, and sequenced [16, 17] but not studied yet the characteristics in the context of genomic contents by comparing these isolates. In the present study, we performed a comparative analysis of the genomes of these isolates.

Methods

Isolation and serotyping of strains

In January 2013, a 32-year-old man was hospitalized in the Intensive Care Unit of a general hospital in Seoul, Korea, two days after suffering burns during a visit to India. K. pneumoniae was isolated from his wound and another patient in the same room became infected with the same strain [18]. The K. pneumoniae isolate was identified as the KP617 strain belonging to the sequence type (ST)14, and found to coproduce NDM-1 and OXA-232, which conferred resistance to ertapenem, doripenem, imipenem, and meropenem (MICs: >32 mg/L). The K. pneumoniae strains PittNDM01 [17], NUHL24835 [19], and ATCC BAA-2146 [20] were used as reference strains for comparative genomic analysis.

Library preparation and whole-genome sequencing

Whole-genome sequencing of KP617 was performed using three platforms: Illumina-HiSeq 2500, PacBio RS II, and Sanger sequencing (GnC Bio: Daejeon, Republic of Korea) [16]. Sanger sequencing was used for the construction of a physical map of the genome.

Genome assembly and annotation

A hybrid assembly was performed using the Celera Assembler (version 8.2) [21] and a fosmid paired-end sequencing map was used to confirm the assembly. The final assembly was revised using proovread (version 2.12) [22]. An initial annotation of the KP617 genome was generated using the RAST (Rapid Annotation using Subsystem Technology, version 4.0) server pipeline [23]. The genomes of three K. pneumoniae strains, PittNDM01, NUHL24835, and ATCC BAA-2146, were annotated using the RAST server pipeline. In order to compare the total coding sequences (CDSs) of KP617 with those of the three K. pneumoniae strains, the sequence-based comparison functionality of the RAST server was utilized.

Phylogenetic analysis

Concatenated whole genomes of 44 K. pneumoniae strains, including KP617, and multi-locus sequence typing (MLST) of seven genes [24, 25] were used for the calculation of evolutionary distances. The seven genes used for MLST were as follows: gapA, infB, mdh, pgi, phoE, rpoB and tonB. Multiple sequence alignments were performed using Mugsy (version 1.2.3) [26]. The generalized time-reversible model [27] + CAT model [28] (FastTree Version 2.1.7) [29] was used to construct approximate maximum-likelihood phylogenetic trees. The resulting trees were visualized using FigTree (version 1.3.1) (http://tree.bio.ed.ac.uk/software/figtree/).

Comparison of genomic structure

The chromosome and plasmids of KP617 and the reference strains were compared using Easyfig (version 2.2.2) [30]. Whole-genome nucleotide alignments were generated using BLASTN to identify syntenic genes. The syntenic genes and genomic structures were visualized using Easyfig. A stand-alone BLAST algorithm was used to analyze the structure of the genes of interest, i.e. the OXA-232- and NDM-1 carbapenemase-encoding genes.

Identification of the antimicrobial resistance genes

We identified the antibiotic resistance genes using complete sequences of chromosomes and plasmids of four K. pneumoniae isolates: KP617, PittNDM01, NUHL24853 and ATCC BAA-2146 using ResFinder 2.1 (https://cge.cbs.dtu.dk/services/ResFinder/) [31].

Analysis of virulence factors and phage-associated regions

The virulence factor-encoding genes were searched against the virulence factor database (VFDB) [32] using BLAST with an e-value threshold of 1e-5. Homologous virulence factor genes with a BLAST Score Ratio (BSR) of ≥0.4 were selected. The BSR score was calculated using our in-house scripts. Phage-associated regions in the genome sequences of the four K. pneumoniae strains were predicted using the PHAST server [33]. Three scenarios for the completeness of the predicted phage-associated regions were defined according to how many genes/proteins of a known phage the region contained: intact (≥90 %), questionable (90–60 %), and incomplete (≤60 %).

Quality assurance

Genomic DNA was purified from a pure culture of a single bacterial isolate of KP617. Potential contamination of the genomic library by other microorganisms was assessed using a BLAST search against the non-redundant database.

Results and discussion

General features

A total of 316,881,346 (32,005,015,946 bp) paired-end reads were generated using Illumina-HiSeq 2500. Using the PacBio RS II platform, 46,134 (421,257,386 bp) raw reads were produced. The complete genome of KP617 consists of a 5,416,282-bp circular chromosome and two plasmids of 273,628 bp and 6141 bp in size. The genomic features of KP617 and the reference strains are summarized in Table 1. Based on a RAST analysis, 5024 putative open reading frames (ORFs) and 110 RNA genes on the circular chromosome (Figs. 1, 2; Additional file 1: Table S1), 342 putative ORFs on plasmid 1, and 9 putative ORFs on plasmid 2 were identified.
Table 1

Genomic features of Klebsiella pneumoniae KP617 and other strains

Strain

KP617

PittNDM01

NUHL24835

ATCC BAA-2146

Genome (Mb)

5.69

5.81

5.53

5.78

% GC (chromosome)

57.4

57.5

57.4

57.3

Total open reading frames

5375

4940

5191

5883

Plasmids

2

4

2

4

Fig. 1

Subsystem category distribution of KP617 based on SEED databases

Fig. 2

Circular map of the KP617 chromosome. Circular map of the KP617 genome, generated using cgview (version 2.2.2); from outside to inside, the tracks display the following information: CDSs of KP617 on the + strand (1); CDSs of KP617 on the − strand (2); tblastx result against PittNDM01 (3), tblastx result against NUHL24835 (4), tblastx result against ATCC BAA-2146 (5), GC content (6), GC skew with + value (green) and − value (purple) (7)

Comparison of KP617 and the reference strains based on sequence similarity (percent identity ≤80) showed that 32 genes are unique for KP617, and that most of the functional genes of this strain are also conserved in the reference strains. The genes unique to the KP617 strain, such as the SOS-response repressor and protease LexA (EC 3.4.21.88), integrase, and phage-related protein were identified as belonging to the genome of the prophage Salmonella phage SEN4 (GenBank accession: NC_029015). When the KP617 genome was compared with that of the PittNDM01 strain, which represents the closest neighbor of the former strain on the phylogenetic tree (Figs. 3a, b), 94 genes showed a percent similarity of below 80; most of these were phage protein-encoding genes. These results indicate that the presence of prophage DNA is an important feature of the KP617 genome.
Fig. 3

Phylogenetic tree of Klebsiella pneumoniae, a whole-genome phylogenetic tree; b MLSA phylogenetic tree; the scale represents the number of substitutions per site

Phylogenetic analysis

The whole-genome phylogenetic analysis indicated that KP617 is evolutionarily close to PittNDM01 and NUHL24835, and that the strains belong to the WGLW2 group. However, KP617 was found to be evolutionarily distant from ATCC BAA-2146 (Fig. 3). Concordantly, MLST-based phylogenetic analysis revealed that while KP617, PittNDM01, and NUHL24835 belong to the same group [sequence type (ST)14], ATCC BAA-2146 belongs to the HS11286 group, ST 11 [20]. The only difference between the whole-genome phylogenetic tree and the MLST-based phylogenetic tree was the divergence time within the same group; MLST-based phylogeny did not reveal the minor details of genomic evolution such as the divergence between KP617, PittNDM01 and NUHL24835 in the whole-genome phylogeny. The difference was attributed to horizontal gene transfer in regions not covered by the MLST genes.

Comparison of genome structures

The comparison of genomic structures of the chromosome indicated the presence of highly conserved structures in the KP617, NUHL24835, and PittNDM01 strains (Fig. 4a). Interestingly, a 1-Mb region (233,805–1,517,597) of the KP617 chromosome was inverted relative to its arrangement in the chromosome of PittNDM01 (1,500,972–225,619). Despite this inversion, KP617 and PittNDM01 exhibited a lower substitution rate (score 20) than NUHL24835 (score 30) (Fig. 3). However, the chromosomal structure of the ATCC BAA-2146 strain, which consisted of two large inverted regions, was significantly different from that of the other strains. In addition, a 71 Kb inversion was found in the sequence of plasmid 1 of KP617 (18,633–90,686) relative to plasmid 1 of PittNDM01 (91,507–19,453); however, the two plasmids were highly homologous to each other (Fig. 4b).
Fig. 4

Comparative analysis of genome structures between KP617 and the reference strains PittNDM01, NUHL24835, and ATCC BAA-2146. a Comparison of chromosome structure between KP617 and the reference strains. An inversion spanning 233,805 bp to 1,517,597 bp (1 Mb in size) in the KP617 chromosome is shown. b Comparison between the structure of plasmid 1 of KP617 and plasmid 4 of PittNDM01. There was a 71 kb inversion, from 18,633 bp to 90,686 bp, in plasmid 1 of the KP617 strain

Antimicrobial resistance genes

Nineteen antibiotic resistance genes were identified in the genome of KP617, 39 in the genome of PittNDM01, 29 in that of ATCC BAA-2146, and nine in that of the NUHL24385 strain (Table 2). The β-lactam resistance genes in the KP617 genome were bla OXA-1 and bla SHV-28 in the chromosome, bla NDM-1 in plasmid 1, and bla OXA-232 in plasmid 2; however, genes conferring resistance to colistin and tetracycline were not found (Table 2). Plasmid 2 of KP617, which includes the OXA-232-encoding gene, consists of a 6141-bp sequence; the sequence of this plasmid was identical to that of plasmid 4 of PittNDM01 (100 % coverage and similarity) and the plasmid of E. coli (coverage: 100 %, similarity: 99.9 %). Plasmid 2 of KP617, plasmid 4 of PittNDM01 and E. coli Mob gene cluster (GenBank accession: JX423831) [12] carried the OXA-232-encoding gene, and pKF-3 of K. pneumoniae carried the OXA-181-encoding gene. However, pKF-3 was identical to plasmid 2 of KP617, except in that the insertion sequence ISEcp1 was inserted upstream of OXA-181 and included in the transposon Tn2013 [12, 34].
Table 2

Antimicrobila resistance genes of KP617 and the reference strains

Antibiotics

Resistance gene

% identity

Query/HSP length

Predicted phenotype

Accession number

Positiona

KP617

PittNDM01

BAA-2146

NHUL24385

Aminoglycosides

aacA4

100

555/555

Aminoglycoside resistance

KM278199

  

P3_115183..115737

 

aac(3)-IIa

99.77

861/861

X51534

  

P2_41114..41974

 

aac(3)-IId

99.88

861/861

EU022314

 

P3_64003..64863

  

aac(6′)-Ib

100

606/606

M21682

 

P3_2456..3061

P2_82742..83347

 

aadA1

100

789/789

JQ480156

 

P3_3131..3919

  

99.75

792/798

JQ414041

 

P3_44412..45203

  

aadA2

100

792/792

JQ364967

P1_261911..262702

P1_271654..272445

 

P1_53050..53841

100

780/780

X68227

  

C_2297697..2298476

 

aph(3′)-VIa

98.46

780/780

X07753

P1_4558..5337

P1_4558..5337

  

armA

100

774/774

AY220558

P1_267391..268164

P1_277134..277907

  

rmtC

100

 

AB194779

  

P3_120100..120945

 

strA

99.88

804/804

AF321551

 

P3_29207..30010

  

100

  

P2_53242..54045

 

strB

99.88

837/837

M96392

 

P3_30010..30846

  

100

  

P2_52406..53242

 

aac(6′)Ib-cr

100

600/600

Fluoroquinolone and aminoglycoside resistance

DQ303918

C_612688..613287

C_1122863..1123462

  
 

P1_136163..136762

P2_38111..38710

 

Beta-lactams

blaOXA-1

100

831/831

Beta-lactam resistance

J02967

C_613418..614248

C_1121902..1122732

  
 

P1_136893..137723

P2_38841..39671

 

blaOXA-9

100

840/840

JF703130

 

P3_3964..4803

  

blaOXA-232

100

798/798

JX423831

P2_3878..4675

P4_3878..4675

  

blaNDM-1

100

813/813

FN396876

P1_7770..8582

P1_7770..8582

P3_122191..123003

 

blaNDM-5

100

813/813

JN104597

   

P2_10716..11528

blaCTX-M-15

100

876/876

DQ302097

  

C_5407907..5408782

 
 

P3_68389..69264

P2_47128..48003

P1_47694..48569

blaTEM-1A

100

861/861

HM749966

 

P3_5503..6363

  

blaTEM-1B

100

861/861

JF910132

  

P2_50825..51685

 

595/861

   

P1_49351..49945

blaSHV-11

100

861/861

GQ407109

 

P3_57446..58306

C_2612965..2613825

 

99.88

  

P2_36311..37171

 

blaSHV-28

100

861/861

HM751101

   

C_1087615..1088475

99.88

C_1078475..1079335

C_656815..657675

  

blaCMY-6

100

1146/1146

AJ011293

  

P3_72203..73348

 

Fluoroquinolones

aac(6′)Ib-cr

100

600/600

Fluoroquinolone and aminoglycoside resistance

DQ303918

C_612688..613287

C_1122863..1123462

  
 

P1_136163..136762

P2_38111..38710

 

99.42

519/519

EF636461

 

P3_2543..3061

P2_82742..83260

 

99.61

  

P3_115219..115737

 

QnrB1

99.85

682/681

Quinolone resistance

EF682133

P1_130519..131200

P1_130247..130928

  

QnrB58

98.68

681/681

JX259319

  

P2_26062..26742

 

oqxA

100

1176/1176

EU370913

  

C_4169699..4170874

 

99.23

C_4847144..4848319

C_4793024..4794199

 

C_4849531..4850706

oqxB

98.83

3153/3153

EU370913

C_4843968..4847120

C_4789848..4793000

C_4170898..4174050

 

98.79

   

C_4846355..4849507

Fosfomycin

fosA

97.38

420/420

Fosfomycin resistance

NZ_AFBO01000747

C_2957629..2958048

C_2903507..2903926

 

C_2946180..2946599

97.14

  

C_667959..668378

 

MLS—macrolide, lincosamide and streptogramin B

ere(A)

95.11

1227/1227

Macrolide resistance

AF099140

 

P3_45289..46515

  

mph(A)

100

906/906

D16251

  

P1_16503..17408

 

mph(E)

99.89

885/885

EU294228

P1_271994..272878

P1_281737..282621

  

msr(E)

100

1476/1476

Macrolide, Lincosamide and Streptogramin B resistance

EU294228

P1_270463..271938

P1_280206..281681

  

Phenicol

catB3

100

442/633

Phenicol resistance

AJ009818

 

P1_137861..138302

P2_39809..40250

 

C_614386..614827

C_1121323..1121764

  

cmlA1

99.13

 

AB212941

 

P3_42931..44190

  

Rifampicin

ARR-2

100

453/453

Rifampicin resistance

HQ141279

 

P3_46791..47243

  

ARR-3

  

CP002151

  

C_2298894..2299820

 

Sulphonamides

sul1

100

927/927

Sulphonamide resistance

CP002151

P1_263120..264046

P1_272863..273789

P3_116160..117086

 

sul1

100

837/837

JN581942

 

P3_41559..42395

  

sul2

100

816/816

GQ421466

 

P3_28331..29146

  

Tetracyclines

tet(A)

100

1200/1200

Tetracycline resistance

AJ517790

  

P1_19168..20367

 

Trimethoprim

dfrA1

100

474/474

Trimethoprim resistance

X00926

C_3627607..3628080

C_3573485..3573958

  

dfrA12

100

498/498

AB571791

P1_261006..261503

P1_270749..271246

 

P1_52145..52642

dfrA14

99.59

483/483

DQ388123

 

P1_144525..145007

P2_8272..8754

 

KP617: C, CP012753.1; P1, CP012754.1; P2, CP012755.1

PittNDM01: C, CP006798.1; P1, CP006799.1; P2, CP006800.1; P3, CP006801.1; P4, CP006802.1

ATCC BAA-2146: C, CP006659.2; P1 (PCuAs), CP006663.1; P2 (PHg), CP006662.2; P3, CP006660.1; P4, CP006661.1

NUHL24385: C, CP014004.1; P1, CP014005.1; P2, CP014006.1

a C chromosome, P plasmid

The structure of plasmid 1 (273,628 bp in size) of the KP617 strain was similar to that of plasmid 1 (283,371 bp in size) of PittNDM01. A region of about 40 kb in size within plasmid 1 of the KP617 strain, which included the NDM-1-encoding gene, was composed of various resistance genes such as aadA2, armA, aac(3″)-VI, dfrA12, msrE, mphE, sul1 and qnrB1, and identical (coverage: 100 %, homology: 100 %) to a 40-kb sequence of plasmid 1 of PittNDM01 (Fig. 4b). Adjacent to the NDM-1-encoding gene, a region of about 70 kb in size was inverted in plasmid 1 of KP617 relative to plasmid 1 of PittNDM01. In addition, the OXA-1-encoding gene was identified in PittNDM01 but not in KP617. Transposases were found in a part of the NDM-1-encoding gene cluster (about 10 kb) in plasmid 1 of KP617. Gram-negative bacteria are known to possess a diverse range of transposases; moreover, the sequence of the NDM-1-encoding gene cluster includes a transposon [35, 36]. The partial, or complete, transfer of NDM-1-harboring plasmids between K. pneumoniae and E. coli, via conjugation, has been shown to result in the emergence of strains resistant to several antimicrobial agents [11, 32, 36, 37].

Following the initial identification of NDM-1 in a K. pneumoniae isolate from a patient who had travelled to India in 2008, most NDM-1-producing K. pneumoniae isolates have been recovered from patients associated with India; however, in some cases, these strains have been isolated from patients with no history of travelling abroad, or any association with India [38]. These observations suggest that the transfer of the NDM-1- and OXA-232-harboring plasmids between Gram-negative bacteria has resulted in the spread of carbapenem resistance and emergence of strong carbapenem-resistant strains outside the Indian subcontinent.

Virulence factors

Klebsiella pneumoniae, a significant pathogen of human hosts, causes urinary tract infections, pneumonia, septicemia, and soft tissue infections [1]. The clinical features of K. pneumoniae infections depend on the virulence factors expressed by the infecting strain [39]. Therefore, we investigated the virulence factors of the present strain and compared these with those of KP617 and the reference strains. A BLAST search was performed against VFDB to identify 117 virulence factors harbored by the KP617 strain (Table 3). All 117 virulence genes of KP617 were also harbored by the reference strains; KP617 did not possess any unique virulence factors. The PittNDM01 strain was also found to possess no unique virulence factors; however, NUHL24835 and ATCC BAA-2146 possessed 3 and 7 unique virulence factors, respectively. The 117 virulence genes of KP617 were classified into 31 the following categories: Iron uptake (30 genes), Immune evasion (12 genes), Endotoxin (11 genes), Adherence (10 genes), Fimbrial adherence determinants (8 genes), Toxin (7 genes), Antiphagocytosis (6 genes), Regulation (5 genes), Acid resistance (3 genes), Anaerobic respiration (2 genes), Cell surface components (2 genes) and Secretion system (2 genes). Among the 117 virulence genes identified, 8 genes were lipopolysaccharide [40]-related genes and 4 genes were capsular polysaccharide [41]-related.
Table 3

Virulence genes of KP617 and the reference strains

Strains

Category

Subcategory

Name

KP617, PittNDM01, NUHL24385 and ATCC BAA-2146

Acid resistance

Urease

ureA, ureB, ureF, ureG, ureH

Adherence

Cell wall associated fibronectin binding protein

ebh

Adherence

CFA/I fimbriae

ibeB

Adherence

Flagella

fleN, fleR, fleS

Adherence

Hsp60

htpB

Adherence

Intercellular adhesin

icaA, icaR

Adherence

Listeria adhesion protein

lap

Adherence

OapA

oapA

Adherence

Omp89

omp89

Adherence

P fimbriae

papX

Adherence

PEB1/CBF1

pebA

Adherence

Phosphoethanolamine modification

lptA

Adherence

Type I fimbriae

fimB, fimE, fimG

Adherence

Type IV pili

comE/pilQ

Adherence

Type IV pili biosynthesis

pilM, pilW

Adherence

Type IV pili twitching motility related proteins

chpD, chpE

Adhesin

Laminin-binding protein

lmb

Adhesin

Streptococcal lipoprotein rotamase A

slrA

Adhesin

Streptococcal plasmin receptor/GAPDH

plr/gapA

Adhesin

Type IV pili

pilD, pilN, pilR, pilR, pilS, pilT

Amino acid and purine metabolism

Glutamine synthesis

glnA1

Amino acid and purine metabolism

Leucine synthesis

leuD

Amino acid and purine metabolism

Lysine synthesis

lysA

Amino acid and purine metabolism

Proline synthesis

proC

Amino acid and purine metabolism

Purine synthesis

purC

Amino acid and purine metabolism

Tryptophan synthesis

trpD

Anaerobic respiration

Nitrate reductase

narG, narH, narI, narJ

Anaerobic respiration

Nitrate/nitrite transporter

narK2

Anti-apoptosis factor

NuoG

nuoG

Antimicrobial activity

Phenazines biosynthesis

phzE1, phzF1, phzG1phzS

Antiphagocytosis

Alginate regulation

algQ, algR, algU, algW, algZ, mucB, mucC, mucD, mucP

Antiphagocytosis

Capsular polysaccharide

cpsB, wbfT, wbfV/wcvB, wbjD/wecB, wza, wzc

Antiphagocytosis

Capsule

cpsF

Antiphagocytosis

Capsule I

gmhA, wcbN, wcbP, wcbR, wcbT, wzt2

Cell surface components

GPL locus

fadE5, fmt, rmlB

Cell surface components

MymA operon

adhD, fadD13, sadH, tgs4

Cell surface components

PDIM (phthiocerol dimycocerosate) and PGL (phenolic glycolipid) biosynthesis and transport

ddrA, mas, ppsC, ppsE

Cell surface components

Potassium/proton antiporter

kefB

Cell surface components

Proximal cyclopropane synthase of alpha mycolates

pcaA

Cell surface components

Trehalose-recycling ABC transporter

lpqY, sugA, sugB, sugC

Chemotaxis and motility

Flagella

flrA, flrB

Efflux pump

FarAB

farA, farB

Efflux pump

MtrCDE

mtrC, mtrD

Endotoxin

LOS

gmhA/lpcA, kdtA, kpsF, lgtF, licA, lpxH, msbA, opsX/rfaC, orfM, rfaD, rfaE, rfaF, wecA, yhbX

Endotoxin

LPS

bplA, bplC, bplF, wbmE, wbmI

Endotoxin

LPS-modifying enzyme

pagP

Exoenzyme

Cysteine protease

sspB

Exoenzyme

Streptococcal enolase

eno

Fimbrial adherence determinants

Agf/Csg

csgD

Fimbrial adherence determinants

Fim

fimA, fimC, fimD, fimF, fimH, fimI

Fimbrial adherence determinants

Lpf

lpfB, lpfC

Fimbrial adherence determinants

Stg

stgA

Fimbrial adherence determinants

Sth

sthA, sthB, sthC, sthD, sthE

Fimbrial adherence determinants

Sti

stiB

Glycosylation system

N-linked protein glycosylation

pglJ

Host immune evasion

Exopolysaccharide

galE, galU, manA, mrsA/glmM, pgi

Host immune evasion

LPS glucosylation

gtrB

Host immune evasion

Polyglutamic acid capsule

capD

Immune evasion

LPS

acpXL, htrB, kdsA, lpxA, lpxB, lpxC, lpxD, lpxK, pgm, wbkC

Intracellular survival

LigA

ligA

Intracellular survival

Lipoate protein ligase A1

lplA1

Intracellular survival

Mip

mip

Intracellular survival

Oligopeptide-binding protein

oppA

Intracellular survival

Post-translocation chaperone

prsA2

Intracellular survival

Sugar-uptake system

hpt

Invasion

Ail

ail

Invasion

Cell wall hydrolase

iap/cwhA

Iron acquisition

Cytochrome c muturation (ccm) locus

ccmA, ccmB, ccmC, ccmE, ccmF

Iron acquisition

Ferrous iron transport

feoA, feoB

Iron acquisition

Iron acquisition/assimilation locus

iraB

Iron and heme acquisition

Haemophilus iron transport locus

hitA, hitB, hitC

Iron and heme acquisition

Heme biosynthesis

hemA, hemB, hemC, hemD, hemE, hemG, hemH, hemL, hemM, hemN, hemX, hemY

Iron uptake

ABC transporter

fagD

Iron uptake

ABC-type heme transporter

hmuT, hmuU, hmuV

Iron uptake

Achromobactin biosynthesis and transport

acsB, cbrB, cbrD

Iron uptake

Aerobactin transport

iutA

Iron uptake

ciu iron uptake and siderophore biosynthesis system

ciuD

Iron uptake

Enterobactin receptors

irgA

Iron uptake

Enterobactin synthesis

entE, entF

Iron uptake

Enterobactin transport

fepA, fepB, fepC, fepD, fepG

Iron uptake

Heme transport

shuV

Iron uptake

Hemin uptake

chuS, chuT, chuY

Iron uptake

Iron-regulated element

ireA

Iron uptake

Iron/managanease transport

sitA, sitB, sitC, sitD

Iron uptake

Periplasmic binding protein-dependent ABC transport systems

viuC

Iron uptake

Pyochelin

pchA, pchB, pchR

Iron uptake

Pyoverdine

pvdE, pvdH, pvdJ, pvdM, pvdN, pvdO

Iron uptake

Salmochelin synthesis and transport

iroE, iroN

Iron uptake

Vibriobactin biosynthesis

vibB

Iron uptake

Vibriobactin utilization

viuB

Iron uptake

Yersiniabactin siderophore

ybtA, ybtP

Iron uptake systems

Ton system

exbB, exbD

Lipid and fatty acid metabolism

FAS-II

kasB

Lipid and fatty acid metabolism

Isocitrate lyase

icl

Lipid and fatty acid metabolism

Pantothenate synthesis

panC, panD

Lipid and fatty acid metabolism

Phospholipases C

plcD

Macrophage inducible genes

Mig-5

mig-5

Magnesium uptake

Mg2+ transport

mgtB

Mammalian cell entry (mce) operons

Mce3

mce3B

Metal exporters

Copper exporter

ctpV

Metal uptake

ABC transporter

irtB

Metal uptake

Exochelin (smegmatis)

fxbA

Metal uptake

Heme uptake

mmpL11

Metal uptake

Magnesium transport

mgtC

Metal uptake

Mycobactin

fadE14, mbtH, mbtI

Motility and export apparatus

Flagella

flhF, flhG, fliY

Nonfimbrial adherence determinants

SinH

sinH

Other adhesion-related proteins

EF-Tu

tuf

Other adhesion-related proteins

PDH-B

pdhB

Others

MsbB2

msbB2

Others

Nuclease

nuc

Others

VirK

virK

Phagosome arresting

Nucleoside diphosphate kinase

ndk

Protease

Trigger factor

tig/ropA

Proteases

Proteasome-associated proteins

mpa

Quorum sensing

Autoinducer-2

luxS

Quorum sensing systems

Acylhomoserine lactone synthase

hdtS

Quorum sensing systems

N-(butanoyl)-l-homoserine lactone QS system

rhlR

Regulation

Alternative sigma factor RpoS

rpoS

Regulation

AtxA

atxA

Regulation

BvrRS

bvrR

Regulation

Carbon storage regulator A

csrA

Regulation

DevR/S

devR/dosR

Regulation

GacS/GacA two-component system

gacA, gacS

Regulation

LetA/LetS two component

letA

Regulation

LisR/LisK

lisK

Regulation

MprA/B

mprA, mprB

Regulation

PhoP/R

phoR

Regulation

RegX3

regX3

Regulation

RelA

relA

Regulation

SenX3

senX3

Regulation

Sigma A

sigA/rpoV

Regulation

Two-component system

bvgA, bvgS

Secreted proteins

Antigen 85 complex

fbpB, fbpC

Secretion system

Accessory secretion factor

secA2

Secretion system

Bsa T3SS

bprC

Secretion system

Flagella (cluster I)

fliZ

Secretion system

Mxi-Spa TTSS effectors controlled by MxiE

ipaH, ipaH2.5

Secretion system

P. aeruginosa TTSS

exsA

Secretion system

P. syringae TTSS

hrcN

Secretion system

P. syringae TTSS effectors

hopAJ2, hopAN1, hopI1

Secretion system

TTSS secreted proteins

bopD

Secretion system

Type III secretion system

bscS

Secretion system

Type VII secretion system

essC

Secretion system

VirB/VirD4 type IV secretion system & translocated effector Beps

bepA

Serum resistance

BrkAB system

brkB

Stress adaptation

AhpC

ahpC

Stress adaptation

Catalase-peroxidase

katG

Stress adaptation

Pore-forming protein

ompA

Stress protein

Catalase

katA

Stress protein

Manganese transport system

mntA, mntB, mntC

Stress protein

Recombinational repair protein

recN

Stress protein

SodCI

sodCI

Surface protein anchoring

Lipoprotein diacylglyceryl transferase

lgt

Surface protein anchoring

Lipoprotein-specific signal peptidase II

lspA

Toxin

Beta-hemolysin/cytolysin

cylG

Toxin

Enterotoxin

entA, entB, entC, entD

Toxin

Hydrogen cyanide production

hcnC

Toxin

Phytotoxin phaseolotoxin

argD, argK, cysC1

Toxin

Streptolysin S

sagA

Toxins

Alpha-hemolysin

hlyA

Toxins

Enterotoxin SenB/TieB

senB

Two-component system

PhoPQ

phoP, phoQ

Type I secretion system

ABC transporter for dispersin

aatC

KP617, PittNDM01 and NUHL24385

Antiphagocytosis

Capsular polysaccharide

cpsA

Cell surface components

GPL locus

pks

Cell surface components

Mycolic acid trans-cyclopropane synthetase

cmaA2

Endotoxin

LOS

lgtA

Iron uptake

Pyoverdine receptors

fpvA

Iron uptake

Vibriobactin biosynthesis

vibA

Iron uptake

Yersiniabactin siderophore

irp1, irp2, ybtE, ybtQ, ybtS, ybtT, ybtU, ybtX

Secretion system

EPS type II secretion system

epsG

Secretion system

Trw type IV secretion system

trwE

Secretion system

VirB/VirD4 type IV secretion system & translocated effector Beps

virB11, virB4, virB9

Toxin

RTX toxin

rtxB, rtxD

KP617 and PittNDM01

Adhesin

Streptococcal collagen-like proteins

sclB

Chemotaxis and motility

Flagella

flrC

Iron uptake

Yersiniabactin siderophore

fyuA

KP617 and PittNDM01 were found to possess two virulence factors that were not present in the other two strains: invasion (encoded by ail, attachment invasion locus protein) [42] and Iron uptake (encoded by fyuA, Yersiniabactin siderophore) [43].

Phage-associated regions

Prophages contribute to the genetic and phenotypic plasticity of their bacterial hosts [44] and act as vehicles for the transfer of antimicrobial resistance genes [45] or virulence factors [46]. Six phage-associated regions (KC1–KC5) of the KP617 chromosome and one phage-associated region (KP1) in plasmid 1 of the KP617 strain were identified using the PHAST algorithm (Table 4). With regard to the reference strains, six phage-associated regions were identified in the PittNDM01 strain, six in NUHL24835, and 12 in ATCC BAA-2146.
Table 4

Phage-associated regions of KP617 and the reference strains

Strain

Chromosome/plasmid

Region

Region_length (Kb)

Completeness

Score

#CDS

Region_position

Possible phage

GC_percentage (%)

ATCC BAA-2146

Chromosome

AC1

23.3

Questionable

75

14

596765–620097

Entero_P4

43.01

Chromosome

AC2

52

Intact

100

70

1293924–1345940

Cronob_ENT47670

53.06

Chromosome

AC3

37.5

Intact

150

48

1785522–1823022

Entero_Fels_2 

51.11

Chromosome

AC4

25.7

Incomplete

50

31

2283748–2309524

Entero_mEpX1

52.98

Chromosome

AC5

45.6

Intact

110

62

2342458–2388075

Salmon_SEN34

51.79

Chromosome

AC6

7

Incomplete

30

7

3543581–3550658

Shigel_SfIV

48.73

Chromosome

AC7

45.1

Intact

106

57

3969834–4015015

Salmon_SPN1S

54.61

Chromosome

AC8

24.7

Intact

150

31

4128565–4153295

Salmon_RE_2010

56.56

Chromosome

AC9

25.7

Questionable

90

26

4910621–4936374

Salmon_ST64B

52.32

Plasmid1

AP1-1

16

Questionable

70

13

5385–21439

Staphy_SPbeta_like

57.65

Plasmid2

AP2-1

46

Intact

130

38

3924–49935

Stx2_converting_1717

51.29

Plasmid2

AP2-2

18.1

Questionable

70

23

37308–55427

Staphy_SPbeta_like

50.68

Plasmid2

AP2-3

18.7

Incomplete

30

21

66337–85097

Entero_P1

51.85

KP617

Chromosome

KC1

59.4

Intact

140

78

187337–246765

Salmon_E1

53.99

Chromosome

KC2

52.2

Intact

150

51

1148902–1201105

Entero_HK140

54.02

Chromosome

KC3

37.3

Intact

150

39

1524848–1562220

Salmon_SEN4

50.97

Chromosome

KC4

43.1

Questionable

90

52

4912300–4955407

Escher_HK639

52.40

Chromosome

KC5

20

Incomplete

30

17

5015118–5035178

Entero_phiP27

51.93

Plasmid1

KP1-1

20.7

Incomplete

50

25

123005–143753

Escher_Av_05.

0.4718

NUHL24835

Chromosome

NC1

41.6

Intact

140

47

132925–174606

Entero_HK140

50.75

Chromosome

NC2

12.8

Incomplete

30

14

1481474–1494341

Thermu_phiYS40

58.36

Chromosome

NC3

34.7

Intact

150

32

1524859–1559640

Entero_c_1

52.15

Chromosome

NC4

41.9

Intact

150

52

4283813–4325722

Entero_Fels_2

53.26

Chromosome

NC5

38.7

Intact

150

45

5082826–5121566

Entero_mEp235

50.24

Plasmid1

NP1-1

21.4

Incomplete

30

6

65638–87083

Entero_P1

49.29

PittNDM01

Chromosome

PC1

50.8

Intact

130

63

209103–259953

Vibrio_pYD38_A

53.35

Chromosome

PC2

49.9

Intact

120

65

4847596–4897574

Salmon_SPN3UB

51.59

Chromosome

PC3

20

Incomplete

30

19

4961006–4981067

Entero_P4

51.92

Plasmid1

PP1-1

30.8

Questionable

70

22

124082–154939

Vibrio_pYD38_A

48.18

Plasmid2

PP2-1

34.3

Questionable

70

27

556–34952

Entero_P1

52.30

Plasmid3

PP3-1

50.3

Intact

150

56

8885–59236

Entero_P1

53.90

Three of the six phages, KC1, KC2 and KC3, in the KP617 strain were intact, whereas the remaining prophages were incomplete (KC5 and KP1) or questionable (KC4) and had a low PHAST score of below 90. Based on the sequence similarity of their genomes, KP617 and PittNDM01 were found to have high similarity to each other (Figs. 2, 3a, b). Concordantly, the profile of prophage DNA in their genomes, as determined via a BLAST search, was similar, and the two strains shared four of the six prophages, whereas two phage regions, KC2 (Entero_HK140) and KC3 (Salmon_SEN4), were specific to the KP617 genome. Furthermore, it was found that one phage-associated region of KP617, namely KC2 (Entero_HK140), exhibited a high similarity to the phage-associated region of the NUHL24835 strain, NC1, with 60 % query coverage and 99 % identity. It should be noted that the strains compared in the present study, i.e. KP617 and the reference strain, ATCC BAA-2146, had no prophages in common.

Investigation of the antimicrobial resistance genes harbored by the strains, which was performed using ResFinder, and comparison with the prophage-associated region, as predicted using PHAST, did not reveal the presence of a prophage-delivered beta-lactamase-encoding gene in the KP617 genome, indicating that prophages did not act as a vehicle for the transfer of antimicrobial resistance genes in this strain. This finding is consistent with previous observations that beta-lactamase-encoding genes are borne by transposons [35, 36]. Bacteriophages are applicable to phage therapy. In particular, bacteriophages have been used as a potential therapeutic agent to treat patients infected with multidrug resistant bacteria [47] and have been used for serological typing for diagnostic and epidemiological typing in K. pneumoniae [48]. However, because we did not characterize the phages in KP617, we are not sure whether or not they are active.

Future directions

Klebsiella pneumoniae subsp. pneumoniae KP617, which is strongly pathogenic, is known to cause severe nosocomial infections. This strain, as well as the PittNDM01 and NUHL24835 strains in the WGLW2 group, belongs to the sequence type ST14. In this study, we investigated specific antimicrobial resistance genes, virulence factors, and prophages related to pathogenicity and drug resistance in K. pneumoniae subsp. pneumoniae KP617 via a comparative analysis of the genome of this strain and those of PittNDM01, NUHL24835, and ATCC BAA-2146. Significant homology was observed in terms of the genomic structure, gene content, antimicrobial resistance genes and virulence factors between KP617 and the reference strains; phylogenetic analysis indicated that KP617 is next to PittNDM01, despite the presence of large inversions. Moreover, KP617 shares 98.3 % of its genes with PittNDM01. Despite the similarity in genome sequences and content, there were differences in phage-related genes, plasmids, and plasmid-harbored antimicrobial resistance genes. PittNDM01 harbors two more plasmids and 21 more antimicrobial resistance genes than KP617. In order to elucidate the precise role of these factors in the pathogenicity of KP617, further studies are required.

Availability of supporting data

Nucleotide sequence accession numbers The complete genome sequence of K. pneumoniae KP617 has been deposited in DDBJ/EMBL/GenBank under the accession numbers CP012753, CP012754, and CP012755 [49].

Notes

Abbreviations

BSR: 

BLAST score ratio

CDS: 

coding DNA sequences

HGT: 

horizontal gene transfer

MLST: 

multi-locus sequence typing

NDM-1: 

New Delhi metallo-β-lactamase 1

RAST: 

Rapid Annotation using Subsystem Technology

ST: 

sequence type

str: 

strain

substr: 

substrain

Declarations

Authors’ contributions

DWK and WK designed and led the project and contributed to the interpretation of the results. DWK drafted the manuscript. YHJ and TK interpreted the results. YHJ, SHL, MRY, and TK performed the gene annotation and bioinformatics analysis. TK and YHJ wrote the manuscript. All authors read and approved the final manuscript before submission.

Acknowledgements

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by a grant from the Marine Biotechnology Program (Genome Analysis of Marine Organisms and Development of Functional Applications) funded by the Ministry of Oceans and Fisheries.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
School of Biological Sciences, Seoul National University
(2)
Division of Antimicrobial Resistance, Korea National Institute of Health
(3)
Division of Biosafety Evaluation and Control, Korea National Institute of Health

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