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

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.

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 [14-16]. Recently, K. pneumoniae isolates coproducing two carbapenemases, bla and bla, 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

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 and bla in the chromosome, bla in plasmid 1, and bla 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].