Comparative genomic analysis of Klebsiella pneumoniae subsp. pneumoniae KP617 and PittNDM01, NUHL24835, and ATCC BAA-2146 reveals unique evolutionary history of this strain
verfasst von:
Taesoo Kwon, Young-Hee Jung, Sanghyun Lee, Mi-ran Yun, Won Kim, Dae-Won Kim
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: blaOXA-1 and blaSHV-28 in the chromosome, blaNDM-1 in plasmid 1, and blaOXA-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.
The online version of this article (doi:10.1186/s13099-016-0117-1) contains supplementary material, which is available to authorized users.
Taesoo Kwon and Young-Hee Jung contributed equally to this work
Won Kim and Dae-Won Kim contributed equally to the work
Abkürzungen
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
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].
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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, blaNDM-1 and blaOXA-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.
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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
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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.
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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
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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 blaOXA-1 and blaSHV-28 in the chromosome, blaNDM-1 in plasmid 1, and blaOXA-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
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.
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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
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.
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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].
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.
Comparative genomic analysis of Klebsiella pneumoniae subsp. pneumoniae KP617 and PittNDM01, NUHL24835, and ATCC BAA-2146 reveals unique evolutionary history of this strain
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