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Complete Nucleotide Sequence of pKOI-34, an IncL/M Plasmid Carrying blaIMP-34 in Klebsiella oxytoca Isolated in Japan Norimitsu Shimada,a,b,c Shizuo Kayama,a,b Norifumi Shigemoto,a,d Junzo Hisatsune,a,b Ryuichi Kuwahara,a,b,e Hisaaki Nishio,f Katsutoshi Yamasaki,g Yasunao Wada,h Taijiro Sueda,c Hiroki Ohge,a,d Motoyuki Sugaia,b Project Research Center for Nosocomial Infectious Diseases, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima City, Hiroshima, Japana; Department of Bacteriology, Hiroshima University Graduate School of Biomedical & Health Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima City, Hiroshima, Japanb; Department of Surgery I, Hiroshima University Graduate School of Biomedical & Health Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima City, Hiroshima, Japanc; Department of Infectious Diseases, Hiroshima University Hospital, 1-2-3 Kasumi, Minami-ku, Hiroshima City, Hiroshima, Japand; Clinical Laboratory, Hiroshima General Hospital of West Japan Railway Company, Futaba-no-sato 3-1-36, Higashi-ku, Hiroshima City, Hiroshima, Japane; Clinical Laboratory, Shiga Medical Center for Adults, 5-4-30, Moriyama, Moriyama City, Shiga, Japanf; Department of Clinical Laboratory, Wakayama Rosai Hospital, 93-1 Kinomoto, Wakayama City, Wakayama, Japang; Clinical Laboratory, Hyogo Medical University Hospital, 1-1 Mukogawa-cho, Nishinomiya City, Hyogo, Japanh
We determined the complete nucleotide sequence of a self-transmissible IncL/M plasmid, pKOI-34, from a Klebsiella oxytoca isolate. pKOI-34 possessed the core structure of an IncL/M plasmid found in Erwinia amylovora, pEL60, with two mobile elements inserted, a transposon carrying the arsenic resistance operon and a Tn21-like core module (tnp and mer modules) piggybacking blaIMP-34 as a class 1 integron, In808, where blaIMP-34 confers a resistance to carbapenems in K. oxytoca and Klebsiella pneumoniae.
C
arbapenem-resistant Enterobacteriaceae (CRE) infections are increasing, which is one of the most important issues in health care facilities around the world (1, 2). In Japan, IMP-6, a variant of IMP-1 with one amino acid substitution (Ser196Gly), has been detected sporadically in clinical isolates of the family Enterobacteriaceae (3). IMP-6 shows a paradoxical effect on carbapenem resistance, i.e., imipenem susceptible but meropenem resistant (4), where a plasmid pKPI-6 harboring blaIMP-6 and blaCTX-M-2 confers this stealth-type carbapenem-resistant phenotype that is not detectable when testing for imipenem resistance (5). (Preliminary data from this study were presented at the 62nd Annual Meeting of the Japanese Society of Chemotherapy, 2014.) During a screening for MBL-producing Enterobacteriaceae showing the paradoxical resistance to carbapenems, we found IMP-34 in five MBL-positive isolates of Klebsiella oxytoca (MS5279,
MS5280) and Klebsiella pneumoniae (MS5284, MS5285, MS5286) in the Kinki region, which is the geographic center of Japan. The
Received 27 October 2015 Returned for modification 17 November 2015 Accepted 16 February 2016 Accepted manuscript posted online 22 February 2016 Citation Shimada N, Kayama S, Shigemoto N, Hisatsune J, Kuwahara R, Nishio H, Yamasaki K, Wada Y, Sueda T, Ohge H, Sugai M. 2016. Complete nucleotide sequence of pKOI-34, an IncL/M plasmid carrying blaIMP-34 in Klebsiella oxytoca isolated in Japan. Antimicrob Agents Chemother 60:3156 –3162. doi:10.1128/AAC.02507-15. Address correspondence to Motoyuki Sugai, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.02507-15. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
FIG 1 Structural features of IncL/M plasmid pKOI-34 (87,343 bp) in comparison with pEL60, which is considered the ancestral strain of the IncL/M plasmid. Two mobile gene elements of 8.8 and 19.5 kb, respectively, are inserted into the IncL/M backbone. Arrows show annotated coding sequences. The 104 identified ORFs are color coded on the basis of function as follows: blue, replication-related genes; green, antibiotic-resistant genes; red, transposases; yellow, conjugation-related genes. The lower schema shows the result of PCR scanning for plasmids carrying blaIMP-34 recovered from K. oxytoca and K. pneumoniae. The 22 sets of PCR primers were designed from the complete sequence of pKOI-34. All plasmids without pKOI-34 showed the same results: set primers 3 and 4 were not detected, and the area corresponding to the mobile gene element containing the arsenic resistance genes is missing. PCR using the forward primer of set 3 and the reverse primer of set 4 showed a DNA fragment of 600 bp (see Table S1 in the supplemental material).
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Complete Nucleotide Sequence of pKOI-34
TABLE 1 Features of pKOI-34 ORFs Position (bp)
Identity (%)
Overlap (no. of aa)
Accession no.
99
248/249
WP_020277896
99
673/677
YP_006958848
100
117/117
YP_002287003
100
120/120
YP_002287003
99
582/583
YP_002287002
Arsenic pump membrane protein Arsenate reductase
100
429/429
YP_002287001
100
141/141
YP_002287001
Arsenic transporter ATPase TnpR, site-specific recombinases Transposase, Tn3 family
100 100
124/124 190/190
WP_004118313 WP_012291338
100
1002/1002
YP_008003449
Protein disulfide isomerase
91
300/329
YP_002333315
Conjugal transfer protein
98
428/435
YP_002333316
Soluble lytic murein transglycosylase and related regulatory proteins Archaeal fructose-1,6-bisphosphatase Transfer inhibition protein
100
131/131
YP_006958778
100
116/116
WP_004187434
98
214/217
YP_006964687
Growth regulator, antitoxin involved in plasmid maintenance Growth inhibitor, toxin involved in plasmid maintenance SOS response transcriptional repressors Nucleotidyltransferase Transposase of IS4321R
100
85/85
YP_007878506
100
110/110
YP_008090835
99
143/144
YP_006958786
99 100
411/412 334/334
AHE47434 YP_005352261
100
990/990
YP_006501624
100
185/185
YP_008725240
100
337/337
YP_006961973
99
109/110
AHN60087
100
184/184
YP_002791400
99
245/246
ABF70513
99
109/110
ACN22644
100
115/115
YP_001965784
ORFs
Start
Stop
Strand
Gene
Length (no. of aaa)
Source
Description
1
1
750
⫹
repA
249
K. pneumoniae
2
2,120
4,153
⫺
trbC
677
3
4,510
4,863
⫹
arsR
117
4
4,911
5,273
⫹
arsD
120
5
5,291
7,041
⫹
arsA
583
6
7,091
8,380
⫹
arsB
429
7
8,393
8,818
⫹
arsC
141
8 9
8,849 9,262
9,223 9,834
⫺ ⫺
arsA tnpR
124 190
10
9,998
13,006
⫹
tnpA
1,002
K. pneumoniae (pOXA-48) K. pneumoniae (plasmid 12) K. pneumoniae (plasmid 12) K. pneumoniae (plasmid 12) K. pneumoniae (plasmid 12) K. pneumoniae (plasmid 12) Enterobacteriaceae Yersinia enterocolitica K. pneumoniae (pKP1433)
RepA, IncL/M type replicase protein Protein involved in plasmid transfer Arsenic-resistant operon repressor Arsenic-resistant operon trans-acting repressor Arsenic pump driving ATPase
11
13,003
13,992
⫺
trbB
329
12
14,003
15,310
⫺
trbA
435
13
15,310
15,705
⫺
trbN
131
14
15,810
16,160
⫺
15
16,273
16,926
⫹
16
17,019
17,276
17
17,278
18
K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae (pOXA-48)
116
K. pneumoniae
tir
217
⫹
pemI
85
E. cloacae (pNE1280) K. pneumoniae (pJEG011)
17,610
⫹
pemK
110
K. pneumoniae (pKPoxa-48N1)
17,703
18,137
⫹
mucA
144
19 20
18,152 19,989
19,390 20,993
⫹ ⫺
mucB tnpA
412 334
K. pneumoniae (pOXA-48) K. pneumoniae K. pneumoniae (pNDM-MAR)
21
21,072
24,044
⫺
tnpA
990
22
24,047
24,604
⫺
tnpR
185
23
24,910
25,923
⫺
intl1
337
24
26,174
26,506
⫹
qacF
110
25
26,595
27,149
⫹
aacA4
184
26
27,226
27,966
⫹
blaIMP-34 246
27
28,192
28,524
⫹
qacE2
110
28
28,735
29,082
⫹
qacE⌬1
115
K. oxytoca (pKOX_R1) K. pneumoniae (pIMP-PH114) K. oxytoca (pINCan01) Stenotrophomonas maltophilia E. cloacae (pEC-IMP) Pseudomonas aeruginosa Uncultured bacterium K. pneumoniae (pK29)
Transposase, Tn3 family protein Resolvase domain containing protein Integrase Ethidium bromide resistance protein Aminoglycoside N(6=)-acetyltransferase Metallo--lactamase Ethidium bromide resistance protein Ethidium bromide resistance protein
(Continued on following page)
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TABLE 1 (Continued) Position (bp) ORFs
Start
Stop
Strand
Gene
Length (no. of aaa)
29
29,076
29,915
⫹
sul1
279
30
30,043
30,543
⫹
orf5
166
31
30,512
31,504
⫺
tniB
330
32
31,507
33,186
⫺
tinA
559
33
33,261
33,968
⫺
urf2
235
34
33,965
34,201
⫺
merE
78
35
34,198
34,560
⫺
merD
120
36
34,578
36,272
⫺
merA
564
37
36,324
36,785
⫺
merC
153
38
36,782
37,057
⫺
merP
91
39
37,071
37,421
⫺
merT
116
40
37,493
37,927
⫹
merR
144
41
38,006
39,010
⫹
tnpA
334
42 43 44
39,844 40,309 41,015
40,164 40,947 41,224
⫺ ⫺ ⫺
106 212 69
45
41,227
41,445
⫺
72
46 47 48 49 50
41,490 42,208 44,068 44,402 44,982
42,191 43,539 44,424 44,980 45,389
⫺ ⫺ ⫹ ⫹ ⫹
233 443 118 192 135
51
45,542
46,087
⫺
181
52
46,225
46,683
⫹
152
53
46,680
46,928
⫹
82
54
46,921
47,508
⫹
195
55
47,505
47,990
⫹
161
56
47,987
48,235
⫹
82
57
48,254
48,994
⫹
resD
246
58
49,235
50,209
⫹
parA
324
59
50,212
50,655
⫹
parB
147
Source
Description
Identity (%)
Overlap (no. of aa)
Accession no.
K. pneumoniae (pK29) P. aeruginosa (Rms149)
Dihydropteroate synthase
100
279/279
YP_001965785
Acetyltransferase (GNAT) family protein
100
166/166
YP_245437
Salmonella enterica (pOU7519) K. pneumoniae (pR55) S. enterica (pOU7519) K. pneumoniae (pKpQIL) K. pneumoniae (pKpQIL) K. pneumoniae (pKpQIL) K. pneumoniae (pKPHS2) K. pneumoniae (pR55) K. pneumoniae (pKP048) E. coli
NTPb binding protein
100
330/330
YP_209341
Transposase and inactivated derivatives Tn21 protein of unknown function Urf2 Mercuric transport protein
100
559/559
YP_005352048
100
235/235
YP_001598123
100
78/78
YP_003560404
Transcriptional regulator
100
120/120
YP_003560405
Mercuric ion reductase
100
564/564
YP_003560406
Mercury transport protein
100
153/153
YP_005229641
Mercury transport protein
100
91/91
YP_005352054
Mercury transport protein
100
116/116
YP_003754062
Mercuric resistance operon regulatory protein
100
144/144
KKA59047
K. pneumoniae (pNDM-MAR) Enterobacteriaceae E. coli K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae (pJEG011)
Transposase of IS4321R
100
334/334
YP_005352261
Hypothetical protein Hypothetical protein Hypothetical protein
99 100 100
106/106 212/212 69/69
WP_021561451 WP_021561450 YP_002333329
Hypothetical protein
97
71/72
YP_002333330
Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein
92 93 92 93 98
219/233 414/443 113/118 184/192 134/135
WP_023302501 WP_023302502 WP_023302503 WP_023302504 YP_007878517
K. pneumoniae (pJEG011) Erwinia amylovora (pEL60) K. pneumoniae (pNDM-OM) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae (pKPoxa-48N2) K. pneumoniae (pKP048) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360)
Hypothetical protein
98
178/181
YP_007878518
Hypothetical protein
90
141/152
NP_943212
Hypothetical protein
98
82/82
YP_007195536
Hypothetical protein
98
191/195
YP_002333340
Hypothetical protein
99
160/161
YP_002333341
Hypothetical protein
93
81/82
YP_008110943
Resolvase
99
245/246
YP_006958800
StbA family protein
100
324/324
YP_002333344
Plasmid stability protein
100
147/147
YP_002333345
(Continued on following page)
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Complete Nucleotide Sequence of pKOI-34
TABLE 1 (Continued) Position (bp) ORFs
Start
Stop
Strand
Gene
Length (no. of aaa)
60
50,665
51,216
⫹
nuc
61
51,334
51,840
62
51,833
63
Identity (%)
Overlap (no. of aa)
Accession no.
Catalytic domain of EDTAresistant nuclease
100
183/183
YP_002333346
Hypothetical protein
100
168/168
YP_002333347
Hypothetical protein
100
159/159
YP_002333348
Hypothetical protein
99
136/136
YP_002333349
Hypothetical protein
100
87/87
YP_002333350
Hypothetical protein
100
120/120
YP_002333351
Hypothetical protein
100
149/149
NP_774994
Putative transcriptional repressor protein Hypothetical protein
100
88/88
YP_002333353
100
427/427
YP_002333354
Hypothetical protein
100
59/59
YP_006965388
Hypothetical protein
99
265/266
YP_002333355
Hypothetical protein
100
112/112
YP_006958814
Hemolysin expression modulating protein Hypothetical protein
100
76/76
YP_006958815
99
71/71
YP_008090866
Hypothetical protein
100
136/136
YP_002333358
Hypothetical protein
100
101/101
YP_006952492
Antirestriction protein
100
146/146
YP_007195464
Hypothetical protein
100
94/94
NP_775004
hypothetical protein
100
73/73
YP_006952495
Single-stranded DNA binding protein Hypothetical protein
100
144/144
YP_006958822
99
103/103
YP_006958823
Hypothetical protein
100
178/178
YP_006964602
MobC protein involved in plasmid mobilization MobB protein involved in plasmid mobilization MobA protein involved in plasmid mobilization Conjugative transfer protein
98
119/121
YP_002333286
99
105/105
YP_002333287
99
659/659
YP_008090802
99
165/166
YP_002333289
Conjugative transfer protein
99
259/259
YP_002333290
Tfp pilus assembly protein
100
387/387
WP_020805653
Source
Description
183
K. pneumoniae (pCTXM360)
⫹
168
52,312
⫹
159
52341
52,751
⫹
136
64
52,869
53,132
⫹
87
65
53,154
53,516
⫹
120
66
53,638
54,087
⫹
149
67
54,132
54,398
⫹
68
54,462
55,745
⫹
69
56,352
56,531
⫹
70
56,664
57,464
⫹
266
K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) C. freundii (pCTX-M3) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) E. cloacae (pEI1573) K. pneumoniae (pCTXM360)
71
57,655
57,993
⫹
112
72
58,091
58,321
⫹
73
58,653
58,868
⫹
71
74
58,940
59,350
⫹
136
75
59,412
59,717
⫹
101
76
59,918
60,358
⫹
77
60,402
60,686
⫹
94
78
60,841
61,062
⫹
73
79
61,134
61,568
⫹
80
61,624
61,935
⫹
103
81
62,068
62,604
⫹
178
82
63,106
63,471
⫺
mobC
121
83
63,746
64,063
⫹
mobB
105
84
64,050
66,029
⫹
mobA
659
85
66,043
66,543
⫹
traH
166
86
66,540
67,319
⫹
traI
259
87
67,330
68,493
⫹
traJ
387
korC
88 427
ccgA1
rmoA
klcA
ssb
59
76
146
144
K. pneumoniae (pOXA-48) K. pneumoniae (Kp11978) K. pneumoniae (pKPoxa-48N1) K. pneumoniae (pOXA-48) K. pneumoniae (pNDM-HK) K. pneumoniae (pNDM-OM) C. freundii (pCTX-M3) E. coli (pNDM-HK) K. pneumoniae (pOXA-48) K. pneumoniae (pOXA-48) Serratia marcescens (R830b) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae (pKPoxa-48N1) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae
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TABLE 1 (Continued) Position (bp) ORFs
Start
Stop
Strand
Gene
Length (no. of aaa)
88
68,483
68,743
⫹
traK
86
89 90
68,768 72,060
72,118 72,596
⫹ ⫹
pri traL
1,116 178
91
72,562
73,212
⫹
92
73,190
73,972
⫹
traM
260
93
73,981
75,132
⫹
traN
383
94
75,144
76,493
⫹
traO
449
95
76,505
77,209
⫹
traP
234
96
77,233
77,763
⫹
traQ
176
97
77,780
78,169
⫹
traR
129
98
78,215
78,709
⫹
99
78,706
81,756
⫹
traU
1,016
100
81,753
82,961
⫹
traW
402
101 102
82,958 83,508
83,608 85,781
⫹ ⫹
traX traY
216 757
103
85,784
86,437
⫹
excA
217
104
86,511
86,741
⫹
repC
76
216
164
Source
Description
Identity (%)
Overlap (no. of aa)
Accession no.
K. pneumoniae (pCTXM360) K. oxytoca K. pneumoniae (pKPoxa-48N1)
Conjugative transfer protein
100
86/86
YP_002333292
DNA primase Conjugative transfer protein
96 100
1077/1116 178/178
WP_031942492 YP_008090807
Hypothetical protein
97
213/216
YP_008090808
Conjugative transfer protein
98
257/260
YP_002333295
Conjugative transfer protein
98
380/383
YP_002333296
Conjugative transfer protein
100
449/449
YP_002333297
Conjugative transfer protein
98
232/234
YP_007195483
Conjugative transfer protein
100
176/176
YP_007195484
Conjugative transfer protein
100
129/129
YP_006964622
Hypothetical protein
100
164/164
YP_007195486
Conjugative transfer protein
99
1014/1016
YP_002333302
Conjugative transfer protein
100
402/402
YP_006964668
Conjugative transfer protein Conjugative transfer protein
100 100
216/216 757/757
WP_004187488 YP_006964669
Phosphoglycerol transferase
100
216/217
YP_002333306
Replication regulatory protein
99
76/76
YP_006964671
K. pneumoniae (pKPoxa-48N1) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae (pNDM-OM) K. pneumoniae (pNDM-OM) K. pneumoniae (pNDM-OM) K. pneumoniae (pNDM-OM) K. pneumoniae (pCTXM360) E. cloacae (pNE1280) Enterobacteriaceae E. cloacae (pNE1280) K. pneumoniae (pCTXM360) E. cloacae
a
aa, amino acids. b NTP, nucleoside triphosphate.
isolates showed intermediate resistance to imipenem but resistance to meropenem (6). IMP-34 has a single amino acid substitution, Glu87Gly, compared to IMP-1. Conjugal transfer experiments showed that blaIMP-34 is located on a self-transmissible plasmid designated pKOI-34. We sequenced the plasmid DNA of pKOI-34 purified from the E. coli BL21 transconjugant (BL21_pKOI-34) from K. oxytoca strain MS5279. The draft sequences of pKOI-34 were generated using Illumina MiSeq (Nextera paired-end library; 2,354,946 bp), assembled using CLC Genomics Workbench (CLC bio, Cambridge, MA), and sorted using OSLay (7). Gap closing was performed using direct sequencing of PCR products amplified with oligonucleotide primers designed to anneal each end to the neighboring contigs. pKOI-34 is an 87,343-bp plasmid with an average GC content of 53%, and 104 open reading frames (ORFs) (Fig. 1; Table 1). The BLASTP program showed that the amino acid sequence of RepA in pKOI-34 conforms to the IncL/M group. Replicon typing of plasmid preparations from the five strains positive for blaIMP-34 showed that they all belong to the IncL/M group. Sequence comparisons of pKOI-34 with the plasmids registered in the GenBank database show extensive similarity to IncL/M plasmids (see Fig. S1a in the supplemental material). The plasmid pEL60 (60,145 bp) from the plant pathogen Er-
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winia amylovora is considered the ancestral IncL/M plasmid. It does not possess any resistance genes and shares its entire sequence with other IncL/M plasmid backbones (8, 9) (see Fig. S1a in the supplemental material). The nucleotide sequence identity of pKOI-34 with pEL60 is 53% (46,561 bp), and the common backbone sequence possesses genes for replication (rep), a toxinantitoxin system (pemIK) that elevates mutation frequency conferring a UV-resistant phenotype (mucAB) (10), plasmid stability (parAB), endonuclease (nuc), primase (pri), and mobility/conjugal transfer genes (trbCBAN, tir, mobAB, traHIJK, and traLMNOPQUWXY) (Fig. 1). The pKOI-34 repA gene shows 93% nucleotide identity with pEL60 (53 nucleotide substitutions resulting in 13 amino acid variants) and 99% nucleotide identity with the majority of the other IncL/M plasmids (5 to 8 nucleotide substitutions, resulting in 1 or 2 amino acid substitutions). pKOI-34 possesses two mobile genetic elements. One is an 8.8-kb element consisting of tnpA, tnpR, and the arsenic resistance operon (10); and the other is a Tn21-like transposon, including two IS4321R (19.5 kb) (Fig. 2). The 8.8-kb element was inserted at the 5= end of trbC that was related to recombination, bacterial conjugation, and DNA transfer. This region showed 99% homology to the plasmid pZA1001,
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Complete Nucleotide Sequence of pKOI-34
FIG 2 The schema for the insertion region of pKOI-34 was based on the IncL/M backbone of pEL60. pKOI-34 possesses two mobile gene elements. One is an 8.8-kb region containing transposase (tnpA), resolvase (tnpR), and arsenic resistance genes. Another is aTn21-like transposon, consisting of tnpAR, an integron cassette structure of the class I integron (intl1, qacF, aacA4, blaIMP-34, qacE2, qacE⌬1, sul1, orf5), tniAB, urf2, and the mercury resistance operon. This transferable structure (Tn21-like transposon and IR4321R) was inserted into orf6, and the direct repeat sequence TTAAA (green) was generated. The Tn21-like transposon has an incomplete inverted repeat site, IRtnp and IRmer, on the left and right sides, respectively. The IRtnp of the Tn21-like transposon (orange) is truncated on the left side of IS4321R, and the IRmer of the Tn21-like transposon (pink) is truncated on the right side of IS4321R. Black, inverted repeat of IS4321R; green, direct repeat sequence of Tn21-like transposon; orange, IRtnp of the Tn21-like transposon; light blue, additional sequence; black, inverted repeat of IS4321R; pink, IRmer of Tn21-like transposon. The arsenic resistance operon has an incomplete inverted repeat (underlined).
R46, and pCC416 (accession nos. CP001723, AY046276, and AJ704863, respectively). The 19.5-kb element has a Tn21-like structure that divides orf6 into two truncated sequences (Fig. 2). The sites of insertion, defined by a 6-bp duplication of the target DNA TTAAAG, is AT rich, similar to Tn21 (11) (Fig. 2, green box). The core element of the Tn21-like structure is a composite of the Tn21 tnp and mer modules forming Tn21⌬. Both sides of the 38-bp Tn21⌬ terminal inverted repeat (TIR) are interrupted by the 1,327-bp IS4321R. IS4321 insertion creates 11-bp inverted repeats (IRl and IRr of IS4321) and additional flanking bases on both sides. IS4321R is a member of the IS1111 family that targets a specific position in the 38-bp complete TIR in the family of Tn21 transposons (12). A similar IS insertion into a 38-bp TIR is observed in many plasmids
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carrying TIR from the family of Tn21 transposons, Tn21/Tn501 (12). In Tn21, In2 is integrated between tnpR and urf2 in Tn21⌬, generating an imperfect inverted repeat of 25 bp and a 5-bp direct repeat TCCAT. However, in this Tn21-like structure, In808 carrying blaIMP-34 is present instead of In2 (Fig. 2). Besides blaIMP-34, In808 possesses other resistance genes, i.e., qacF, qacE2, and aacA4 (4). In808 is flanked by an imperfect inverted repeat of 25 bp (IRl, IRr). The TCCAT sequence was found on the right side but not on the left side, suggesting a later swapping event of an integron module between In2 and In808. The other four plasmids recovered from MS5280, MS5284, MS5285, and MS5286 carrying blaIMP-34 were examined using the PCR scanning method (13). Twenty-two primer sets covering the total DNA sequence with the PCR products of pKOI-34 were used
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(Fig. 1; see Table S1 in the supplemental material). PCR scanning data showed that primer sets 3 and 4 generated no amplicon, but the other 20 sets of the primer pairs yielded PCR products of the expected sizes in all four plasmids. The data suggest that all four plasmids were identical to pKOI-34 but lacked the mobile element carried by the transposon containing the arsenic resistance operon inserted between trbC and trbB. To examine the junction between the sequence corresponding to the plasmid backbone and the transposon, an ⬃600-bp amplicon was generated with PCR using the set 3 forward primer and set 4 reverse primer (see Table S1). The nucleotide sequence of the ⬃600-bp amplicon was identical to the trbC to trbB region of pEL60. This indicates that the four plasmids recovered from MS5280, MS5284, MS5285, and MS5286 are pKOI-34 variants lacking the transposon carrying the arsenic resistance operon. MS5279 and MS5280 were isolated in 2004, MS5285 and MS5286 in 2006, and MS5284 in 2007. Therefore, it may be reasonable to assume that the arsenic operon was lost in MS5280, MS5284, MS5285, and MS5286. In any case, this fact is not strong enough support that this element was lost in these strains. Multilocus sequence typing (MLST) was performed using the protocol published by Larsen et al. (14). MLST showed that K. pneumoniae MS5284, MS5285, and MS5286 belong to ST334, and K. oxytoca MS5279 and MS5280 belong to ST171 (gapA, 3; infB, 4; mdh, 15; pgi, 4; phoE, 18; rpoB, 3; tonB, 4). The number of members of the IncL/M multidrug resistance (MDR) plasmids harboring broad-spectrum -lactam resistance is increasing worldwide. pKOI-34 is the newest member of this group and the first IncL/M drug resistance plasmid found in Japan. Bonnin et al. (15) suggest that the evolution of the IncL/M MDR plasmids was through the acquisition of resistance genes and insertion sequences. There are two integration hot spots in the IncL/M plasmids; one is located between the rep locus and trbC, and the other is near pemIK (15) (see Fig. S1b in the supplemental material) (16, 17). In pKOI-34, two target sites for inserted sequences are different from those previously reported (see Fig. S1a,b in the supplemental material). The arsenic resistance operon was located between trbC and trbB, and the other Tn21-like transposon was located within the orf6 of pEL60. In conclusion, we report the complete sequence of pKOI-34, an IncL/M type conjugal plasmid carrying blaIMP-34. pKOI-34 possesses a pEL60 backbone with two inserted mobile elements, a Tn21-like architecture with a class 1 integron, In808 instead of In2, and a transposon carrying the arsenic resistance operon. We show here that the spread of the blaIMP-34 gene in K. oxytoca and K. pneumoniae is linked to the spread of pKOI-34 or its derivatives. Nucleotide sequence accession number. The complete sequence of pKOI-34 has been deposited in GenBank (accession number AB715422). ACKNOWLEDGMENTS We thank Jim Nelson for editorial assistance. N.S. received the 62nd presentation award in the category of basic research conferred by the Director of the West Japan Branch of the Japanese Society of Chemotherapy. This research was supported by the Research Program on Emerging and Re-emerging Infectious Diseases from the Japan Agency for Medical Research and Development (AMED).
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REFERENCES 1. McKenna M. 2013. Antibiotic resistance: the last resort. Nature 499:394 – 396. http://dx.doi.org/10.1038/499394a. 2. Nordmann P, Naas T, Poirel L. 2011. Global spread of carbapenemaseproducing Enterobacteriaceae. Emerg Infect Dis 17:1791–1798. http://dx .doi.org/10.3201/eid1710.110655. 3. Yano H, Kuga A, Okamoto R, Kitasato H, Kobayashi T, Inoue M. 2001. Plasmid-encoded metallo-beta-lactamase (IMP-6) conferring resistance to carbapenems, especially meropenem. Antimicrob Agents Chemother 45:1343–1348. http://dx.doi.org/10.1128/AAC.45.5.1343-1348.2001. 4. Shigemoto N, Kuwahara R, Kayama S, Shimizu W, Onodera M, Yokozaki M, Hisatsune J, Kato F, Ohge H, Sugai M. 2012. Emergence in Japan of an imipenem-susceptible, meropenem-resistant Klebsiella pneumoniae carrying blaIMP-6. Diagn Microbiol Infect Dis 72:109 –112. http: //dx.doi.org/10.1016/j.diagmicrobio.2011.09.019. 5. Kayama S, Shigemoto N, Kuwahara R, Oshima K, Hirakawa H, Hisatsune J, Jové T, Nishio H, Yamasaki K, Wada Y, Ueshimo T, Miura T, Sueda T, Onodera M, Yokozaki M, Hattori M, Ohge H, Sugai M. 2015. Complete nucleotide sequence of the IncN plasmid encoding IMP-6 and CTX-M-2 from emerging carbapenem-resistant Enterobacteriaceae in Japan. Antimicrob Agents Chemother 59:1356 –1359. http://dx.doi.org/10 .1128/AAC.04759-14. 6. Shigemoto N, Kayama S, Kuwahara R, Hisatsune J, Kato F, Nishio H, Yamasaki K, Wada Y, Sueda T, Ohge H, Sugai M. 2013. A novel metallo--lactamase, IMP-34, in Klebsiella isolates with decreased resistance to imipenem. Diagn Microbiol Infect Dis 76:119 –121. http://dx.doi .org/10.1016/j.diagmicrobio.2013.02.030. 7. Richter DC, Schuster SC, Huson DH. 2007. OSLay: optimal syntenic layout of unfinished assemblies. Bioinformatics 23:1573–1579. http://dx .doi.org/10.1093/bioinformatics/btm153. 8. Partridge SR, Ginn AN, Paulsen IT. 2012. pEl1573 carrying blaIMP-4, from Sydney, Australia, is closely related to other IncL/M plasmids. Antimicrob Agents Chemother 56:6029–6032. http://dx.doi.org/10.1128/AAC.01189-12. 9. Foster GC, McGhee GC, Jones AL, Sundin GW. 2004. Nucleotide sequences, genetic organization, and distribution of pEU30 and pEL60 from Erwinia amylovora. Appl Environ Microbiol 70:7539 –7544. http: //dx.doi.org/10.1128/AEM.70.12.7539-7544.2004. 10. Ho PL, Lo WU, Yeung MK, Lin CH, Chow KH, Ang I, Tong AHY, Bao JY-J, Lok S, Lo JYC. 2011. Complete sequencing of pNDM-HK encoding NDM-1 carbapenemase from a multidrug-resistant Escherichia coli strain isolated in Hong Kong. PLoS One 6:e17989. http://dx.doi.org/10.1371 /journal.pone.0017989. 11. Liebert CA, Hall RM, Summers AO. 1999. Transposon Tn21, flagship of the floating genome. Microbiol Mol Biol Rev 63:507–522. 12. Partridge SR, Hall RM. 2003. The IS1111 family members IS4321 and IS5075 have subterminal inverted repeats and target the terminal inverted repeats of Tn21 family transposons. J Bacteriol 185:6371– 6384. http://dx .doi.org/10.1128/JB.185.21.6371-6384.2003. 13. Yamaguchi T, Hayashi T, Takami H, Ohnishi M, Murata T, Nakayama K, Asakawa K, Ohara M, Komatsuzawa H, Sugai M. 2001. Complete nucleotide sequence of a Staphylococcus aureus exfoliative toxin B plasmid and identification of a novel ADP-ribosyltransferase, EDIN-C. Infect Immun 69:7760 –7771. http://dx.doi.org/10.1128/IAI .69.12.7760-7771.2001. 14. Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, Jelsbak L, Sicheritz-Ponten T, Ussery DW, Aarestrup FM, Lund O. 2012. Multilocus sequence typing of total-genome-sequenced bacteria. J Clin Microbiol 50:1355–1361. http://dx.doi.org/10.1128/JCM.06094-11. 15. Bonnin RA, Nordmann P, Carattoli A, Poirel L. 2013. Comparative genomics of IncL/M-type plasmids: evolution by acquisition of resistance genes and insertion sequences. Antimicrob Agents Chemother 57:674 – 676. http://dx.doi.org/10.1128/AAC.01086-12. 16. Gołebiewski M, Kern-Zdanowicz I, Zienkiewicz M, Adamczyk M, Zylinska J, Baraniak A, Gniadkowski M, Bardowski J, Cegłowski P. 2007. Complete nucleotide sequence of the pCTX-M3 plasmid and its involvement in spread of the extended-spectrum beta-lactamase gene blaCTX-M-3. Antimicrob Agents Chemother 51:3789 –3795. http://dx.doi.org/10.1128/AAC.00457-07. 17. Poirel L, Bonnin RA, Nordmann P. 2012. Genetic features of the widespread plasmid coding for the carbapenemase OXA-48. Antimicrob Agents Chemother 56:559 –562. http://dx.doi.org/10.1128/AAC .05289-11.
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Complete Nucleotide Sequence of pKOI-34, an IncL/M Plasmid Carrying blaIMP-34 in Klebsiella oxytoca Isolated in Japan Norimitsu Shimada,a,b,c Shizuo Kayama,a,b Norifumi Shigemoto,a,d Junzo Hisatsune,a,b Ryuichi Kuwahara,a,b,e Hisaaki Nishio,f Katsutoshi Yamasaki,g Yasunao Wada,h Taijiro Sueda,c Hiroki Ohge,a,d Motoyuki Sugaia,b Project Research Center for Nosocomial Infectious Diseases, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima City, Hiroshima, Japana; Department of Bacteriology, Hiroshima University Graduate School of Biomedical & Health Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima City, Hiroshima, Japanb; Department of Surgery I, Hiroshima University Graduate School of Biomedical & Health Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima City, Hiroshima, Japanc; Department of Infectious Diseases, Hiroshima University Hospital, 1-2-3 Kasumi, Minami-ku, Hiroshima City, Hiroshima, Japand; Clinical Laboratory, Hiroshima General Hospital of West Japan Railway Company, Futaba-no-sato 3-1-36, Higashi-ku, Hiroshima City, Hiroshima, Japane; Clinical Laboratory, Shiga Medical Center for Adults, 5-4-30, Moriyama, Moriyama City, Shiga, Japanf; Department of Clinical Laboratory, Wakayama Rosai Hospital, 93-1 Kinomoto, Wakayama City, Wakayama, Japang; Clinical Laboratory, Hyogo Medical University Hospital, 1-1 Mukogawa-cho, Nishinomiya City, Hyogo, Japanh
We determined the complete nucleotide sequence of a self-transmissible IncL/M plasmid, pKOI-34, from a Klebsiella oxytoca isolate. pKOI-34 possessed the core structure of an IncL/M plasmid found in Erwinia amylovora, pEL60, with two mobile elements inserted, a transposon carrying the arsenic resistance operon and a Tn21-like core module (tnp and mer modules) piggybacking blaIMP-34 as a class 1 integron, In808, where blaIMP-34 confers a resistance to carbapenems in K. oxytoca and Klebsiella pneumoniae.
C
arbapenem-resistant Enterobacteriaceae (CRE) infections are increasing, which is one of the most important issues in health care facilities around the world (1, 2). In Japan, IMP-6, a variant of IMP-1 with one amino acid substitution (Ser196Gly), has been detected sporadically in clinical isolates of the family Enterobacteriaceae (3). IMP-6 shows a paradoxical effect on carbapenem resistance, i.e., imipenem susceptible but meropenem resistant (4), where a plasmid pKPI-6 harboring blaIMP-6 and blaCTX-M-2 confers this stealth-type carbapenem-resistant phenotype that is not detectable when testing for imipenem resistance (5). (Preliminary data from this study were presented at the 62nd Annual Meeting of the Japanese Society of Chemotherapy, 2014.) During a screening for MBL-producing Enterobacteriaceae showing the paradoxical resistance to carbapenems, we found IMP-34 in five MBL-positive isolates of Klebsiella oxytoca (MS5279,
MS5280) and Klebsiella pneumoniae (MS5284, MS5285, MS5286) in the Kinki region, which is the geographic center of Japan. The
Received 27 October 2015 Returned for modification 17 November 2015 Accepted 16 February 2016 Accepted manuscript posted online 22 February 2016 Citation Shimada N, Kayama S, Shigemoto N, Hisatsune J, Kuwahara R, Nishio H, Yamasaki K, Wada Y, Sueda T, Ohge H, Sugai M. 2016. Complete nucleotide sequence of pKOI-34, an IncL/M plasmid carrying blaIMP-34 in Klebsiella oxytoca isolated in Japan. Antimicrob Agents Chemother 60:3156 –3162. doi:10.1128/AAC.02507-15. Address correspondence to Motoyuki Sugai, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.02507-15. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
FIG 1 Structural features of IncL/M plasmid pKOI-34 (87,343 bp) in comparison with pEL60, which is considered the ancestral strain of the IncL/M plasmid. Two mobile gene elements of 8.8 and 19.5 kb, respectively, are inserted into the IncL/M backbone. Arrows show annotated coding sequences. The 104 identified ORFs are color coded on the basis of function as follows: blue, replication-related genes; green, antibiotic-resistant genes; red, transposases; yellow, conjugation-related genes. The lower schema shows the result of PCR scanning for plasmids carrying blaIMP-34 recovered from K. oxytoca and K. pneumoniae. The 22 sets of PCR primers were designed from the complete sequence of pKOI-34. All plasmids without pKOI-34 showed the same results: set primers 3 and 4 were not detected, and the area corresponding to the mobile gene element containing the arsenic resistance genes is missing. PCR using the forward primer of set 3 and the reverse primer of set 4 showed a DNA fragment of 600 bp (see Table S1 in the supplemental material).
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Complete Nucleotide Sequence of pKOI-34
TABLE 1 Features of pKOI-34 ORFs Position (bp)
Identity (%)
Overlap (no. of aa)
Accession no.
99
248/249
WP_020277896
99
673/677
YP_006958848
100
117/117
YP_002287003
100
120/120
YP_002287003
99
582/583
YP_002287002
Arsenic pump membrane protein Arsenate reductase
100
429/429
YP_002287001
100
141/141
YP_002287001
Arsenic transporter ATPase TnpR, site-specific recombinases Transposase, Tn3 family
100 100
124/124 190/190
WP_004118313 WP_012291338
100
1002/1002
YP_008003449
Protein disulfide isomerase
91
300/329
YP_002333315
Conjugal transfer protein
98
428/435
YP_002333316
Soluble lytic murein transglycosylase and related regulatory proteins Archaeal fructose-1,6-bisphosphatase Transfer inhibition protein
100
131/131
YP_006958778
100
116/116
WP_004187434
98
214/217
YP_006964687
Growth regulator, antitoxin involved in plasmid maintenance Growth inhibitor, toxin involved in plasmid maintenance SOS response transcriptional repressors Nucleotidyltransferase Transposase of IS4321R
100
85/85
YP_007878506
100
110/110
YP_008090835
99
143/144
YP_006958786
99 100
411/412 334/334
AHE47434 YP_005352261
100
990/990
YP_006501624
100
185/185
YP_008725240
100
337/337
YP_006961973
99
109/110
AHN60087
100
184/184
YP_002791400
99
245/246
ABF70513
99
109/110
ACN22644
100
115/115
YP_001965784
ORFs
Start
Stop
Strand
Gene
Length (no. of aaa)
Source
Description
1
1
750
⫹
repA
249
K. pneumoniae
2
2,120
4,153
⫺
trbC
677
3
4,510
4,863
⫹
arsR
117
4
4,911
5,273
⫹
arsD
120
5
5,291
7,041
⫹
arsA
583
6
7,091
8,380
⫹
arsB
429
7
8,393
8,818
⫹
arsC
141
8 9
8,849 9,262
9,223 9,834
⫺ ⫺
arsA tnpR
124 190
10
9,998
13,006
⫹
tnpA
1,002
K. pneumoniae (pOXA-48) K. pneumoniae (plasmid 12) K. pneumoniae (plasmid 12) K. pneumoniae (plasmid 12) K. pneumoniae (plasmid 12) K. pneumoniae (plasmid 12) Enterobacteriaceae Yersinia enterocolitica K. pneumoniae (pKP1433)
RepA, IncL/M type replicase protein Protein involved in plasmid transfer Arsenic-resistant operon repressor Arsenic-resistant operon trans-acting repressor Arsenic pump driving ATPase
11
13,003
13,992
⫺
trbB
329
12
14,003
15,310
⫺
trbA
435
13
15,310
15,705
⫺
trbN
131
14
15,810
16,160
⫺
15
16,273
16,926
⫹
16
17,019
17,276
17
17,278
18
K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae (pOXA-48)
116
K. pneumoniae
tir
217
⫹
pemI
85
E. cloacae (pNE1280) K. pneumoniae (pJEG011)
17,610
⫹
pemK
110
K. pneumoniae (pKPoxa-48N1)
17,703
18,137
⫹
mucA
144
19 20
18,152 19,989
19,390 20,993
⫹ ⫺
mucB tnpA
412 334
K. pneumoniae (pOXA-48) K. pneumoniae K. pneumoniae (pNDM-MAR)
21
21,072
24,044
⫺
tnpA
990
22
24,047
24,604
⫺
tnpR
185
23
24,910
25,923
⫺
intl1
337
24
26,174
26,506
⫹
qacF
110
25
26,595
27,149
⫹
aacA4
184
26
27,226
27,966
⫹
blaIMP-34 246
27
28,192
28,524
⫹
qacE2
110
28
28,735
29,082
⫹
qacE⌬1
115
K. oxytoca (pKOX_R1) K. pneumoniae (pIMP-PH114) K. oxytoca (pINCan01) Stenotrophomonas maltophilia E. cloacae (pEC-IMP) Pseudomonas aeruginosa Uncultured bacterium K. pneumoniae (pK29)
Transposase, Tn3 family protein Resolvase domain containing protein Integrase Ethidium bromide resistance protein Aminoglycoside N(6=)-acetyltransferase Metallo--lactamase Ethidium bromide resistance protein Ethidium bromide resistance protein
(Continued on following page)
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TABLE 1 (Continued) Position (bp) ORFs
Start
Stop
Strand
Gene
Length (no. of aaa)
29
29,076
29,915
⫹
sul1
279
30
30,043
30,543
⫹
orf5
166
31
30,512
31,504
⫺
tniB
330
32
31,507
33,186
⫺
tinA
559
33
33,261
33,968
⫺
urf2
235
34
33,965
34,201
⫺
merE
78
35
34,198
34,560
⫺
merD
120
36
34,578
36,272
⫺
merA
564
37
36,324
36,785
⫺
merC
153
38
36,782
37,057
⫺
merP
91
39
37,071
37,421
⫺
merT
116
40
37,493
37,927
⫹
merR
144
41
38,006
39,010
⫹
tnpA
334
42 43 44
39,844 40,309 41,015
40,164 40,947 41,224
⫺ ⫺ ⫺
106 212 69
45
41,227
41,445
⫺
72
46 47 48 49 50
41,490 42,208 44,068 44,402 44,982
42,191 43,539 44,424 44,980 45,389
⫺ ⫺ ⫹ ⫹ ⫹
233 443 118 192 135
51
45,542
46,087
⫺
181
52
46,225
46,683
⫹
152
53
46,680
46,928
⫹
82
54
46,921
47,508
⫹
195
55
47,505
47,990
⫹
161
56
47,987
48,235
⫹
82
57
48,254
48,994
⫹
resD
246
58
49,235
50,209
⫹
parA
324
59
50,212
50,655
⫹
parB
147
Source
Description
Identity (%)
Overlap (no. of aa)
Accession no.
K. pneumoniae (pK29) P. aeruginosa (Rms149)
Dihydropteroate synthase
100
279/279
YP_001965785
Acetyltransferase (GNAT) family protein
100
166/166
YP_245437
Salmonella enterica (pOU7519) K. pneumoniae (pR55) S. enterica (pOU7519) K. pneumoniae (pKpQIL) K. pneumoniae (pKpQIL) K. pneumoniae (pKpQIL) K. pneumoniae (pKPHS2) K. pneumoniae (pR55) K. pneumoniae (pKP048) E. coli
NTPb binding protein
100
330/330
YP_209341
Transposase and inactivated derivatives Tn21 protein of unknown function Urf2 Mercuric transport protein
100
559/559
YP_005352048
100
235/235
YP_001598123
100
78/78
YP_003560404
Transcriptional regulator
100
120/120
YP_003560405
Mercuric ion reductase
100
564/564
YP_003560406
Mercury transport protein
100
153/153
YP_005229641
Mercury transport protein
100
91/91
YP_005352054
Mercury transport protein
100
116/116
YP_003754062
Mercuric resistance operon regulatory protein
100
144/144
KKA59047
K. pneumoniae (pNDM-MAR) Enterobacteriaceae E. coli K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae (pJEG011)
Transposase of IS4321R
100
334/334
YP_005352261
Hypothetical protein Hypothetical protein Hypothetical protein
99 100 100
106/106 212/212 69/69
WP_021561451 WP_021561450 YP_002333329
Hypothetical protein
97
71/72
YP_002333330
Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein
92 93 92 93 98
219/233 414/443 113/118 184/192 134/135
WP_023302501 WP_023302502 WP_023302503 WP_023302504 YP_007878517
K. pneumoniae (pJEG011) Erwinia amylovora (pEL60) K. pneumoniae (pNDM-OM) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae (pKPoxa-48N2) K. pneumoniae (pKP048) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360)
Hypothetical protein
98
178/181
YP_007878518
Hypothetical protein
90
141/152
NP_943212
Hypothetical protein
98
82/82
YP_007195536
Hypothetical protein
98
191/195
YP_002333340
Hypothetical protein
99
160/161
YP_002333341
Hypothetical protein
93
81/82
YP_008110943
Resolvase
99
245/246
YP_006958800
StbA family protein
100
324/324
YP_002333344
Plasmid stability protein
100
147/147
YP_002333345
(Continued on following page)
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Complete Nucleotide Sequence of pKOI-34
TABLE 1 (Continued) Position (bp) ORFs
Start
Stop
Strand
Gene
Length (no. of aaa)
60
50,665
51,216
⫹
nuc
61
51,334
51,840
62
51,833
63
Identity (%)
Overlap (no. of aa)
Accession no.
Catalytic domain of EDTAresistant nuclease
100
183/183
YP_002333346
Hypothetical protein
100
168/168
YP_002333347
Hypothetical protein
100
159/159
YP_002333348
Hypothetical protein
99
136/136
YP_002333349
Hypothetical protein
100
87/87
YP_002333350
Hypothetical protein
100
120/120
YP_002333351
Hypothetical protein
100
149/149
NP_774994
Putative transcriptional repressor protein Hypothetical protein
100
88/88
YP_002333353
100
427/427
YP_002333354
Hypothetical protein
100
59/59
YP_006965388
Hypothetical protein
99
265/266
YP_002333355
Hypothetical protein
100
112/112
YP_006958814
Hemolysin expression modulating protein Hypothetical protein
100
76/76
YP_006958815
99
71/71
YP_008090866
Hypothetical protein
100
136/136
YP_002333358
Hypothetical protein
100
101/101
YP_006952492
Antirestriction protein
100
146/146
YP_007195464
Hypothetical protein
100
94/94
NP_775004
hypothetical protein
100
73/73
YP_006952495
Single-stranded DNA binding protein Hypothetical protein
100
144/144
YP_006958822
99
103/103
YP_006958823
Hypothetical protein
100
178/178
YP_006964602
MobC protein involved in plasmid mobilization MobB protein involved in plasmid mobilization MobA protein involved in plasmid mobilization Conjugative transfer protein
98
119/121
YP_002333286
99
105/105
YP_002333287
99
659/659
YP_008090802
99
165/166
YP_002333289
Conjugative transfer protein
99
259/259
YP_002333290
Tfp pilus assembly protein
100
387/387
WP_020805653
Source
Description
183
K. pneumoniae (pCTXM360)
⫹
168
52,312
⫹
159
52341
52,751
⫹
136
64
52,869
53,132
⫹
87
65
53,154
53,516
⫹
120
66
53,638
54,087
⫹
149
67
54,132
54,398
⫹
68
54,462
55,745
⫹
69
56,352
56,531
⫹
70
56,664
57,464
⫹
266
K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) C. freundii (pCTX-M3) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) E. cloacae (pEI1573) K. pneumoniae (pCTXM360)
71
57,655
57,993
⫹
112
72
58,091
58,321
⫹
73
58,653
58,868
⫹
71
74
58,940
59,350
⫹
136
75
59,412
59,717
⫹
101
76
59,918
60,358
⫹
77
60,402
60,686
⫹
94
78
60,841
61,062
⫹
73
79
61,134
61,568
⫹
80
61,624
61,935
⫹
103
81
62,068
62,604
⫹
178
82
63,106
63,471
⫺
mobC
121
83
63,746
64,063
⫹
mobB
105
84
64,050
66,029
⫹
mobA
659
85
66,043
66,543
⫹
traH
166
86
66,540
67,319
⫹
traI
259
87
67,330
68,493
⫹
traJ
387
korC
88 427
ccgA1
rmoA
klcA
ssb
59
76
146
144
K. pneumoniae (pOXA-48) K. pneumoniae (Kp11978) K. pneumoniae (pKPoxa-48N1) K. pneumoniae (pOXA-48) K. pneumoniae (pNDM-HK) K. pneumoniae (pNDM-OM) C. freundii (pCTX-M3) E. coli (pNDM-HK) K. pneumoniae (pOXA-48) K. pneumoniae (pOXA-48) Serratia marcescens (R830b) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae (pKPoxa-48N1) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae
(Continued on following page)
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TABLE 1 (Continued) Position (bp) ORFs
Start
Stop
Strand
Gene
Length (no. of aaa)
88
68,483
68,743
⫹
traK
86
89 90
68,768 72,060
72,118 72,596
⫹ ⫹
pri traL
1,116 178
91
72,562
73,212
⫹
92
73,190
73,972
⫹
traM
260
93
73,981
75,132
⫹
traN
383
94
75,144
76,493
⫹
traO
449
95
76,505
77,209
⫹
traP
234
96
77,233
77,763
⫹
traQ
176
97
77,780
78,169
⫹
traR
129
98
78,215
78,709
⫹
99
78,706
81,756
⫹
traU
1,016
100
81,753
82,961
⫹
traW
402
101 102
82,958 83,508
83,608 85,781
⫹ ⫹
traX traY
216 757
103
85,784
86,437
⫹
excA
217
104
86,511
86,741
⫹
repC
76
216
164
Source
Description
Identity (%)
Overlap (no. of aa)
Accession no.
K. pneumoniae (pCTXM360) K. oxytoca K. pneumoniae (pKPoxa-48N1)
Conjugative transfer protein
100
86/86
YP_002333292
DNA primase Conjugative transfer protein
96 100
1077/1116 178/178
WP_031942492 YP_008090807
Hypothetical protein
97
213/216
YP_008090808
Conjugative transfer protein
98
257/260
YP_002333295
Conjugative transfer protein
98
380/383
YP_002333296
Conjugative transfer protein
100
449/449
YP_002333297
Conjugative transfer protein
98
232/234
YP_007195483
Conjugative transfer protein
100
176/176
YP_007195484
Conjugative transfer protein
100
129/129
YP_006964622
Hypothetical protein
100
164/164
YP_007195486
Conjugative transfer protein
99
1014/1016
YP_002333302
Conjugative transfer protein
100
402/402
YP_006964668
Conjugative transfer protein Conjugative transfer protein
100 100
216/216 757/757
WP_004187488 YP_006964669
Phosphoglycerol transferase
100
216/217
YP_002333306
Replication regulatory protein
99
76/76
YP_006964671
K. pneumoniae (pKPoxa-48N1) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae (pCTXM360) K. pneumoniae (pNDM-OM) K. pneumoniae (pNDM-OM) K. pneumoniae (pNDM-OM) K. pneumoniae (pNDM-OM) K. pneumoniae (pCTXM360) E. cloacae (pNE1280) Enterobacteriaceae E. cloacae (pNE1280) K. pneumoniae (pCTXM360) E. cloacae
a
aa, amino acids. b NTP, nucleoside triphosphate.
isolates showed intermediate resistance to imipenem but resistance to meropenem (6). IMP-34 has a single amino acid substitution, Glu87Gly, compared to IMP-1. Conjugal transfer experiments showed that blaIMP-34 is located on a self-transmissible plasmid designated pKOI-34. We sequenced the plasmid DNA of pKOI-34 purified from the E. coli BL21 transconjugant (BL21_pKOI-34) from K. oxytoca strain MS5279. The draft sequences of pKOI-34 were generated using Illumina MiSeq (Nextera paired-end library; 2,354,946 bp), assembled using CLC Genomics Workbench (CLC bio, Cambridge, MA), and sorted using OSLay (7). Gap closing was performed using direct sequencing of PCR products amplified with oligonucleotide primers designed to anneal each end to the neighboring contigs. pKOI-34 is an 87,343-bp plasmid with an average GC content of 53%, and 104 open reading frames (ORFs) (Fig. 1; Table 1). The BLASTP program showed that the amino acid sequence of RepA in pKOI-34 conforms to the IncL/M group. Replicon typing of plasmid preparations from the five strains positive for blaIMP-34 showed that they all belong to the IncL/M group. Sequence comparisons of pKOI-34 with the plasmids registered in the GenBank database show extensive similarity to IncL/M plasmids (see Fig. S1a in the supplemental material). The plasmid pEL60 (60,145 bp) from the plant pathogen Er-
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winia amylovora is considered the ancestral IncL/M plasmid. It does not possess any resistance genes and shares its entire sequence with other IncL/M plasmid backbones (8, 9) (see Fig. S1a in the supplemental material). The nucleotide sequence identity of pKOI-34 with pEL60 is 53% (46,561 bp), and the common backbone sequence possesses genes for replication (rep), a toxinantitoxin system (pemIK) that elevates mutation frequency conferring a UV-resistant phenotype (mucAB) (10), plasmid stability (parAB), endonuclease (nuc), primase (pri), and mobility/conjugal transfer genes (trbCBAN, tir, mobAB, traHIJK, and traLMNOPQUWXY) (Fig. 1). The pKOI-34 repA gene shows 93% nucleotide identity with pEL60 (53 nucleotide substitutions resulting in 13 amino acid variants) and 99% nucleotide identity with the majority of the other IncL/M plasmids (5 to 8 nucleotide substitutions, resulting in 1 or 2 amino acid substitutions). pKOI-34 possesses two mobile genetic elements. One is an 8.8-kb element consisting of tnpA, tnpR, and the arsenic resistance operon (10); and the other is a Tn21-like transposon, including two IS4321R (19.5 kb) (Fig. 2). The 8.8-kb element was inserted at the 5= end of trbC that was related to recombination, bacterial conjugation, and DNA transfer. This region showed 99% homology to the plasmid pZA1001,
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Complete Nucleotide Sequence of pKOI-34
FIG 2 The schema for the insertion region of pKOI-34 was based on the IncL/M backbone of pEL60. pKOI-34 possesses two mobile gene elements. One is an 8.8-kb region containing transposase (tnpA), resolvase (tnpR), and arsenic resistance genes. Another is aTn21-like transposon, consisting of tnpAR, an integron cassette structure of the class I integron (intl1, qacF, aacA4, blaIMP-34, qacE2, qacE⌬1, sul1, orf5), tniAB, urf2, and the mercury resistance operon. This transferable structure (Tn21-like transposon and IR4321R) was inserted into orf6, and the direct repeat sequence TTAAA (green) was generated. The Tn21-like transposon has an incomplete inverted repeat site, IRtnp and IRmer, on the left and right sides, respectively. The IRtnp of the Tn21-like transposon (orange) is truncated on the left side of IS4321R, and the IRmer of the Tn21-like transposon (pink) is truncated on the right side of IS4321R. Black, inverted repeat of IS4321R; green, direct repeat sequence of Tn21-like transposon; orange, IRtnp of the Tn21-like transposon; light blue, additional sequence; black, inverted repeat of IS4321R; pink, IRmer of Tn21-like transposon. The arsenic resistance operon has an incomplete inverted repeat (underlined).
R46, and pCC416 (accession nos. CP001723, AY046276, and AJ704863, respectively). The 19.5-kb element has a Tn21-like structure that divides orf6 into two truncated sequences (Fig. 2). The sites of insertion, defined by a 6-bp duplication of the target DNA TTAAAG, is AT rich, similar to Tn21 (11) (Fig. 2, green box). The core element of the Tn21-like structure is a composite of the Tn21 tnp and mer modules forming Tn21⌬. Both sides of the 38-bp Tn21⌬ terminal inverted repeat (TIR) are interrupted by the 1,327-bp IS4321R. IS4321 insertion creates 11-bp inverted repeats (IRl and IRr of IS4321) and additional flanking bases on both sides. IS4321R is a member of the IS1111 family that targets a specific position in the 38-bp complete TIR in the family of Tn21 transposons (12). A similar IS insertion into a 38-bp TIR is observed in many plasmids
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carrying TIR from the family of Tn21 transposons, Tn21/Tn501 (12). In Tn21, In2 is integrated between tnpR and urf2 in Tn21⌬, generating an imperfect inverted repeat of 25 bp and a 5-bp direct repeat TCCAT. However, in this Tn21-like structure, In808 carrying blaIMP-34 is present instead of In2 (Fig. 2). Besides blaIMP-34, In808 possesses other resistance genes, i.e., qacF, qacE2, and aacA4 (4). In808 is flanked by an imperfect inverted repeat of 25 bp (IRl, IRr). The TCCAT sequence was found on the right side but not on the left side, suggesting a later swapping event of an integron module between In2 and In808. The other four plasmids recovered from MS5280, MS5284, MS5285, and MS5286 carrying blaIMP-34 were examined using the PCR scanning method (13). Twenty-two primer sets covering the total DNA sequence with the PCR products of pKOI-34 were used
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(Fig. 1; see Table S1 in the supplemental material). PCR scanning data showed that primer sets 3 and 4 generated no amplicon, but the other 20 sets of the primer pairs yielded PCR products of the expected sizes in all four plasmids. The data suggest that all four plasmids were identical to pKOI-34 but lacked the mobile element carried by the transposon containing the arsenic resistance operon inserted between trbC and trbB. To examine the junction between the sequence corresponding to the plasmid backbone and the transposon, an ⬃600-bp amplicon was generated with PCR using the set 3 forward primer and set 4 reverse primer (see Table S1). The nucleotide sequence of the ⬃600-bp amplicon was identical to the trbC to trbB region of pEL60. This indicates that the four plasmids recovered from MS5280, MS5284, MS5285, and MS5286 are pKOI-34 variants lacking the transposon carrying the arsenic resistance operon. MS5279 and MS5280 were isolated in 2004, MS5285 and MS5286 in 2006, and MS5284 in 2007. Therefore, it may be reasonable to assume that the arsenic operon was lost in MS5280, MS5284, MS5285, and MS5286. In any case, this fact is not strong enough support that this element was lost in these strains. Multilocus sequence typing (MLST) was performed using the protocol published by Larsen et al. (14). MLST showed that K. pneumoniae MS5284, MS5285, and MS5286 belong to ST334, and K. oxytoca MS5279 and MS5280 belong to ST171 (gapA, 3; infB, 4; mdh, 15; pgi, 4; phoE, 18; rpoB, 3; tonB, 4). The number of members of the IncL/M multidrug resistance (MDR) plasmids harboring broad-spectrum -lactam resistance is increasing worldwide. pKOI-34 is the newest member of this group and the first IncL/M drug resistance plasmid found in Japan. Bonnin et al. (15) suggest that the evolution of the IncL/M MDR plasmids was through the acquisition of resistance genes and insertion sequences. There are two integration hot spots in the IncL/M plasmids; one is located between the rep locus and trbC, and the other is near pemIK (15) (see Fig. S1b in the supplemental material) (16, 17). In pKOI-34, two target sites for inserted sequences are different from those previously reported (see Fig. S1a,b in the supplemental material). The arsenic resistance operon was located between trbC and trbB, and the other Tn21-like transposon was located within the orf6 of pEL60. In conclusion, we report the complete sequence of pKOI-34, an IncL/M type conjugal plasmid carrying blaIMP-34. pKOI-34 possesses a pEL60 backbone with two inserted mobile elements, a Tn21-like architecture with a class 1 integron, In808 instead of In2, and a transposon carrying the arsenic resistance operon. We show here that the spread of the blaIMP-34 gene in K. oxytoca and K. pneumoniae is linked to the spread of pKOI-34 or its derivatives. Nucleotide sequence accession number. The complete sequence of pKOI-34 has been deposited in GenBank (accession number AB715422). ACKNOWLEDGMENTS We thank Jim Nelson for editorial assistance. N.S. received the 62nd presentation award in the category of basic research conferred by the Director of the West Japan Branch of the Japanese Society of Chemotherapy. This research was supported by the Research Program on Emerging and Re-emerging Infectious Diseases from the Japan Agency for Medical Research and Development (AMED).
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