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. 2016 Apr 22;60(5):3156–3162. doi: 10.1128/AAC.02507-15

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 Sugai a,b,✉
PMCID: PMC4862478  PMID: 26902770

Abstract

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.

TEXT

Carbapenem-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 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).

FIG 1.

FIG 1

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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).

TABLE 1.

Features of pKOI-34 ORFs

ORFs Position (bp)
 Strand Gene Length (no. of aaa) Source Description Identity (%) Overlap (no. of aa) Accession no.
Start Stop
1 1 750 + repA 249 K. pneumoniae RepA, IncL/M type replicase protein 99 248/249 WP_020277896
2 2,120 4,153 − trbC 677 K. pneumoniae (pOXA-48) Protein involved in plasmid transfer 99 673/677 YP_006958848
3 4,510 4,863 + arsR 117 K. pneumoniae (plasmid 12) Arsenic-resistant operon repressor 100 117/117 YP_002287003
4 4,911 5,273 + arsD 120 K. pneumoniae (plasmid 12) Arsenic-resistant operon trans-acting repressor 100 120/120 YP_002287003
5 5,291 7,041 + arsA 583 K. pneumoniae (plasmid 12) Arsenic pump driving ATPase 99 582/583 YP_002287002
6 7,091 8,380 + arsB 429 K. pneumoniae (plasmid 12) Arsenic pump membrane protein 100 429/429 YP_002287001
7 8,393 8,818 + arsC 141 K. pneumoniae (plasmid 12) Arsenate reductase 100 141/141 YP_002287001
8 8,849 9,223 − arsA 124 Enterobacteriaceae Arsenic transporter ATPase 100 124/124 WP_004118313
9 9,262 9,834 − tnpR 190 Yersinia enterocolitica TnpR, site-specific recombinases 100 190/190 WP_012291338
10 9,998 13,006 + tnpA 1,002 K. pneumoniae (pKP1433) Transposase, Tn3 family 100 1002/1002 YP_008003449
11 13,003 13,992 − trbB 329 K. pneumoniae (pCTXM360) Protein disulfide isomerase 91 300/329 YP_002333315
12 14,003 15,310 − trbA 435 K. pneumoniae (pCTXM360) Conjugal transfer protein 98 428/435 YP_002333316
13 15,310 15,705 − trbN 131 K. pneumoniae (pOXA-48) Soluble lytic murein transglycosylase and related regulatory proteins 100 131/131 YP_006958778
14 15,810 16,160 − 116 K. pneumoniae Archaeal fructose-1,6-bisphosphatase 100 116/116 WP_004187434
15 16,273 16,926 + tir 217 E. cloacae (pNE1280) Transfer inhibition protein 98 214/217 YP_006964687
16 17,019 17,276 + pemI 85 K. pneumoniae (pJEG011) Growth regulator, antitoxin involved in plasmid maintenance 100 85/85 YP_007878506
17 17,278 17,610 + pemK 110 K. pneumoniae (pKPoxa-48N1) Growth inhibitor, toxin involved in plasmid maintenance 100 110/110 YP_008090835
18 17,703 18,137 + mucA 144 K. pneumoniae (pOXA-48) SOS response transcriptional repressors 99 143/144 YP_006958786
19 18,152 19,390 + mucB 412 K. pneumoniae Nucleotidyltransferase 99 411/412 AHE47434
20 19,989 20,993 − tnpA 334 K. pneumoniae (pNDM-MAR) Transposase of IS4321R 100 334/334 YP_005352261
21 21,072 24,044 − tnpA 990 K. oxytoca (pKOX_R1) Transposase, Tn3 family protein 100 990/990 YP_006501624
22 24,047 24,604 − tnpR 185 K. pneumoniae (pIMP-PH114) Resolvase domain containing protein 100 185/185 YP_008725240
23 24,910 25,923 − intl1 337 K. oxytoca (pINCan01) Integrase 100 337/337 YP_006961973
24 26,174 26,506 + qacF 110 Stenotrophomonas maltophilia Ethidium bromide resistance protein 99 109/110 AHN60087
25 26,595 27,149 + aacA4 184 E. cloacae (pEC-IMP) Aminoglycoside N(6′)-acetyltransferase 100 184/184 YP_002791400
26 27,226 27,966 + blaIMP-34 246 Pseudomonas aeruginosa Metallo-β-lactamase 99 245/246 ABF70513
27 28,192 28,524 + qacE2 110 Uncultured bacterium Ethidium bromide resistance protein 99 109/110 ACN22644
28 28,735 29,082 + qacEΔ1 115 K. pneumoniae (pK29) Ethidium bromide resistance protein 100 115/115 YP_001965784
29 29,076 29,915 + sul1 279 K. pneumoniae (pK29) Dihydropteroate synthase 100 279/279 YP_001965785
30 30,043 30,543 + orf5 166 P. aeruginosa (Rms149) Acetyltransferase (GNAT) family protein 100 166/166 YP_245437
31 30,512 31,504 − tniB 330 Salmonella enterica (pOU7519) NTPb binding protein 100 330/330 YP_209341
32 31,507 33,186 − tinA 559 K. pneumoniae (pR55) Transposase and inactivated derivatives 100 559/559 YP_005352048
33 33,261 33,968 − urf2 235 S. enterica (pOU7519) Tn21 protein of unknown function Urf2 100 235/235 YP_001598123
34 33,965 34,201 − merE 78 K. pneumoniae (pKpQIL) Mercuric transport protein 100 78/78 YP_003560404
35 34,198 34,560 − merD 120 K. pneumoniae (pKpQIL) Transcriptional regulator 100 120/120 YP_003560405
36 34,578 36,272 − merA 564 K. pneumoniae (pKpQIL) Mercuric ion reductase 100 564/564 YP_003560406
37 36,324 36,785 − merC 153 K. pneumoniae (pKPHS2) Mercury transport protein 100 153/153 YP_005229641
38 36,782 37,057 − merP 91 K. pneumoniae (pR55) Mercury transport protein 100 91/91 YP_005352054
39 37,071 37,421 − merT 116 K. pneumoniae (pKP048) Mercury transport protein 100 116/116 YP_003754062
40 37,493 37,927 + merR 144 E. coli Mercuric resistance operon regulatory protein 100 144/144 KKA59047
41 38,006 39,010 + tnpA 334 K. pneumoniae (pNDM-MAR) Transposase of IS4321R 100 334/334 YP_005352261
42 39,844 40,164 − 106 Enterobacteriaceae Hypothetical protein 99 106/106 WP_021561451
43 40,309 40,947 − 212 E. coli Hypothetical protein 100 212/212 WP_021561450
44 41,015 41,224 − 69 K. pneumoniae (pCTXM360) Hypothetical protein 100 69/69 YP_002333329
45 41,227 41,445 − 72 K. pneumoniae (pCTXM360) Hypothetical protein 97 71/72 YP_002333330
46 41,490 42,191 − 233 K. pneumoniae Hypothetical protein 92 219/233 WP_023302501
47 42,208 43,539 − 443 K. pneumoniae Hypothetical protein 93 414/443 WP_023302502
48 44,068 44,424 + 118 K. pneumoniae Hypothetical protein 92 113/118 WP_023302503
49 44,402 44,980 + 192 K. pneumoniae Hypothetical protein 93 184/192 WP_023302504
50 44,982 45,389 + 135 K. pneumoniae (pJEG011) Hypothetical protein 98 134/135 YP_007878517
51 45,542 46,087 − 181 K. pneumoniae (pJEG011) Hypothetical protein 98 178/181 YP_007878518
52 46,225 46,683 + 152 Erwinia amylovora (pEL60) Hypothetical protein 90 141/152 NP_943212
53 46,680 46,928 + 82 K. pneumoniae (pNDM-OM) Hypothetical protein 98 82/82 YP_007195536
54 46,921 47,508 + 195 K. pneumoniae (pCTXM360) Hypothetical protein 98 191/195 YP_002333340
55 47,505 47,990 + 161 K. pneumoniae (pCTXM360) Hypothetical protein 99 160/161 YP_002333341
56 47,987 48,235 + 82 K. pneumoniae (pKPoxa-48N2) Hypothetical protein 93 81/82 YP_008110943
57 48,254 48,994 + resD 246 K. pneumoniae (pKP048) Resolvase 99 245/246 YP_006958800
58 49,235 50,209 + parA 324 K. pneumoniae (pCTXM360) StbA family protein 100 324/324 YP_002333344
59 50,212 50,655 + parB 147 K. pneumoniae (pCTXM360) Plasmid stability protein 100 147/147 YP_002333345
60 50,665 51,216 + nuc 183 K. pneumoniae (pCTXM360) Catalytic domain of EDTA-resistant nuclease 100 183/183 YP_002333346
61 51,334 51,840 + 168 K. pneumoniae (pCTXM360) Hypothetical protein 100 168/168 YP_002333347
62 51,833 52,312 + 159 K. pneumoniae (pCTXM360) Hypothetical protein 100 159/159 YP_002333348
63 52341 52,751 + 136 K. pneumoniae (pCTXM360) Hypothetical protein 99 136/136 YP_002333349
64 52,869 53,132 + 87 K. pneumoniae (pCTXM360) Hypothetical protein 100 87/87 YP_002333350
65 53,154 53,516 + 120 K. pneumoniae (pCTXM360) Hypothetical protein 100 120/120 YP_002333351
66 53,638 54,087 + 149 C. freundii (pCTX-M3) Hypothetical protein 100 149/149 NP_774994
67 54,132 54,398 + korC 88 K. pneumoniae (pCTXM360) Putative transcriptional repressor protein 100 88/88 YP_002333353
68 54,462 55,745 + 427 K. pneumoniae (pCTXM360) Hypothetical protein 100 427/427 YP_002333354
69 56,352 56,531 + ccgA1 59 E. cloacae (pEI1573) Hypothetical protein 100 59/59 YP_006965388
70 56,664 57,464 + 266 K. pneumoniae (pCTXM360) Hypothetical protein 99 265/266 YP_002333355
71 57,655 57,993 + 112 K. pneumoniae (pOXA-48) Hypothetical protein 100 112/112 YP_006958814
72 58,091 58,321 + rmoA 76 K. pneumoniae (Kp11978) Hemolysin expression modulating protein 100 76/76 YP_006958815
73 58,653 58,868 + 71 K. pneumoniae (pKPoxa-48N1) Hypothetical protein 99 71/71 YP_008090866
74 58,940 59,350 + 136 K. pneumoniae (pOXA-48) Hypothetical protein 100 136/136 YP_002333358
75 59,412 59,717 + 101 K. pneumoniae (pNDM-HK) Hypothetical protein 100 101/101 YP_006952492
76 59,918 60,358 + klcA 146 K. pneumoniae (pNDM-OM) Antirestriction protein 100 146/146 YP_007195464
77 60,402 60,686 + 94 C. freundii (pCTX-M3) Hypothetical protein 100 94/94 NP_775004
78 60,841 61,062 + 73 E. coli (pNDM-HK) hypothetical protein 100 73/73 YP_006952495
79 61,134 61,568 + ssb 144 K. pneumoniae (pOXA-48) Single-stranded DNA binding protein 100 144/144 YP_006958822
80 61,624 61,935 + 103 K. pneumoniae (pOXA-48) Hypothetical protein 99 103/103 YP_006958823
81 62,068 62,604 + 178 Serratia marcescens (R830b) Hypothetical protein 100 178/178 YP_006964602
82 63,106 63,471 − mobC 121 K. pneumoniae (pCTXM360) MobC protein involved in plasmid mobilization 98 119/121 YP_002333286
83 63,746 64,063 + mobB 105 K. pneumoniae (pCTXM360) MobB protein involved in plasmid mobilization 99 105/105 YP_002333287
84 64,050 66,029 + mobA 659 K. pneumoniae (pKPoxa-48N1) MobA protein involved in plasmid mobilization 99 659/659 YP_008090802
85 66,043 66,543 + traH 166 K. pneumoniae (pCTXM360) Conjugative transfer protein 99 165/166 YP_002333289
86 66,540 67,319 + traI 259 K. pneumoniae (pCTXM360) Conjugative transfer protein 99 259/259 YP_002333290
87 67,330 68,493 + traJ 387 K. pneumoniae Tfp pilus assembly protein 100 387/387 WP_020805653
88 68,483 68,743 + traK 86 K. pneumoniae (pCTXM360) Conjugative transfer protein 100 86/86 YP_002333292
89 68,768 72,118 + pri 1,116 K. oxytoca DNA primase 96 1077/1116 WP_031942492
90 72,060 72,596 + traL 178 K. pneumoniae (pKPoxa-48N1) Conjugative transfer protein 100 178/178 YP_008090807
91 72,562 73,212 + 216 K. pneumoniae (pKPoxa-48N1) Hypothetical protein 97 213/216 YP_008090808
92 73,190 73,972 + traM 260 K. pneumoniae (pCTXM360) Conjugative transfer protein 98 257/260 YP_002333295
93 73,981 75,132 + traN 383 K. pneumoniae (pCTXM360) Conjugative transfer protein 98 380/383 YP_002333296
94 75,144 76,493 + traO 449 K. pneumoniae (pCTXM360) Conjugative transfer protein 100 449/449 YP_002333297
95 76,505 77,209 + traP 234 K. pneumoniae (pNDM-OM) Conjugative transfer protein 98 232/234 YP_007195483
96 77,233 77,763 + traQ 176 K. pneumoniae (pNDM-OM) Conjugative transfer protein 100 176/176 YP_007195484
97 77,780 78,169 + traR 129 K. pneumoniae (pNDM-OM) Conjugative transfer protein 100 129/129 YP_006964622
98 78,215 78,709 + 164 K. pneumoniae (pNDM-OM) Hypothetical protein 100 164/164 YP_007195486
99 78,706 81,756 + traU 1,016 K. pneumoniae (pCTXM360) Conjugative transfer protein 99 1014/1016 YP_002333302
100 81,753 82,961 + traW 402 E. cloacae (pNE1280) Conjugative transfer protein 100 402/402 YP_006964668
101 82,958 83,608 + traX 216 Enterobacteriaceae Conjugative transfer protein 100 216/216 WP_004187488
102 83,508 85,781 + traY 757 E. cloacae (pNE1280) Conjugative transfer protein 100 757/757 YP_006964669
103 85,784 86,437 + excA 217 K. pneumoniae (pCTXM360) Phosphoglycerol transferase 100 216/217 YP_002333306
104 86,511 86,741 + repC 76 E. cloacae Replication regulatory protein 99 76/76 YP_006964671
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a

aa, amino acids.

b

NTP, nucleoside triphosphate.

The plasmid pEL60 (60,145 bp) from the plant pathogen Erwinia 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 toxin-antitoxin 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).

FIG 2.

FIG 2

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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, qacEΔ1, 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).

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, 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 Tn21Δ. Both sides of the 38-bp Tn21Δ 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 carrying TIR from the family of Tn21 transposons, Tn21/Tn501 (12). In Tn21, In2 is integrated between tnpR and urf2 in Tn21Δ, 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 (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 β-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).

Supplementary Material

Supplemental material
supp_60_5_3156__index.html (806B, html)

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).

Footnotes

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.02507-15.

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