Plasmids Carrying bla VIM-2 in Acinetobacter nosocomialis and A. seifertii Isolates from South Korea

Abstract

We identified nine Verona integron-encoded metallo-β-lactamase-2 (VIM-2)-producing Acinetobacter nosocomialis (n = 8) and Acinetobacter seifertii (n = 1) isolates from South Korea and performed whole-plasmid sequencing for two A. nosocomialis isolates and one A. seifertii isolate. Genotyping, antibiotic resistance profiles, and whole plasmid sequences indicated clonal dissemination of the eight VIM-2–producing A. nosocomialis isolates. The plasmid-bearing bla_VIM-2 in the A. seifertii isolate differed from those in the A. nosocomialis isolates.

Introduction

Verona integron-encoded metallo- β-lactamase-2 (VIM-2) is a metallo-β-lactamase (MBL) that was first identified in Pseudomonas aeruginosa in 2000¹ and subsequently identified in Acinetobacter spp. isolates.² It is primarily located in class 1 integrons in plasmids.^1,2 Although bla_VIM-2 has mostly been identified in Acinetobacter baumannii, it has been reported in other species, including Acinetobacter nosocomialis.³ However, whole sequences of plasmids carrying bla_VIM-2 in Acinetobacter spp. other than A. baumannii have not been evaluated, and thus, the association between plasmids carrying it is unclear.

We identified nine VIM-2–producing Acinetobacter spp. isolates (eight A. nosocomialis and one Acinetobacter seifertii) from South Korea and performed whole-plasmid sequencing in two A. nosocomialis isolates and the A. seifertii isolate to investigate the origin of the plasmids carrying bla_VIM-2.

Materials and Methods

We collected 602 Acinetobacter sp. isolates from patients at Daegu Fatima Hospital (Daegu, South Korea) from 2011 to 2014. Species identification was performed using partial rpoB sequences.⁴ Antimicrobial susceptibility testing was performed on the bla_VIM-2-positive isolates using nine antimicrobial agents (Table 1) based on the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.⁵ For genotyping, multilocus sequence typing was performed according to the Oxford scheme.⁶

Table 1.

Acinetobacter nosocomialis and Acinetobacter seifertii Isolates Carrying the bla_VIM-2 Gene

Isolate no.	Species	Isolation year	Genotype	Carbapenemase gene	MICs (mg/L)
Isolate no.	Species	Isolation year	Genotype	Carbapenemase gene	IMI	MER	ERT	AMK	COL	CIP	CAZ	SXT	PIP/TAZ
FA70	A. nosocomialis	2011	ST854	bla _VIM-2	64	32	64	64	16	1	>64	608/32	8/4
FA172	A. nosocomialis	2012	ST854	bla _VIM-2	64	32	64	>64	8	1	>64	608/32	4/4
FA267^a	A. nosocomialis	2012	ST854	bla _VIM-2	64	32	64	>64	8	1	>64	608/32	8/4
FA648^a	A. nosocomialis	2013	ST854	bla _VIM-2	64	32	64	>64	4	1	>64	>608/32	8/4
FA753	A. nosocomialis	2013	ST854	bla _VIM-2	64	32	64	>64	8	1	>64	608/32	>64/4
FA758	A. nosocomialis	2013	ST854	bla _VIM-2, bla _OXA-23	>256	>256	>256	>64	4	1	>64	608/32	8/4
FA782	A. nosocomialis	2013	ST854	bla _VIM-2	64	32	64	>64	8	1	>64	>608/32	8/4
FA1333	A. nosocomialis	2014	ST854	bla _VIM-2	64	32	64	>64	4	1	>64	608/32	8/4
FA319^a	A. seifertii	2012	ST1558	bla _VIM-2	64	64	64	>64	2	1	>64	608/32	8/4

Isolates of which whole plasmids were sequenced.

AMK, amikacin; CAZ, ceftazidime; CIP, ciprofloxacin; COL, colistin; ERT, ertapenem; IMI, imipenem; MER, meropenem; MIC, minimum inhibitory concentration; PIP/TAZ, piperacillin–tazobactam; ST, sequence type; SXT, trimethoprim–sulfamethoxazole.

Plasmid DNA was extracted from the transconjugants and was sequenced on a PacBio RS II system (Pacific Biosciences, Menlo Park, CA) following the manufacturer's instructions. The sequences were assembled using HGAP algorithm (v3.0). Open reading frames were predicted and annotated using Prokka v1.12b. The sequences of each predicted protein were compared with those in a protein database using BlastP (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with a minimum cut-off of 30% identity and 80% length coverage. The antibiotic resistance genes were verified using CARD. The plasmid replicon type was determined using Plasmid MLST. The complete plasmid sequences were deposited in GenBank database (accession numbers MT771289–MT771291).

Results and Discussion

Among the 602 Acinetobacter spp. isolates, 482 (80.1%) were A. baumannii and 120 were not A. baumannii (non-baumannii Acinetobacter sp.). A. nosocomialis was the most prevalent (94/602 isolates, 15.6%), followed by A. seifertii (7/602, 1.2%). Six A. pittii and one A. calcoaceticus isolates were identified. The remaining 12 isolates belonged to A. soli (n = 3), A. oleivorans (n = 3), A. colistinresistans (n = 3), A. bereziniae (n = 2), and A. dijkshoorniae (n = 1). Twelve A. nosocomialis and one A. seifertii isolates were resistant to imipenem (12.8% and 14.3%, respectively).

We investigated the presence of carbapenemase genes in the imipenem-resistant A. nosocomialis and A. seifertii isolates. The bla_VIM-2 gene was identified in 8 of 12 (66.7%) imipenem-resistant A. nosocomialis isolates and one imipenem-resistant A. seifertii isolate (Table 1), but other carbapenemase genes were not identified. Only one isolate, FA758, possessed bla_OXA-23 as well as bla_VIM-2. All bla_VIM-2-producing A. nosocomialis isolates were from sputum, and one A. seifertii isolate was from a spot urine sample.

All bla_VIM-2-positive isolates were resistant to amikacin, ceftazidime, and trimethoprim–sulfamethoxazole but susceptible to ciprofloxacin. Isolate FA758, which coproduced VIM-2 and OXA-23, exhibited higher carbapenem (imipenem, meropenem, and ertapenem) minimum inhibitory concentrations (MICs; >256 mg/L) than the other bla_VIM-2-positive isolates. Although all A. nosocomialis isolates were resistant to colistin (MIC ≥4 mg/L), the A. seifertii isolate was susceptible to it. Only one isolate (FA753) was resistant to piperacillin–tazobactam (Table 1).

All bla_VIM-2-positive A. nosocomialis isolates had the same sequence types (STs; ST854), and the A. seifertii isolate belonged to ST1558 (Table 1). ST854 was previously identified in a strain isolated from South Korea.⁷ Pulsed-field gel electrophoresis analysis based on ApaI restriction also showed that bla_VIM-2-positive A. nosocomialis isolates were clonal.

We performed whole-plasmid sequencing for two bla_VIM-2-positive A. nosocomialis isolates (FA267 and FA648) and the bla_VIM-2-positive A. seifertii isolate FA319. All three plasmids were conjugative type IncI1. Although bla_VIM-2 has been identified in plasmids of diverse conjugative types, such as IncF, IncHI1, IncHI2, IncA/C, IncN, and IncX3,⁸ it has not been reported in the IncI1 type. To the best of our knowledge, this is the first report of an IncI1-type plasmid bearing bla_VIM-2.

The plasmids of the two bla_VIM-2-positive A. nosocomialis isolates showed the same sequences (140,960 bp, GC content 36.3%) and were the most similar to pAnAC1530 of A. nosocomialis from Malaysia (∼50% similarity) (Fig. 1). However, the structure of pANAC1530, which belongs to IncF1, differed from those of pFA267 and pFA648, and it carried bla_OXA-58 instead of bla_VIM-2. In the plasmids pFA267 and pFA648, the aminoglycoside resistance genes aph(3′)-llb, ant, and aacA(6′)-4 were identified. pFA319 from the A. seifertii isolate (114,739 bp) had a shorter sequence length than the plasmids from the A. nosocomialis isolates, and its GC content was 41.8%. It was most similar to pACI-3569 of Acinetobacter sp. from the United States (75% similarity) but had a different structure (Fig. 1). Furthermore, pACI-3569 lacked bla_VIM-2 and any other resistance genes, and its Inc. type could not be determined based on Plasmid MLST. The structure of the class 1 integrons, including bla_VIM-2 and two aminoglycoside genes ant1 and aacA(6′)-4, In105 type described by Yum et al.² was the same in all three plasmids.

FIG. 1.

Comparison of the structures of the plasmids carrying bla_VIM-2. (A) Comparison of plasmids pFA267, pFA648, and pAnAC1530 (GenBank accession no. CPO45561.1). (B) Comparison of plasmids pFA319 and pACI-3569 (GenBank accession no. CPO26416.1). Regions of homology (95–100%) are indicated by gray-shaded regions between sequences. Resistance genes, site-specific recombinase genes, transposon-related genes, and other backbone genes are indicated, respectively. Vertical arrows indicate bla_VIM-2.

The genotyping and antibiotic susceptibility profiles suggest that the eight bla_VIM-2-positive A. nosocomialis isolates resulted from the clonal dissemination of a single strain. Two randomly selected plasmids carrying bla_VIM-2 showed exactly the same sequences, supporting the clonal dissemination of VIM-2–producing A. nosocomialis strains. Because bla_OXA-23 was identified in one strain, it was inferred to have been introduced into its chromosome. The bla_VIM-2-positive A. nosocomialis isolates were collected over a period of 3 years from a single hospital, which could indicate that the VIM-2–producing carbapenem-resistant A. nosocomialis isolates may have settled down at least in the hospital. Although A. nosocomialis infections have a relatively more favorable outcome than A. baumannii infections,⁹ they should not be neglected.¹⁰

We identified a VIM-2–producing A. seifertii isolate for the first time. A. seifertii belongs to Acinetobacter calcoaceticus–A. baumannii complex and is a recently described species.¹¹ It is isolated relatively frequently in South Korea and shows intrinsic colistin resistance.¹² The plasmid bearing bla_VIM-2 in the A. seifertii isolate differed from that in the A. nosocomialis isolates, indicating that the bla_VIM-2-carrying plasmid was not transferred between the two Acinetobacter species. Although whole plasmid sequences of A. baumannii isolates from Korea have not been reported, we speculate that two more plasmids carrying bla_VIM-2 contribute to carbapenem resistance in Acinetobacter sp. due to VIM-2 carbapenemase. Carbapenem resistance in Acinetobacter sp. in South Korea has mainly been attributed to OXA-23 oxacillinase.¹³ As demonstrated in A. nosocomialis isolate FA758 in our study (Table 1), the coproduction of VIM-2 and OXA-23 increases carbapenem resistance. Thus, the dissemination of several plasmids carrying carbapenem resistance would be of significant concern. However, plasmid conjugation or electroporation experiments was not successful, which may limit the horizontal transfer of bla_VIM-2-carrying plasmids.

This study identified the clonal dissemination of several VIM-2–producing A. nosocomialis isolates and one case of VIM-2–producing A. seifertii in a hospital in South Korea. Whole-plasmid sequencing showed that the plasmids carrying bla_VIM-2 were not previously reported, and they were not transferred between the two species. The occurrence and dissemination of MBL-producing non-baumannii Acinetobacter sp. and A. baumannii should be monitored continuously.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

This research was supported partly by the Bio & Medical Technology Development Program and the Basic Science Research Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (NRF-2018M3A9H4055197 and NRF-2019R1A2C2004879).

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