Complete DNA Sequence of an IncM1 Plasmid Bearing the Novel qnrE1 Plasmid-Mediated Quinolone Resistance Variant and bla_CTX-M-8 from Klebsiella pneumoniae Sequence Type 147

LETTER

The recent article by Albornoz et al. (1) reported a member of a new family of plasmid-mediated quinolone resistance genes, named qnrE1, originating from the chromosome of Enterobacter spp. We hereby report the identification of qnrE1 in a Klebsiella pneumoniae isolate from a parrot with respiratory disease. Its complete DNA sequence revealed the presence of a multidrug resistance (MDR) IncM1 plasmid.

In January 2016, an MDR K. pneumoniae strain was isolated from nasal secretion of a diseased blue-fronted parrot (Amazona aestiva). The isolate showed resistance to broad-spectrum cephalosporins, fluoroquinolones, and aminoglycosides by the broth microdilution method (Table 1) (2, 3).

TABLE 1.

Resistance profiles of the parental K. pneumoniae Kp41, transconjugant TcKp41, and recipient E. coli C600 strains

Antibiotic	MIC (μg/ml) for:
	Kp41a	TcKp41 (pKp41 M)b	E. coli C600
Nalidixic acid	>528	16	2
Ciprofloxacin	>16	0.25	0.03
Enrofloxacin	>16	1	0.03
Marbofloxacin	>16	0.5	0.03
Cefotaxime	>128	>128	0.125
Ceftiofur	>128	>128	0.125
Ceftazidime	128	2	0.25
Cefepime	>128	>128	0.125
Amikacin	8	32	4
Gentamicin	>128	64	2

^aKp41 presents mutations in gyrA (Ser83Ile) and parC (Ser80Ile) and harbors bla_CTX-M-15 in an IncF-type plasmid (data not shown).

^bTransconjugants were selected on MacConkey agar containing streptomycin (2,000 μg/ml), cefotaxime (2 μg/ml), and ciprofloxacin (0.06 μg/ml).

The genomic DNA of Kp41 was sequenced using 150-bp paired-end libraries on the Illumina NextSeq platform. Illumina reads were then assembled using Velvet 1.1.12 (4), and the gaps were filled in by PCR and Sanger sequencing. Open reading frames (ORFs) were predicted using Glimmer. Automatic annotation was carried out using RAST (http://rast.nmpdr.org) and manually curated using Artemis 16.0.0 software (5). Resistance genes, plasmid classification (plasmid-based replicon typing [PBRT]), and multilocus sequence typing (MLST) were determined in silico using ResFinder, PlasmidFinder, and MLST databases (https://cge.cbs.dtu.dk/). Differentiation of IncL and IncM plasmids was assessed as previously described (6). Alignments with other sequences were carried out using MAFFT.

Conjugation was performed by broth mating experiments using Escherichia coli C600 as the recipient. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) was carried out for the parental strain and its respective transconjugants. Both strains were shown to have an ∼70-kb plasmid.

K. pneumoniae Kp41 belonged to sequence type 147 (ST147) and harbored qnrE1, bla_CTX-M-8, bla_TEM-1, bla_OXA-9, aac(6′)-Ib, and aadA1 on an IncM1 plasmid. This plasmid, named pKp41M, is 70,354 bp and has a 51% G+C content and 91 predicted ORFs (Fig. 1A). The backbone of pKp41M consists of 55,047 bp, with genes encoding plasmid replication, maintenance, and transfer functions and two resistance regions encoding mobile elements and resistance genes (Fig. 1A). The first resistance region (RR-1) (Fig. 1B) is an 11,747-bp structure consisting of Tn1331-like, truncated by a 4,108-bp insertion of ISEcp1-qnrE1-araJ-Δahp. Tn1331 is a multidrug resistance transposon in a human clinical K. pneumoniae strain from Argentina described by Tolmasky and Crosa (7). Both the Tn1331-like and ISEcp1-qnrE1 inserts have intact inverted repeats (Fig. 1B). The 4,108-bp insertion has high nucleotide identity (95%) with the genetic environment of qnrE1 in pKp1130, described by Albornoz et al. (1), differing by a deletion of 152 bp in pKp1130 (GenBank accession number KY073238), and has 100% similarity with pKp145-11b from Brazil (GenBank accession number KX118608, unpublished). In the second resistance region (RR-2; 3,560 bp), the bla_CTX-M-8 gene was bracketed by two copies of IS26 and truncated IS10 upstream (Fig. 1B). pKp41M has high nucleotide identity (96%) with pKp145-11b, indicating that it may be an epidemic plasmid circulating in K. pneumoniae from humans and animals in Brazil. Differences between these plasmids are in a bla_CTX-M-8 genetic environment, intergenic regions in Tn1331-like, and backbone regions (Fig. 1A).

FIG 1.

(A) Linear genetic map of pKp41M IncM1 plasmid (GenBank accession no. KY781949) and comparison with pKp145-11b and Tn1331. Coding regions are indicated by blue arrows pointing in the direction of transcription. Green arrows indicate qnrE1 and bla_CTX-M-8, blue arrows indicate other resistance genes, yellow arrows indicate genes related to mobile elements, purple arrows indicate genes of plasmid replication and maintenance, light-gray arrows indicate genes related to transfer and mobilization, red arrows indicate other genes with known functions, and white arrows indicate genes with unknown functions. The two resistance regions are indicated by dotted lines. (B) Schematic representation of the two resistance regions (RR-1 and RR-2) in the pKp41M IncM1 plasmid. Arrows point in the direction of transcription. Red arrows indicate qnrE1 and bla_CTX-M-8, green arrows indicate resistance genes, and white arrows indicate genes related to mobile elements or other functional genes. Black lines indicate mobile elements, and boxes indicate inverted repeats (IRR, inverted repeat right; IRL, inverted repeat left). Scale bar, 2,000 bp.

IncM plasmids are considered an epidemic resistance plasmid family detected in Enterobacteriaceae worldwide (8). Here, we present the complete sequence of an IncM1 plasmid harboring the recently described qnrE1 gene, indicating that this gene may be present not only in human clinical isolates but also in clinically significant Enterobacteriaceae of animal origin.

Accession number(s).

The complete nucleotide sequence of pKp41M was deposited in GenBank under accession number KY781949.