Description of a 2,683-Base-Pair Plasmid Containing qnrD in Two Providencia rettgeri Isolates

Abstract

qnr genes are plasmid-mediated quinolone resistance genes mainly harbored on large conjugative multiresistant plasmids. The qnrD gene was recently observed in Salmonella enterica on a small nonconjugative plasmid (p2007057). We describe two strains of Providencia rettgeri harboring qnrD on nonconjugative plasmids. The plasmids were 99% identical, with 2,683 bp and four open reading frames, including qnrD, but exhibited only 53% identity with the plasmid found in S. enterica.

TEXT

The first qnr gene was observed in the late 1990s and described as a plasmid-mediated quinolone resistance gene (PMQR). Since then, five types of qnr genes have been reported: qnrA, qnrB, qnrC, qnrD, and qnrS. Their sequences are deposited at the following website (http://www.lahey.org/qnrStudies/). Most were observed in Enterobacteriaceae, located on large conjugative multiresistance plasmids, such as pMG252 seen in the original qnrA1-positive strain (14). qnrD was recently reported in four Salmonella enterica isolates (7) and one Escherichia coli isolate (22). In S. enterica, the gene was located on a small nonconjugative plasmid of 4,270 bp (p2007057), a location greatly different from that of the other qnr genes. We describe two clinical isolates of Providencia rettgeri harboring qnrD. We investigated the genetic support for qnrD in these two strains and showed that qnrD was harbored on a small nonconjugative plasmid which was unlike that found in the Salmonella isolates.

P. rettgeri DIJ09-518 was isolated from a stool specimen of a patient admitted in 2008 to the hematology ward of the university hospital in Dijon, France. P. rettgeri GHS09-09 was isolated from three urine specimens of a patient hospitalized in 2009 in the emergency unit at Hôpital Pitié-Salpétrière (Paris, France). Quinolone susceptibility phenotypes are shown in Table 1. bla_CTX-M-15 was detected in DIJ09-518. Random amplification of polymorphic DNA confirmed that the two isolates were non-clonally related strains (4). qnrD was detected by using real-time PCR (10). No transconjugant was obtained by either liquid or filter mating-out assays with the following recipient strains and selection conditions: sodium azide-resistant E. coli J53, Proteus mirabilis ATCC 29906 Rif^r (8), and E. coli C600 Rif^r selected on brain heart infusion agar plates containing sodium azide (100 μg/ml) or rifampin (250 μg/ml) and cefpirome (0.25 μg/ml) or ampicillin (100 μg/ml) or nalidixic acid (50 μg/ml) or ciprofloxacin (0.03 μg/ml and 0.06 μg/ml) (12).

Table 1.

Quinolone MICs determined by Etest for P. rettgeri and E. coli DH10B strains carrying qnrD

Strain	MIC (μg/ml)a
	NAL	NOR	CIP	OFX	LVF	MXF
P. rettgeri DIJ09-518	> 256	6	3	8	3	12
P. rettgeri GHS09-09	3	0.19	0.125	0.5	0.25	0.5
E. coli DH10B	1	0.032	0.002	0.016	0.006	0.03
E. coli DH10B/pDIJ09-518a	2	0.38	0.125	0.25	0.094	0.125
E. coli DH10B/pGHS09-09a	2	0.38	0.125	0.25	0.094	0.125
E. coli DH10B/p2007057b	2	0.125	0.125	0.25	ND	ND

^aMIC assays were performed with Etest according to EUCAST guidelines. NAL, nalidixic acid; NOR, norfloxacin; CIP, ciprofloxacin; OFX, ofloxacin; LVF, levofloxacin; MXF, moxifloxacin; ND, no data.

^bMICs reported by Cavaco et al. (7).

Southern blot hybridization studies with plasmid DNA extracts showed that qnrD was present on a small (about 2.5 kb) plasmid in the two strains (data not shown). After electroporation of the plasmid DNA extracts and selection on brain heart infusion (BHI) agar plates containing 0.06 μg/ml ciprofloxacin, two transformants harboring qnrD were obtained, E. coli DH10B/pDIJ09-518a and E. coli DH10B/pGHS09-09a.

Etest data were interpreted by following the EUCAST guidelines (http://www.eucast.org). The MICs of fluoroquinolones for the two transformants were similar and were comparable to MICs previously reported for E. coli DH10B transformed with p2007057, the qnrD-containing plasmid found in S. enterica isolates (7) (Table 1).

The entire nucleotide sequences of pDIJ09-518a and pGHS09-09a were determined (GenBank accession numbers HQ834472 and HQ834473). Both plasmids were 2,683 bp long. Their sequences were identical except for eight randomly distributed differences. They both contained four putative open reading frames (ORFs) with qnrD, which we arbitrarily named Orf1. The sequences were aligned with that of p2007057. pDIJ09-518a and pGHS09-09a exhibited 53% identity with p2007057. Of the five ORFs found on the latter plasmid, only two were similar to that of our plasmids, the qnrD ORF with 100% identity and Orf2 with 97% (Fig. 1). These results suggest that these plasmids might originate from the same parental strain or from a similar genetic event but that they were not simply transmitted from Salmonella to Providencia or vice versa.

Fig 1.

Map of plasmids pDIJ09-518a and pGHS09-09a (this study) compared to that of p2007057 (7). qnrD (positions 1,081 to 1,725) shares 100% sequence identity with qnrD harbored by p2007057; Orf2, spanning from position 1,924 to 2,107, is 97% identical to the Orf2 carried by p2007057; Orf3 (positions 2,546 to 121) and Orf4 (positions 407 to 790) are hypothetical proteins with no significant matches. Three regions located on the plasmid backbone share identity with Proteeae genomes: (i) the sequence spanning from position 1743 to 1964 shares 86% identity with the genome of P. mirabilis HI4320; (ii) the sequence from position 1796 to 2243 shares 89% identity with a nucleotide sequence encoding a conserved hypothetical protein in the Providencia rustigianii DSM 4541 genome; and (iii) the sequence from position 1 to 124 shares 92% identity with a nucleotide sequence encoding a hypothetical protein of Providencia stuartii ATCC 25827.

Since qnrD was not found to be self-transmissible, we speculated that qnrD could have spread either on mobilizable plasmids or on a transferable structure integrated into conjugative plasmids, such as those carrying other Qnr determinants (5, 6, 13, 15, 17, 19–21). Cavaco et al. described one of the ORFs in p2007057 as a putative mob gene (7), and so we searched for signatures of mobilizable plasmids. Apart from qnrD, no mob-like gene, R391-like element (1, 3), insertion sequence, or oriT sequence was identified in the P. rettgeri plasmids.

In silico analyses of Orf4 showed that the upstream sequence had several features of theta-type replicons (Fig. 2) (9): (i) an AT-rich region, (ii) iterons, (iii) three DnaA boxes, and (iv) two pairs of inverted repeats were identified using REPFIND (2). In addition, a putative promoter and transcription start site have been identified for Orf4 using BPROM (Softberry) and the promoter prediction by neural network method (18). In silico secondary structure prediction of Orf4 failed to identify any significant leucine zipper and helix-turn-helix motifs, which are characteristic of most Rep proteins. We propose that Orf4 encodes a Rep protein even though it has no similarity with the currently known Rep proteins. However, further direct evidence is needed to support this hypothesis.

Fig 2.

Sequence analysis of pGHS09-09a origin of replication. The %A+T content of each line (60 bp) is shown on the right side, and nucleotide numbering is shown on the left side. Putative −35 and −10 promoter regions, as well as the transcription start site (+1) of Orf4 are in bold. Start and stop codons are underlined. DnaA boxes are boxed. Inverted repeat 1 and 2 are boxed in arrows, shaded in gray and not shaded, respectively. Four direct repeats considered putative iterons are indicated by arrows.

BLAST comparison to sequenced microbial genomes revealed that three regions of the plasmids pDIJ09-518a and pGHS09-09a matched with genomes of Proteus and Providencia (Fig. 1). To our knowledge, qnr genes have rarely been observed in Proteeae, the only report being that of one strain of P. mirabilis carrying qnrA6 (5). We thus speculated that qnrD could be more closely related to the Proteeae bacteria than the other qnr genes and perhaps originated from them. Several factors lend weight to our suggestion that the plasmids belong to the Proteeae or have resided in Proteeae for a considerable length of time: (i) the high homology between two plasmids found in two clonally nonrelated strains of P. rettgeri, (ii) genomic BLAST results with three sequences matching with different Proteeae chromosomes, (iii) a plasmid backbone with no mobilization structure, and (iv) the fact that the G+C contents of pDIJ09-518a, pGHS09-09a, and qnrD (41.89%, 41.74%, and 38%, respectively) are close to those of Providencia spp. (38 to 40%) and Proteus spp. (38 to 39%) but not to that of S. enterica (48%). We investigated several reference type strains of Proteeae (Table 2) for qnrD and other known qnr genes using a previously described multiplex real-time PCR (10). They were all negative. However, since the taxonomy of the Proteeae species is still evolving (16), we suggest that qnrD could be harbored by one other as-yet-untested species or by a subspecies that has not so far been classified. Consequently, further testing will be necessary and should include clinical strains isolated after the 1980s, since the emergence of qnr genes is recent (11).

Table 2.

List of type strains of the tribe Proteeae tested for presence of qnr genes^a

Organism	Culture collection strain designation(s)
Providencia alcalifaciens	CIP 82.90, ATCC 9886
Providencia rettgeri	CIP 103182, ATCC 29944
Providencia rustigianii	CIP 103032
Providencia stuartii	CIP 104687, ATCC 29914
Providencia vermicola	CIP 108829, DSM 17385
Providencia heimbachae	CIP 103031, ATCC 35613
Morganella morganii subsp. morganii	CIP A231, ATCC 25830
Morganella morganii subsp. sibonii	CIP 103648, ATCC 49948
Proteus mirabilis	CIP 103181, ATCC 29906
Proteus hauseri	CIP 106868, ATCC 700826
Proteus myxofaciens	CIP 106872, ATCC 19692
Proteus penneri	CIP 103030, ATCC 33519
Proteus vulgaris	CIP 104989, ATCC 29905

^aAll of the type strains of the tribe Proteeae were acquired from the Pasteur Institute strain collection (Paris, France).

Nucleotide sequence accession numbers.

The nucleotide sequences reported in this work were deposited in the GenBank nucleotide database under accession number HQ834472 for pDIJ09-518a and HQ834473 for pGHS09-09a.