Prevalence and Plasmid Characterization of the qnrD Determinant in Enterobacteriaceae Isolated from Animals,Retail Meat Products,and Humans

Introduction

Fluoroquinolones have been used to the treatment of bacterial infections for several decades, and the spread of quinolone resistance mediated by chromosome- or plasmid-mediated elements has occurred around the world in a variety of Enterobacteriaceae. ²⁰ The main mechanism of quinolone resistance is the accumulation of mutations in the genes encoding the subunits of the drug's target enzymes, DNA gyrase and DNA topoisomerase. ⁵ However, quinolone resistance carried by mobile genetic elements is increasingly recognized.^17,20 As of yet, three types of plasmid-mediated quinolone resistance (PMQR) determinants have been identified: Qnr (QnrA, QnrB, QnrC, QnrD, and QnrS),^2,4,7,8,25 QepA, ²⁸ and AAC(6′)-Ib-cr. ¹⁶ qnrD, unlike other qnr genes, is mainly located on small nonconjugative plasmids.^2,3,11 Since the first report of qnrD on a 4,270-bp plasmid in Salmonella, ² the qnrD determinant has been reported by several other studies.^6,13,30,31 Recently, a novel qnrD-carrying plasmid around 2,680-bp was identified in Providencia rettgeri, ³ Proteus mirabilis, and Morganella morganii. ¹¹ As of yet, most of the qnrD-positive strains were isolated from human clinical specimens, and there is still a paucity of information on qnrD gene of animal origin. In this study, we investigated 1,373 Enterobacteriaceae isolates from birds, swine, retail meat products, companion animals, and humans to understand the prevalence and dissemination of qnrD and its contribution to bacterial antimicrobial resistance.

Materials and Methods

Results and Discussion

PMQR has become a significant concern for clinical treatments, as these determinants reduce bacterial susceptibility to fluoroquinolone antibiotics and promote the emergence of resistant mutants. The PMQR determinants have been identified in a number of clinically important pathogens and environmental bacteria in different geographic regions of the world.^{10,17,18,20,24} qnrD, as a new qnr determinant, is increasingly reported. However, a large-scale investigation of qnrD distribution among different host species has not been reported. In this study, we screened qnrD-positive strains from 1,373 Enterobacteriaceae isolates from birds, swine, retail meat products, companion animals, and humans and characterized the qnrD-carrying plasmids. Our results identified 12 qnrD-positive strains (0.87%) among the 1,373 isolates (Table 1), with nalidixic acid MICs ranging from 8 to 512 (mg/L) and ciprofloxacin MICs from 0.25 to 2 (mg/L) (Table 2). The 12 strains positive for qnrD included five P. mirabilis, four E. coli, one Klebsiella pneumoniae, one Citrobacter freundii, and one M. morganii. PFGE analysis of four E. coli and five P. mirabilis revealed that these strains were divergent and not clone-related (Fig. 1), suggesting that the dissemination of qnrD was not due to clonal dissemination of qnrD-positive strains. These findings provide new data about PMQR distribution and improved understanding of prevalence and dissemination of qnrD.

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FIG. 1.

PFGE-fingerprinting patterns of total DNA preparations. Lanes: M, PFGE marker; 1, Proteus mirabilis CGP180; 2, P. mirabilis CGP248; 3, P. mirabilis CGS49; 4, P. mirabilis CGH15; 5, P. mirabilis CGH40; 6, Escherichia coli CGP246; 7, E. coli CGP169; 8, E. coli CGB40; 9, E. coli CGS13; PFGE,pulsed-field gel electrophoresis.

Unlike other qnr determinants that are usually located on large and variable plasmids, the qnrD-carrying plasmids typically have a simple backbone and are stably maintained. As observed in previous studies, the qnrD gene was found on plasmids of 2.7^3,11 or 4.2 kbp², respectively. Of the 12 qnrD-harboring plasmids identified in this study, the 2.7- and 4.2-kbp plasmids account for 41.7% and 58.3% of all plasmids, respectively. No relationship was seen between plasmid sizes and bacterial species (Table 1). The complete sequences of the qnrD-positive plasmids matched with p2007057² (4,270 bp) and pT80¹¹ (2,687 bp) (accessions numbers FJ228229 and JN183060). The results showed that the qnrD genes in the 12 plasmids were identical. Among the seven 4.2k-bp plasmids harboring the qnrD gene, pCGP246, pCGP169, pCGP180, pCGH25, and pCGH40 exhibited 100% identity with p2007057, whereas pCGB40 and pCGF41 exhibited 99% identity with p2007057. Of the five qnrD-positive 2.7-kbp plasmids, pCGS13 exhibited 100% identity with pT80, while pCGP248, pCGS49, pCGH15, and pCGH69 were identical and exhibited 99% identity with pT80.

Although only two qnrD-positive-harboring plasmids (4.2 and 2.7 kbp) have been reported, the qnrD plasmids were detected in bacterial strains isolated from a variety of sources, including human-, animal-, and environmental origin,^{3,11,13,30,31} in which other qnr plasmids were also identified. Interestingly, qnrD plasmids seem to be more common in Proteeae. Zhao and Dang investigated the prevalence of qnr determinants in coastal seawater and found that qnrD was the predominantly prevalent qnr determinants (27%), which were all carried by P. vulgaris. ³¹ The present study revealed that six (50%) qnrD-positive strains were Proteeae, further supporting the notion that Proteeae are an essential source or carrier in the spreading of qnrD-harboring plasmids. In this study, five of the qnrD-positive strains also carried the gene aac(6′)-Ib-cr, and one was also positive for qnrS, while qnrA, qnrB, and qepA were absent in all of them (Table 1). The gyrA and parC mutations were detected in 3 and 1 strains, respectively (Table 1). In the study of Zhao and Dang, however, all 22 Proteus vulgaris isolates carrying qnrD had a single mutation in the gyrA gene, but not other PMQR determinants. ³¹

For cross-environmental transmission of different plasmids, mobility is considered one of the most important factors.^1,9,15,20 Due to lack of their own set of mating pair formation (MPF) genes, the qnrD plasmids were nonconjugative. No transconjugants were obtained despite of several attempts in this study, which is in accordance with previous studies on the qnrD gene using the liquid- or filter-mating assays.^3,31 Cavaco et al. have speculated that ORF4 in the 4.2-kbp qnrD plasmid might function as a mob-like gene, ² and if it obtains other MPF genetic elements present in the same cell, it may gain the mobility. It is surprising that no mob-like genes or oriT sequence were identified in the 2.7-kbp plasmids. ³ Nevertheless, 12 qnrD-positive transformants were obtained on SOB agar plates containing 0.06 mg/L ciprofloxacin in the electroporation experiments conducted in this study. The qnrD-positive transformants showed 4-fold to 8-fold, 64-fold to 128-fold, and 16-fold increases in the MICs of nalidixic acid, ciprofloxacin, and ofloxacin, respectively, when compared with the recipient strain (Table 2). This result suggests that the qnrD-carrying plasmids can be transferred by nonconjugative means and may be spread by natural transformation or transduction. Under certain stressful environments, bacteria are able to establish naturally a physiological state to take-up DNA,^12,14 which may be responsible for the spread of the qnrD-carrying plasmids.