Occurrence of the Plasmid-Mediated Fluoroquinolone Resistance qepA1 Gene in Two Clonal Clinical Isolates of CTX-M-15-Producing Escherichia coli from Algeria

Introduction

Apart from chromosomal mutations in the genes encoding DNA gyrase and Topoisomerase IV (mainly gyrA and parC) and mutations leading to decreased permeability or overexpression of efflux pumps in quinolone resistance, plasmid-encoded resistance emerged in the 2000s with three mechanisms known so far: Qnr peptides (QnrA, QnrB, QnrC, QnrD, QnrS, and QnrVC), the aminoglycoside acetyltransferase variant AAC(6′)-Ib-cr, and QepA and OqxAB efflux pumps. ¹ The QepA determinant was first discovered in 2007 in two Escherichia coli clinical isolates from Japan ² and Belgium. ³ This is a plasmid-mediated efflux pump in the major facilitator superfamily that reduces susceptibility to hydrophilic fluoroquinolones, specifically ciprofloxacin and norfloxacin.^2,3 After the initial discovery, a variant of qepA possessing two amino acid substitutions has been identified and named QepA2. ⁴ Wang et al. recently identified a new qepA allele, qepA3, in China. ⁵

Worldwide, the QepA1 determinant has rarely been reported in human isolates. In North Africa, the qepA gene has been described in two (6.6%) clinical E. coli isolates from Egypt ⁶ ; no qepA-positive isolates have been reported to date in Algeria. Indeed, no QepA determinant has been reported among a collection of E. coli ciprofloxacin-resistant strains isolated from chickens, their farmers, and patients from 2011 to 2012 in northeast Algeria. ⁷ This study is the first report of the plasmid-mediated fluoroquinolone efflux pump, QepA, found in clinical isolates of E. coli with high-level fluoroquinolone resistance in the university hospital center of Oran (northwest Algeria). The genetic environment of qepA is also characterized.

Materials and Methods

Forty-nine E. coli isolates nonsusceptible to ciprofloxacin were collected at the university hospital of Oran (Algeria) in 2011 and studied. The isolates were recovered from various clinical samples (urine [n = 22], pus [n = 12], blood cultures [n = 8], sperm [n = 2], bronchial [n = 2], central catheter [n = 1], pleural fluid [n = 1], and ocular fluid [n = 1]) and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF Biotyper 3.1; Microflex BRUKER, Madrid, Spain). Antibiotic susceptibility was determined by the disk diffusion method on Mueller–Hinton agar plates; minimum inhibitory concentrations (MICs) of fluoroquinolones were determined by the microdilution method. The results were interpreted according to CLSI guidelines. ⁸

Screening for plasmid-mediated quinolone resistance (PMQR) genes: qnr (A, B, S, C, D), aac(6′)Ib-cr, qepA, and oqxAB, was carried out by PCR using specific primers, described previously. ⁹ Mutations in gyrA and parC genes were analyzed by amplification and sequencing of the quinolone resistance-determining region (QRDR). ¹⁰

Detection of beta-lactamases was by the double-disk synergy test (DDST) ¹¹ on Mueller–Hinton agar with and without cloxacillin ¹² ; simplex PCR was used for the detection of bla_CTX-M-1, bla _CTX-M-9 , bla_TEM, and bla_SHV genes ¹³ and multiplex PCR for ampC genes. ¹⁴ The presence of the rmtB gene was determined by PCR, as previously described. ¹⁵ All amplicons were sequenced.

Conjugation experiments were performed in liquid culture media. Transconjugants were selected on Trypticase Soy (TS) agar plates containing sodium azide (150 mg/L) and ciprofloxacin (0.03 mg/L). Transformation experiments were performed by electroporation, and recombinant strains were selected onto ciprofloxacin-containing (0.03 mg/L) TS agar plates. Azide-resistant E. coli J53 and streptomycin-resistant E. coli DH10B were used as recipient strains for conjugation and transformation assays, respectively. To determine the clonal relatedness of qepA-positive E. coli isolates, XbaI pulsed-field gel electrophoresis (PFGE) analysis was performed using a CHEF-DR-II system (Bio-Rad, Hemel Hempstead, United Kingdom) (www.pulsenetinternational.org/protocols/pfge.asp).

The genetic structures surrounding the qepA gene in the E. coli isolates were cloned by restricting total DNA with XbaI for the O59 isolate and with XhoI for the O80 isolate overnight at 37°C, then ligated into the pBK-CMV vector, and the recombinant plasmids were transformed into E. coli DH10B by electroporation. Recombinant plasmids were selected onto TS agar plates containing ciprofloxacin (0.03 mg/L) and kanamycin (30 mg/L), and the recombinant plasmid DNA was extracted with a Qiafilter Plasmid Maxi Kit (Qiagen). Primer walking was performed to identify the genetic structures surrounding the qepA gene using universal primers T3 and T7 (Macrogen Europe Laboratory, the Netherlands).

Results

Among 49 E. coli clinical isolates analyzed, 20 (41%) were positive for at least 1 PMQR gene, including qnrS1-like (n = 1), aac(6′)Ib-cr (n = 16), aac(6′)Ib-cr + qnrB1-like (n = 1), qepA1 (n = 1), and qepA1 + qnrB1-like (n = 1). To the best of our knowledge, this is the first report of the occurrence of qepA gene in isolates from Algeria. This study focuses on two qepA1-positive E. coli isolates, O59 and 080, recovered from the urine of 48- and 60-year old female patients who were hospitalized in nephrology and surgical intensive care unit services, respectively. These multidrug-resistant isolates harbored bla_TEM-1, bla_CTX-M-15, and rmtB genes (Fig. 1). They were resistant to cefoxitin, with no increase in susceptibility on Mueller–Hinton agar with cloxacillin in the DDST, and were negative for plasmid-mediated AmpC genes tested by PCR, suggesting chromosomal cephalosporinase production. The QRDR sequences of gyrA and parC showed that the qepA-positive E. coli isolates carried two substitutions in GyrA (Ser83leu and Asp87Asn) and one substitution in ParC (Ser80Ile) with a high MIC of ciprofloxacin of >256 mg/L. Cluster analysis of the PFGE image of restriction fragments revealed high genetic similarity (89%) between qepA-positive E. coli isolates, showing clonal relatedness. These qepA genes were not transferable by conjugation or transformation, despite several attempts. Ciprofloxacin MIC values for clones obtained from the E. coli DH10B recipient were increased by at least 60-fold (Table 1). Sequence analysis revealed that the IS26 element followed by a truncated intI1 and dfr2 gene (class 1 integron-integrase and trimethoprim resistance genes, respectively) was located upstream of the qepA1 gene. Downstream, this gene was associated with the ISCR3C-like element.

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FIG. 1.

Dendrogram of patterns generated by pulsed-field gel electrophoresis of qepA-positive Escherichia coli isolates. PMQR, plasmid-mediated quinolone resistance.

Discussion

In this study, the QepA efflux pump was reported for the first time in Algeria in two (4% prevalence) clinical E. coli strains from 2011. This PMQR determinant has been described worldwide with low prevalence in humans, animals, and the environment in E. coli isolates^6,16 and has been associated with other PMQR determinants or antibiotic markers, such as bla_TEM, bla_CTX-M, and rmtB. In this study, qepA1 gene was found in TEM-1- and CTX-M-15-producing E. coli isolates; these isolates and their clones were also resistant to aminoglycosides with the presence of the rmtB gene. Inclusion of these isolates in ST131 clone was discarded by O25B allele characterization (data not shown). Previous studies have confirmed that the spread of rmtB and qepA is mainly mediated by similar F2:A-:B- plasmids. ¹⁶ The presence of the qepA gene is very rare in human samples. ¹⁷ In our report, the two qepA-positive E. coli isolates, which were isolated in different services of the same hospital in the same year but at an interval of 1 month, were found to be clonally related. The genetic structures surrounding the qepA1 gene revealed that this determinant was flanked by IS26 and ISCR3C elements associated with the bla_TEM-1 gene, previously described by Yamane et al. ² and Périchon et al. ³ in 2007. Wang et al., ⁵ however, reported that the qepA3-positive isolates contained either bla_CTX-M-14 or bla_TEM-12 genes, rather than the bla_TEM-1 gene.

The qepA gene is described for the first time in two clonally related ESBL-positive E. coli clinical isolates from Algeria. The clonal spread of these multidrug-resistant isolates is a major public health concern.