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First Case of Qnr B6 and Qnr B7 Genes in Enterobacteriaceae Producing Extended-Spectrum Beta-Lactamases in Abidjan, Côte D'ivoire

Aims: The aim of this study was to characterize fluoroquinolone resistance genes in enterobacteriaceae that produce extended-spectrum β-lactamases, isolated in Abidjan. Place and Duration of Study: Pasteur Institute of Côte d'Ivoire and research unit on emerging tropical infectious diseases of Aix-Marseille University from January 2017 at July 2017. Methodology: The study included 90 enterobacteriaceae producing extended-spectrum βlactamases isolated from biological products from various hospital services in Abidjan. These strains have been pre-identified and stored at the Center for Biological Resources (CeReB) of Pasteur Institute of Côte d'Ivoire. The identification of the strains was confirmed using the mass Original Research Article Victoire et al.; SAJRM, 3(2): 1-6, 2019; Article no.SAJRM.48002 2 spectrometry MALDI-TOF (MS) and the antibiotic sensitivity test was performed using Müeller Hinton's agar diffusion method. The fluoroquinolone resistant genes were detected by conventional PCR and then, sequenced. Results: The strains studied were Escherichia coli (44), Klebsiella pneumoniae (31) and Enterobacter cloacae (15). High resistance rates to ceftriaxone (96.7%), cefotaxime (95.6%), aztreonam (95.6%) and cefoxitin (72.2%) were observed in all strains producing broad spectrum βlactamases. The resistance rate to fluororquinolones represented by ciprofloxacin was 86.7%. The fluoroquinolone resistance genes detected were qnr A (3.3%) and qnr B (42.2%). Sequencing identified the qnr A1 (3.3%), qnr B1 (31.1%), qnr B6 (2.2%) and qnr B7 (1.1%) genes. Conclusion: This study made it possible to identify fluoroquinolone resistance genes in enterobacteriaceae producing β-lactamases which have an extended spectrum in Abidjan.


INTRODUCTION
Quinolones are widely used antibiotics in the treatment of various infections [1]. Quinolones are generally characterized by a broad spectrum of activity, a good oral bioavailability and a good tissue penetration [2] while fluoroquinolones, are characterized by the presence of a fluorine atom in position 6 and a nitrogen ring, and most often by the presence of a piperazine in position 7 [3]. Their main targets are DNA gyrase and topoisomerase IV DNA [4].
Fluoroquinolones interact with the DNA-enzyme complex, i.e. with the DNA gyrase which is bound to bacterial DNA or with the topoisomerase IV, bound to bacterial DNA to create conformational changes. The new fluoroquinolone-enzyme-DNA complex blocks the progression of the replication fork, resulting in the inhibition of enzymatic activity and DNA synthesis [5,6]. Several mechanisms are involved in fluoroquinolone resistance. These are the mutational modifications of target enzymes, the reduction of membrane's permeability, the reduction of intracellular antibiotic concentration by efflux systems and the action of the QNR protein [7]. The qnr gene that codes for the QNR protein is the genetic determinant of plasmid resistance to fluoroquinolones [8]. The importance of this genetic support is its transferability and its ability to accelerate the spread of fluoroquinolone resistance. Qnr genes have been identified in different strains of enterobacteriaceae and often associated with the production of extended-spectrum betalactamases [4]. This situation is at the root of therapeutic failures and the increase in morbidity and mortality rates worldwide [9]. The objective of this study was to characterise fluoroquinolone resistance genes in enterobacteriaceae producing extended-spectrum β-lactamases isolated in Abidjan, Côte d'Ivoire.

Confirmation of the Identity of Strains by MALDI-TOF
The strains preserved in deep agars were revived using a enrichment broth which were incubated at 37°C for 24 hours in an oven (Thermo Fisher). The strains' isolation was performed on Mac-Conkey agar and their re-identification was confirmed by mass spectrometry (MALDI-TOF) at the laboratory of the Emerging Tropical Infectious Diseases Research Unit at Aix-Marseille University in France.

Research of Fluoroquinolone Resistance Genes by PCR
The strains' DNA was extracted using the EZ1 extraction kit (Qiagen) as recommended by the manufacturer. The search for the qnr A and qnr B genes was carried out by conventional PCR. The amplification reaction was performed in a reaction volume of 25 μL composed of 12.5 μL Master Mix (Quantitect Probe PCR Master mix, Qiagen), 1 μL forward and reverse primer (Eurogentec), 5 μL total DNA and 6.5 μL ultrapure water (Invitrogen).The primers of the fluoroquinolone resistance genes used in this work have been summarized in Table 1.
The amplification of genes by conventional PCR consisted of an initial DNA denaturation step at 95°C for 15 min. This step was followed by 35 amplification cycles including a denaturation at 94°C for 1 min, a hybridization at 55°C for 50 s, an elongation at 72°C for 2 min and a final elongation step of 7 min at 72°C. The amplification products were analyzed by 1.5% agarose gel electrophoresis prepared with 0.5% Tris-Borate-EDTA (TBE) and 3.75% SYBR SAFE. The DNA bands of the amplicons were visualized on a transilluminator.

DNA Sequencing
The amplicons were purified and sequenced using the BigDye® kit (Life technologies) as recommended the manufacturer in an automate ABI PRISM 3730xl Genetic Analyser PLC. In addition, genes' identification was carried out in the ARG-ANNOT (Antibiotic Resistance Gene Annotation) database of the IHU-Marseille in France.
The analysis of the results of the susceptibility testing of Escherichia coli strains to antibiotics showed that for antibiotics of the β-lactam family, 100% of the strains were resistant for amoxicillin and for amoxicillin-clavulanic acid. The cephalosporin resistance rate was 98% and 100% for cefotaxime and ceftriaxone respectively. All strains were susceptible to imipenem, however 27.3% of strains were resistant to ertapenem. The ciprofloxacin resistance rate was 95.4%.
In Klebsiella pneumoniae strains, cephalosporins resistance rate was 71% for cefoxitin, 96.8% for cefotaxime and ceftriaxone respectively. In this species too, all strains were sensitive to imipenem, however 35.5% of strains were resistant to ertapenem. The resistance rate of K. pneumoniae strains was 100% to amoxicillin clavulanic acid. In addition, the ciprofloxacin resistance rate was 74.2%.
For Enterobacter cloacae strains, the resistance rate to amoxicillin clavulanic acid and cefoxitin was 100% while 86.7% of strains were resistant to cefotaxime and ceftriaxone. All strains of E. cloacae were susceptible to imipenem, however 33.3% of the strains were resistant to ertapenem. Table 2 summarizes the antibiotic resistance rates in the different species studied.

DISCUSSION
Fluoroquinolones act at the time of DNA replication. Their targets are DNA gyrase and topoisomerase IV, which regulate the topology of DNA to allow replication [11]. The resistance to fluoroquinolones in enterobacteriaceae is generally the result of a chromosomal mutation causing the alteration of bacterial target enzymes [4]. However, resistance caused by plasmids has also been reported as a result of the acquisition of resistance genes qnr, qepA, and aac(6')-Ib-cr [12,13]. Plasmids carrying the qnr A and qnr B genes frequently carry resistance genes to βlactam, aminoglycosides, and tetracycline [4].
In this work, the fluoroquinolones resistance rate represented by ciprofloxacin in enterobacteriaceae producing extended spectrum β-lactamases was 86.7%. This rate is higher than that reported by Guessennd et al. [14] who, in their work, showed that70.2% of the strains producing extended spectrum βlactamases were resistant to ciprofloxacin. Ouattara et al. [15] reported a 93.2% ciprofloxacin resistance rate in strains producing broad spectrum β-lactamases.
The high resistance rates could be explained by the fact that fluoroquinolones are the most prescribed molecules after β-lactam in Africa and particularly in Côte d'Ivoire [16]. These results are agree with those obtained by some authors in Africa. Indeed, in the Central African Republic, the results of the work of Rafai et al. [17] showed that 84.8% of the broad spectrum strains, producing β-lactamases tested were   [18] reported that 80% of the strains producing broad spectrum βlactamases were resistant to ciprofloxacin. In Algeria, Mathlouthi et al. [19] reported that 80% of strains producing extended spectrum βlactamases tested in their work were resistant to ciprofloxacin.
The qnr genes detected included qnr B gene which was detected at a rate of 42.2% followed by the qnr A gene (3.3%) in strains producing extended spectrum β-lactamases. The sequencing carried out made it possible to identify in addition to the qnr A1 (3.3%) and qnr B1 (31.1%) genes, the qnr B6 (2.2%) and qnr B7 (1.1%) genes which are involved in the resistance to fuoroquinolone. Moreover, the qnr A1 and qnr B1 genes were reported in 2008 in Côte d'Ivoire [14]; however, this study is the first to report the presence of the qnr B6 and qnr B7 genes involved in fluoroquinolone resistance.
Elsewhere in the world, the qnr B6 gene has been detected in South Korea in a strain of Enterobacter aerogenes producing broad spectrum β-lactamases from a collection of 644 enterobacteriaceae from 12 clinical laboratories [20]. Similarly, in Argentina, Cruz et al. [21] reported the presence of qnr B6 in 5% of enterobacteriaceae that produce broad spectrum β-lactamase. Also, in Morocco, the qnr B6 gene was detected in 0.9% of strains producing extended spectrum β-lactamasess tested in the work of Jamali et al. [22].
The qnr B6 and qnr B7 genes were found in South Korea in a study of 347 enterobacteriaceae from two hospitals. These genes were detected respectively in a strain of K. pneumoniae and a strain of Citrobacter freundii [23].

CONCLUSION
This study showed a high level of resistance to fluoroquinolones in enterobacteriaceae producing extended-spectrum β-lactamases. The qnr B gene was the most detected (42.2%) followed by the qnr A gene (3.3%). The study showed as well the presence of the qnr B6 and qnr B7 genes for the first time in Côte d'Ivoire. Given the importance of fluoroquinolones in the treatment of many bacterial infections, the presence of resistance genes is a concern. Therefore, monitoring the prescription of antibiotic is necessary to limit the risk of spreading resistance genes.