JAC Advance Access published online on February 16, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkl547
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Prevalence of qnr genes in Salmonella in France
1 Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI, K.-Bicêtre, France 2 Service de Bactériologie-Virologie-Hygiène, Hôpital Henri Mondor, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine de Créteil, Université Paris XII, Créteil, France 3 Centre National de Référence des Salmonella, Unité Biodiversité des Bactéries Pathogènes Emergentes, Institut Pasteur, Paris, France
* Correspondence address. Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78, rue du Général Leclerc, 94275 Le Kremlin-Bicêtre, France. Tel: +33-1-45-21-36-32; Fax: +33-1-45-21-63-40; E-mail: nordmann.patrice{at}bct.ap-hop-paris.fr
Received 30 November 2006; returned 11 December 2006; revised 14 December 2006; accepted 15 December 2006
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OBJECTIVES: To detect the qnrA, qnrB and qnrS genes among Salmonella isolates received at the French National Reference Centre for Salmonella in Paris, France.
METHODS: Antibiotic susceptibility was determined by disc diffusion for 499 Salmonella isolates including 320 Salmonella Typhimurium, 100 Salmonella Enteritidis and 79 Salmonella Hadar collected in 2002. Amplification with specific primers of qnrA, qnrB and qnrS genes was performed for all Salmonella Typhimurium, Salmonella Enteritidis and Salmonella Hadar isolates resistant to quinolones and for 17 additional isolates that produced expanded-spectrum ß-lactamases (ESBLs).
RESULTS: Prevalence of quinolone resistance was 3.75%, 11% and 79.7% for Salmonella Typhimurium, Salmonella Enteritidis and Salmonella Hadar serovars, respectively. A single isolate (0.2%) was qnrA-positive (QnrA1 determinant) being a Salmonella serovar Concord carrying also the ESBL gene blaCTX-M-15. This strain was probably from East Africa. No qnrB or qnrS genes were identified.
CONCLUSIONS: Whereas plasmid-mediated quinolone resistance of the Qnr type is emerging in Enterobacteriaceae worldwide, it remains rare in Salmonella in France.
Key Words: serovar Concord , qnrA1 , CTX-M-15
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Salmonella constitutes an important cause of human food-borne gastroenteritis, which may be treated with fluoroquinolones in the case of immunocompromised patients or systemic infections. Although resistance to fluoroquinolones remains rare in Salmonella, reduced susceptibility is increasing worldwide.1,2 It has been suggested that this may be the result of selection of animal isolates and their transfer to humans via the food chain.3 A European survey performed in 2000 showed that resistance to ciprofloxacin remains rare in Salmonella (0.5%) but resistance to nalidixic acid associated with decreased susceptibility to ciprofloxacin (MICs from 0.25 to 1.0 mg/L) was found in 14% of isolates (n = 27 059).4
Acquired resistance to quinolones mainly results from chromosomal mutations responsible from modifications of DNA gyrase and topoisomerase IV and from a decrease in drug accumulation in bacteria mainly as a result of the overexpression of efflux pumps (e.g. AcrAB-TolC system).2 Recently, plasmid-mediated resistance of Qnr type (QnrA, B and S) has been identified in several enterobacterial species including Salmonella spp.5–8 Qnr are pentapeptide repeat proteins that may prevent quinolones from binding to DNA topoisomerases. These proteins confer resistance to nalidixic acid and reduced susceptibility to fluoroquinolones.8
The aim of this study was to determine the spread of qnrA, qnrB and qnrS genes in human Salmonella isolates obtained from the French National Reference Centre for Salmonella (NRC-Salm) collected in 2002. We report here a Salmonella enterica serovar Concord isolate harbouring the QnrA determinant.
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Bacterial isolates
In 2002, a total of 11 775 human Salmonella isolates were registered at the NRC-Salm and 74.9% of them belonged to the three most frequently encountered serovars in France: Enteritidis (37.9%, n = 4469), Typhimurium (33.9%, n = 3998) and Hadar (2.4%, n = 282).9 Out of those isolates, 499 representative strains (320 Salmonella Typhimurium, 100 Salmonella Enteritidis and 79 Salmonella Hadar) were available in the NRC-Salm and were studied. Moreover, because of frequent association of genes coding for expanded-spectrum ß-lactamases (ESBLs) and qnr genes, all Salmonella isolates resistant to extended-spectrum cephalosporins (n = 17) were also tested. These 17 additional isolates collected from 2001 to 2005 belonged to the following serovars: Typhimurium (n = 3), Virchow (n = 3), Newport (n = 2), Concord (n = 2), Paratyphi B (n = 1), Livingstone (n = 1), Bareilly (n = 1), Enteritidis (n = 1), Agona (n = 1), Panama (n = 1) and Westhampton (n = 1). Escherichia coli ATCC 25922 and azide-resistant E. coli J53 were used as the control for susceptibility testing and as the host in conjugation experiments, respectively. E. coli Lo, Klebsiella pneumoniae Kp25 and E. coli (pBCH2.6) were used as qnrA-, qnrB- and qnrS-positive controls, respectively.
Antibiotic susceptibility was determined by disc diffusion on Mueller–Hinton agar according to CLSI guidelines.10 The discs were supplied by Bio-Rad Laboratories (Marnes-la-Coquette, France) and the following antibiotics were tested: amoxicillin (25 µg), ticarcillin (75 µg), amoxicillin/clavulanate (20/10 µg), ticarcillin/clavulanate (75/10 µg), piperacillin (75 µg), piperacillin/tazobactam (75/10 µg), cefalotin (30 µg), cefuroxime (30 µg), cefoxitin (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), aztreonam (30 µg), latamoxef (30 µg), cefepime (30 µg), imipenem (10 µg), kanamycin (30 IU), tobramycin (10 µg), gentamicin (15 µg), netilmicin (30 µg), amikacin (30 µg), nalidixic acid (10 µg), norfloxacin (5 µg), ofloxacin (5 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), tetracycline 30 (IU), fosfomycin (50 µg), rifampicin (30 µg), sulfamethoxazole (200 µg), trimethoprim (5 µg) and colistin (50 µg). MICs of quinolones, fluoroquinolones and ß-lactams were determined by using the Etest method according to the manufacturer's recommendations (AB Biodisk, Solna, Sweden). MIC breakpoints used for susceptibility and resistance to nalidixic acid and ciprofloxacin were 
8 and 
32 mg/L and 1 and 4 mg/L, respectively, as recommended by the CLSI.10
PCR amplification and sequencing
Amplification of the qnrA, qnrB and qnrS genes was performed for all Salmonella Typhimurium, Salmonella Enteritidis and Salmonella Hadar isolates categorized as intermediate or resistant to nalidixic acid and/or ciprofloxacin and for the 17 ESBL-positive isolates. Genomic DNA was extracted using the InstaGene matrix kit (Bio-Rad, Marnes-la-Coquette, France) in accordance with the manufacturer's recommendations. PCR experiments were carried out according to standard conditions (annealing temperature at 55°C, extension 1 min at 72°C, 35 cycles) using primers synthesized by Proligo France SAS. Primers used were as follows: for qnrA, QnrA-A (5'-GGGTATGGATATTATTGATAAAG-3') and QnrA-B (5'-CTAATCCGGCAGCACTATTA-3') to give a 660 bp product; for qnrB, QnrBm-F (5'-GGMATHGAAATTCGCCACTG-3') and QnrBm-R (5'-TTTGCYGYYCGCCAGTCGAA-3') (able to amplify the qnrB1 to qnrB6 variants) to give a 264 bp product; and for qnrS, QnrS-A2 (5'-AGTGATCTCACCTTCACCGC-3') and QnrS-B2 (5'-CAGGCTGCAATTTTGATACC-3') to give a 550 bp product. The quinolone-resistance determining regions (QRDRs) of gyrA, gyrB, parC and parE genes of the qnrA-positive isolate were amplified using previously described primers.11 Detection and identification of ESBL was performed for the qnrA-positive strain (and its transconjugant) using specific primers of sequences encoding TEM-, SHV- and CTX-M-type ESBLs, as previously described.9, 12 After PCR amplification, DNA fragments were purified with the Qiaquick PCR purification kit (Qiagen, Courtaboeuf, France). Both strands of the amplification products obtained were sequenced with an ABI 3100 sequencer (Applied Biosystems, Foster City, CA, USA). The nucleotide and deduced protein sequences were analysed with software available over the Internet at the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov).
Conjugation, plasmid analysis and PCR mapping
Conjugation experiments using an azide-resistant E. coli J53 (AziR) as recipient were performed in liquid and solid culture media. Transconjugants were selected on Trypticase soy (TS) agar plates containing sodium azide (100 mg/L) plus amoxicillin (50 mg/L) or nalidixic acid (16 mg/L). Plasmid analysis of the qnrA-positive strain and its E. coli transconjugants was performed by using the Kieser technique followed by agarose gel electrophoresis analysis.13 The sequence of the DNA adjacent to the qnrA gene was determined by PCR mapping using different primers sets. Primers specific for the qnrA gene (see above) were used in combination with primers 3'CS (5'-AAGCA GACTTGACCTGA-3'), 5'CS (5'-GGCATCCAAGCAGCA AG-3'), ORF513-D3 (5'-CTCACGCCCTGGCAAGGTTT-3'), ORF513-D5 (5'-CTTTTGCCCTAGCTGCGGT-3') and ampR- 5'ext (5'- GCGGGTAAAACTGAGATGAC-3').
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Prevalence of quinolone resistance
Twelve out of the 320 isolates of Salmonella Typhimurium isolates (3.75%) were resistant to nalidixic acid (with MICs of ciprofloxacin ranging from 0.25 to 0.5 mg/L) and only a single isolate (0.3%) was resistant to fluoroquinolones (MIC > 32 mg/L). Out of the 100 isolates of Salmonella Enteritidis and 79 of Salmonella Hadar, 11 (11%) and 63 (79.7%) isolates were resistant to nalidixic acid, respectively. Among the 17 isolates resistant to extended-spectrum cephalosporins, only three were resistant to nalidixic acid and none to ciprofloxacin.
As reported in the literature, we found that resistance to nalidixic acid is mostly in Salmonella Hadar (57%) and is less frequently observed in the two most frequently isolated serovars Enteritidis and Typhimurium (13% and 8%, respectively).4 We also reported herein that high-level resistance to fluoroquinolones remains very uncommon in Salmonella, generally lower than 1% of isolates.2,4
For all quinolone-resistant isolates of Salmonella Typhimurium (n = 13), Salmonella Enteritidis (n = 11) and Salmonella Hadar (n = 63), screening gave negative results for qnrA, qnrB and qnrS genes. Among the 17 strains resistant to extended-spectrum cephalosporins, one isolate had a qnrA-like gene but none of these was positive for the qnrB and qnrS genes. This isolate (05-3728) was obtained from two children from the same family and belonged to the serovar Concord. Isolate 05-3728 was recovered in June 2005 from stool of a 2.5-year-old child hospitalized for severe acute gastroenteritis. This episode appeared 2 months after the arrival in his family of an adopted 7-month-old girl from Ethiopia. Even though she was asymptomatic, a rectal swab screening was performed that led to isolation of an isolate presenting an antibiotic resistance pattern identical to that of the isolate 05-3728. Molecular typing by PFGE gave similar patterns for these two isolates and confirmed clonal relatedness between them (data not shown). Interestingly, no quinolone-based treatment had been previously given to these children.
Out of the 516 tested isolates, only a single isolate was positive for qnrA (0.2%). An earlier study, performed in South Korea, did not identify the qnrA gene among 261 nalidixic acid-resistant and community-acquired Salmonella spp. isolates.1 In the second study, 10 out of 335 Salmonella human clinical isolates from the USA were positive for either qnrB or qnrS but none was qnrA-positive.5 An that latter study, several qnrB variants were detected in seven Salmonella Berta isolates (qnrB5) and one Salmonella Mbandaka isolate (qnrB2), whereas qnrS variants were found in isolates of Salmonella Anatum (qnrS2) and Salmonella Bovismorbificans (qnrS1).5 All of these 10 isolates were resistant to nalidixic acid with decreased susceptibility to ciprofloxacin. In addition, QnrA-like determinants were detected in four unrelated clinical isolates of Salmonella Enteritidis in Hong Kong that harboured a blaCTX-M-14 gene and QnrS1 determinant was recovered in a Salmonella Infantis of avian origin in Germany.6,7
Characterization of the qnrA-positive Salmonella Concord isolate
Isolate 05-3728 was resistant to all ß-lactams, except carbapenems and cephamycins, and exhibited an ESBL phenotype. PCR and sequencing revealed that it harboured the qnrA1 and the blaCTX-M-15 genes. Thus, like most qnrA-positive enterobacterial isolates reported worldwide, this qnrA-positive isolate expressed an ESBL.8,12 It was resistant to several aminoglycosides (kanamycin, gentamicin and tobramycin) but remained susceptible to amikacin and netilmicin. In addition, it was also resistant to chloramphenicol, tetracycline, rifampicin, sulphonamide and trimethoprim, and remained susceptible to fosfomycin. Finally, it was resistant to nalidixic acid (MIC >32 mg/L) but remained susceptible to ciprofloxacin (MIC 0.38 mg/L) (Table 1).
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Interestingly, no mutation linked to quinolone resistance was detected in the QRDRs of gyrA, gyrB, parC and parE genes of this isolate. Quinolone resistance was transferred to E. coli J53 AziR by conjugation. The transconjugant exhibited a similar pattern of resistance for almost all antimicrobial agents, in particular to quinolones (Table 1), but was susceptible to aminoglycosides. PCR and sequencing confirmed that the transconjugant carried blaCTX-M-15 and qnrA1 genes. Plasmid analysis revealed that isolate 05-3728 contained five plasmids with estimated sizes of approx. 200, 100, 50, 15 and 5 kb whereas the transconjugant contained a single qnrA1(+) 200 kb plasmid (data not shown). The qnrA1 gene was located on a sul1-type class 1 integron that was identical to the In36 integron identified in E. coli isolates from Shanghai.14 Briefly, the orf513 gene was found immediately upstream of the qnrA1 gene, and an ampR sequence coding for a LysR-type regulatory element was identified downstream.
We report here the prevalence of plasmid-mediated quinolone resistance in Salmonella in France. Only a single isolate (0.2%) was positive for the qnrA gene and none was positive for qnrB and qnrS genes. These results may indicate a low-level rate of dissemination of Qnr determinants among human Salmonella isolates.
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None to declare.
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We thank all the corresponding laboratories of the French National Reference Centre for Salmonella network. This work was funded by a grant from the Ministère de l'Education Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, France and mostly by grants from the European Community (6th PCRD, LSHM-CT-2005-018705). L. P. is a researcher from the INSERM (Paris, France).
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4 . Threlfall EJ, Fisher IS, Berghold C, et al. (2003) Antimicrobial drug resistance in isolates of Salmonella enterica from cases of salmonellosis in humans in Europe in 2000: results of international multi-centre surveillance. Euro Surveill 8:41–5.[Medline]
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