JAC Advance Access originally published online on February 6, 2007
Journal of Antimicrobial Chemotherapy 2007 59(4):804-805; doi:10.1093/jac/dkl554
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Correspondence |
First detection of plasmid-mediated quinolone resistance (qnrA and qnrS) in Escherichia coli strains isolated from humans in Scandinavia
1 National Food Institute, Technical University of Denmark, Bülowsvej 27, DK-1790 Copenhagen V, Denmark 2 Faculty of Life Sciences, University of Copenhagen, Institut for Veterinary Pathobiology, Stigbojlen 4, DK-1870 Frederiksberg C, Denmark 3 Statens Serum Institut (SSI), Artillerivej 5, DK-Copenhagen S, Denmark 4 Department for Clinical Microbiology, Hvidovre Hospital, Denmark
* Corresponding author. Tel: +45-72346269; Fax: +45-72346341; E-mail: lcaxx{at}food.dtu.dk
Keywords: E. coli , qnrB , UTIs , septicaemia
Plasmid-mediated quinolone resistance linked to qnr genes (A, B and S) has recently been discovered.1–4 These encode Qnr proteins that are members of the pentapeptide family and are able to protect topoisomerases and thus reduce their susceptibility to fluoroquinolones and increase the mutant selection window, therefore increasing the likelihood of selection of mutations.4,5 Plasmid-mediated fluoroquinolone resistance associated with qnr genes was first detected in the USA in 1994 in an isolate of Klebsiella pneumoniae; later it has been found also in Asia and in several countries in Europe.4,5 These resistance determinants might be a threat, allowing fast spread of resistance. Besides, qnr genes are often linked to resistance to cephalosporins.4,6
During a study that was performed to screen an Escherichia coli strain collection including 83 nalidixic-acid-resistant and 5 susceptible isolates from humans and 39 nalidixic-acid-resistant and 3 susceptible isolates from pigs in Denmark for characterization of quinolone resistance mechanisms, we found two isolates that showed reduced susceptibility to ciprofloxacin (MICs = 0.5 mg/L), but were susceptible to nalidixic acid (MICs = 4 and 8 mg/L). Both isolates were from patients at Hvidovre Hospital, Denmark: one from a urine sample and one from a blood sample.
In the first case, the patient was an 88-year-old woman hospitalized in mid-December 2005 with recurrent pneumonia, sepsis and bilateral crural ulcers. She was successfully treated with first mecillinam orally, then later cefuroxime and gentamicin iv and finally dicloxacillin orally due to a positive culture of Staphylococcus aureus from her crural ulcers. From a urine specimen, an E. coli resistant to ampicillin, cefuroxime, gentamicin, trimethoprim, sulfamethoxazole, ciprofloxacin and nitrofurantoin was cultured. This isolate, which showed no difference detected by antibiogram to the isolate later found, was, however, not further investigated or kept. In January 2006, the patient was hospitalized with pneumonia and dysuria and from a urine culture, 105 cfu/mL of an extended-spectrum ß-lactamase (ESBL)-producing E. coli was isolated (H88). This isolate showed the same susceptibility pattern as the first isolate at the hospital laboratory. By re-testing at the central lab, it was also considered resistant towards ceftriaxone, cefpodoxime and cefotaxime, but was ciprofloxacin-susceptible. The patient was treated successfully (clinically) with mecillinam 400 mg three times a day for 5 days.
In the second case, the patient was a 72-year-old male with disseminated colon cancer. His recent medical history included three abdominal operations and during the last month, he had received several antibiotics: ampicillin, gentamicin, metronidazole and meropenem. In April 2002, he showed clinical sepsis and an ESBL-producing E. coli was cultured from the blood (H93). This isolate was initially found to be resistant towards ampicillin, cefuroxime, ceftazidime and cefpodoxime, but intermediate to ceftriaxone and susceptible towards cefoxitin, cefotaxime, piperacillin + tazobactam and ciprofloxacin. The patient was initiated on iv piperacillin + tazobactam and ciprofloxacin, on which he improved clinically and became afebrile. During the subsequent 6 months, he deteriorated and finally succumbed to his cancer disease.
There were no indications in either case of acquisition of the strains outside Denmark. Previous to the current investigation, both E. coli strains were classified as ESBL producers. H88 harboured genes for the CTX-M-9 and TEM-1B ß-lactamases and was, in this study, found to be resistant to ampicillin, cefalotin, cefpodoxime, ceftiofur, gentamicin, streptomycin, tetracyclines, sulfamethoxazole and trimethoprim. It was positive by PCR for aac(3'')II, strA, strB, sul2 and tet(A) genes. Strain H93 harboured genes for the SHV-12 and TEM-1B ß-lactamases and was, in this study, found to be resistant to ampicillin, cefalotin, cefpodoxime, chloramphenicol, spectinomycin, gentamicin, streptomycin, sulfamethoxazole and trimethoprim. It was positive by PCR for strA, strB, int2, sul1 and sul2 genes.
The isolates were examined for quinolone resistance conferring mutations in the topoisomerase genes (gyrA, gyrB, parC and parE), by PCR and sequencing, but no mutations were found. Plasmid-mediated quinolone resistance was screened for by PCR for qnrA and qnrS using primers based on published sequences and for qnrB using primers previously described as FQ1 and FQ2.2
As positive control strains, we used E. coli J53 pMG252, positive for qnrA; E. coli J53 pMG298, positive for qnrB (both strains kindly provided by Dr George Jacoby) and for qnrS E. coli MT102 pbc-H2.6, obtained by electroporating the plasmid pH2.1 (obtained from Dr M. Hata, through the Rikken DNA Bank) into E. coli MT102.1 H88 tested positive for qnrS and H93 for qnrA. The identification of the gene was confirmed by sequencing of the fragments obtained.
Conjugative transfer was not successful for both strains. Transformation was performed with plasmid DNA from both strains by electroporation into TG1 competent cells (Stratagene). In the case of strain H93, no transformants were obtained.
Hybridization assays on Southern blots showed hybridization of a qnrS probe to a plasmid in strain H88, namely, to an 
20 kb EcoRI fragment or an 10 kb SmaI fragment of the donor strain and its transformants. In the case of strain H93, hybridization with the qnrA probe was observed to the chromosomal DNA. Although qnrA is generally referred to as plasmid-mediated, this gene is normally found on class I integrons that might jump to the chromosome, which is a possible explanation for the findings in this strain.
This is the first description of plasmid-mediated fluoroquinolone resistance in Scandinavia and in both isolates, the quinolone resistance was found in ESBL-producing multiresistant strains.
None to declare.
Acknowledgements
We thank Inge Hansen, Frank Hansen and Jane Larsen for their technical assistance. We also thank Dr George Jacoby for kindly providing qnrA- and qnrB-positive control strains and Dr Mami Hata for providing qnrS gene DNA through Rikken DNA Bank. This study was supported by a grant from the EU Marie Curie Programme (MEST-CT-2004-007819).
References
1
Hata M, Suzuki M, Matsumoto M, et al. (2005) Cloning of a novel gene for quinolone resistance from a transferable plasmid in Shigella flexneri 2b. Antimicrob Agents Chemother 49:801–3.
2
Jacoby GA, Walsh KE, Mills DM, et al. (2006) qnrB, another plasmid-mediated gene for quinolone resistance. Antimicrob Agents Chemother 50:1178–82.
3 Martinez-Martinez L, Pascual A, Jacoby GA. (1998) Quinolone resistance from a transferable plasmid. Lancet 351:797–9.[CrossRef][Web of Science][Medline]
4 Robicsek A, Jacoby GA, Hooper DC. (2006) The worldwide emergence of plasmid-mediated quinolone resistance. Lancet Infect Dis 6:629–40.[CrossRef][Web of Science][Medline]
5 Li XZ. (2005) Quinolone resistance in bacteria: emphasis on plasmid-mediated mechanisms. Int J Antimicrob Agents 25:453–63.[CrossRef][Web of Science][Medline]
6
Lavigne JP, Marchandin H, Delmas J, et al. (2006) qnrA in CTX-M-producing Escherichia coli from France. Antimicrob Agents Chemother 50:4224–8.
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