This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jonas, D.
Right arrow Articles by Daschner, F. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jonas, D.
Right arrow Articles by Daschner, F. D.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, February 2005, p. 773-775, Vol. 49, No. 2
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.2.773-775.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Plasmid-Mediated Quinolone Resistance in Isolates Obtained in German Intensive Care Units

Daniel Jonas,* Klaus Biehler, Doris Hartung, Bettina Spitzmüller, and Franz D. Daschner

Institute of Environmental Medicine and Hospital Epidemiology, Freiburg University Hospital, Freiburg, Germany

Received 21 June 2004/ Returned for modification 28 July 2004/ Accepted 11 October 2004


arrow
ABSTRACT
 
Screening of 703 isolates of Enterobacteriaceae, obtained from 34 German intensive care units (ICUs), revealed qnr-positive, integron-containing isolates of Enterobacter sp. and Citrobacter freundii from four patients in 2 German ICUs. This is one of the first reports of qnr-positive strains obtained from patients in Europe.


arrow
TEXT
 
Integron-carrying isolates are statistically associated with multidrug resistance against various classes of antimicrobials (7). These episomal transmissible genetic elements transfer resistance to various antimicrobials, which is encoded by so-called "cassettes." Despite the association of the integrons with a fluoroquinolone-resistant phenotype, no corresponding resistance cassette is known.

Usually, quinolone resistance results from mutational changes in the chromosomally encoded type II topoisomerases and the expression of efflux pumps or porins (2, 13). However, recent reports indicate that this resistance can also be mediated by plasmids (9). This first valid quinolone resistance (qnr) plasmid encodes a 218-amino-acid immune protein of the pentapeptide family, which directly protects the Escherichia coli gyrase from quinolone inhibition (16). Molecular characterizations of plasmids isolated from different clinical strains showed that the qnr gene was located together with other resistance determinants near class I integrons between a duplication of the 3' conserved sequence of integrons and orf513 (16, 18, 19), a putative recombinase involved in site-specific acquisition of resistance genes (17).

The low level of resistance of qnr has been shown to contribute additively to the high level of resistance in qnr-positive strains (8). The presence of qnr facilitates the selection of chromosomal mutations causing high-level quinolone resistance (4, 9).

The occurrence of this novel plasmid-mediated quinolone resistance has been reported from few areas around the world. This transmissible quinolone resistance gene was detected in Klebsiella pneumoniae strains in different U.S. states, six E. coli strains from China, and one Providencia stuartii isolate from Egypt (4, 15, 18-20). The aim of this study was to determine the occurrence of the qnr gene in integron-positive strains in German intensive care units (ICUs).

Participants of the German surveillance system "Spread of Nosocomial Infections and Resistant Pathogens" were asked to collect fluoroquinolone- or cephalosporin-resistant Enterobacteriaceae (10). From February 2000 to December 2003, 703 strains were isolated from patients in 34 ICUs throughout Germany. All of the strains were analyzed for the presence of integron cassettes and integrase by use of PCR (6). One hundred thirty-six strains contained both integron-defining structures. All of them were screened for the presence of the qnr gene.

PCR was performed as described previously with the primers QP1 and QP2, which results in a 543-bp amplicon (4). E. coli strain J53 containing the plasmid pMG252 (kindly provided by G. Jacoby, Lahey Clinic, Burlington, Mass.) served as a positive control (16). Six strains from four patients in two ICUs located in two different states were PCR positive. Sequence identities were confirmed by DNA sequencing of the six PCR products by use of the amplification primers and comparison with the published qnr coding sequence (16).

Biochemical species differentiation by the API 20E (bioMérieux, Marcy l'Etoile, France) identified one strain as Enterobacter cloacae, with just 67% identity. Subsequently, the 16S rRNA gene sequence of this isolate was determined with the universal 16S rRNA primers 806R, 8F, 13R, and 515F (3, 14). The sequence comparison by means of the BLASTN algorithm revealed closest similarity to one environmental isolate of Enterobacter, strain B901-2 (GenBank accession no. AB114268), with 99% sequence identity (1, 11). Therefore, this strain was designated as Enterobacter sp. The remaining five isolates originating from the second ICU were unambiguously identified as Citrobacter freundii. The suggested genotypic identity of these five strains isolated from three patients in a single ICU within 2 months was confirmed by XbaI macrorestriction analysis (data not shown) as described previously (5).

In all of the strains of C. freundii and Enterobacter sp., PCR detection of integron cassettes with primers hybridizing to the 5'- and 3'-conserved regions revealed PCR products 1,000 and 1,600 bp in size, respectively.

Antimicrobial susceptibility testing was performed according to the NCCLS (12). All of the strains were ciprofloxacin resistant, and none was susceptible to cefoxitin, cefotaxime, or ceftazidime (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Properties of the clinical isolates examined in this study

The PCR-detectable qnr gene of C. freundii could be mobilized together with the antimicrobial-resistant phenotype into an E. coli J53AzR recipient by transconjugation and subsequent selection on 100-µg/ml Na-azide in combination with either 10-µg/ml tetracycline or 10-µg/ml gentamicin after overnight filter mating (Table 2). Similar experiments with the Enterobacter sp. strain failed, even when 10 µg of ceftazidime or piperacillin per ml or 0.25 µg of ciprofloxacin per ml was employed for selection.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Resistance profile of strains used in transconjugation experiments

The plasmid preparation from Enterobacter sp. and C. freundii C39 as well as all three transconjugants revealed covalently closed circular DNA larger or equal to the qnr-positive plasmid pMG252, which is 180 kb in size (18). By use of a nonradioactively labeled qnr PCR fragment, these plasmids showed the presence of qnr DNA sequences in Southern hybridization experiments (data not shown).

The patients' records did not show that any of them had been abroad in the months preceding ICU admission. The patient carrying the Enterobacter sp. strain had been in a coma vigil state for 1 year prior to admission from a nursing home to the ICU because of cardiac arrhythmia; he stayed in the unit for 4 days. With regard to the small outbreak of qnr-positive C. freundii, the tracheal secretion of the presumptive index case patient was found to be colonized with C. freundii 10 days after admission because of craniocerebral injury. The second patient was admitted to the ICU the same day, and the identical strain was isolated 1 week later from the patient's tracheal secretion, from abdominal swabs taken during surgery two and a half weeks later, and finally from surgical sutures. Therefore, this strain was taken to be the infectious agent of a surgical site infection. The third patient was admitted to the ICU on the same day as the first patient. However, after just 1 month of stay in the unit, this patient's tracheal secretion and catheter urine turned out to be colonized with the outbreak strain. Only some of the strains were made available for investigation (Table 1). As far as the patients' records showed, none of the three patients colonized or infected with C. freundii had been abroad in the months preceding admission.

The importance of the qnr transmissible resistance gene may be its additive effect in raising the quinolone MIC and subsequent greater ease in selecting high quinolone resistance from qnr-expressing strains (8, 9). It may present one possible explanation for a link between integron carriage and quinolone resistance. This is one of the first reports on qnr-positive strains obtained from patients in Europe. Considering recent reports on the detection of this plasmid-mediated resistance gene in isolates originating from the United States, China, and Egypt, the spread of this emerging resistance might be underscored in multidrug-resistant, integron-carrying Enterobacteriaceae.


arrow
ACKNOWLEDGMENTS
 
The members of the Study Group on Spread of Nosocomial Infections and Resistant Pathogens contributed the bacterial isolates. G. Schwarzkopf-Steinhauser and M. Lapatscheck (Städtisches Krankenhaus München Harlaching, Munich, Germany) contributed epidemiological data on the patients. We are grateful to G. A. Jacoby (Lahey Clinic, Burlington, Mass.) for providing the qnr-positive E. coli control strain, J53(pMG252), and the E. coli recipient strain, J53AzR, for conjugation experiments. Deborah Lawrie-Blum assisted with preparation of the manuscript.

This study was supported by a grant from the German Bundesministerium für Bildung und Forschung (01 KI 9907).


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Environmental Medicine and Hospital Epidemiology, University Hospital Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany. Phone: 49 761 270 5445. Fax: 49 761 270 5485. E-mail: djonas{at}IUK3.UKL.uni-Freiburg.de. Back


arrow
REFERENCES
 
    1
  1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.[Abstract/Free Full Text]
    2. 2
  2. Drlica, K., and X. Zhao. 1997. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. Mol. Biol. Rev. 61:377-392.[Abstract]
    3. 3
  3. Fredricks, D. N., and D. A. Relman. 1996. Sequence-based identification of microbial pathogens: a reconsideration of Koch's postulates. Clin. Microbiol. Rev. 9:18-33.[Abstract]
    4. 4
  4. Jacoby, G. A., N. Chow, and K. B. Waites. 2003. Prevalence of plasmid-mediated quinolone resistance. Antimicrob. Agents Chemother. 47:559-562.[Abstract/Free Full Text]
    5. 5
  5. Jalaluddin, S., J. M. Devaster, R. Scheen, M. Gerard, and J.-P. Butzler. 1998. Molecular epidemiological study of nosocomial Enterobacter aerogenes isolates in a Belgian hospital. J. Clin. Microbiol. 36:1846-1852.[Abstract/Free Full Text]
    6. 6
  6. Lévesque, C., L. Piché, C. Larose, and P. H. Roy. 1995. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob. Agents Chemother. 39:185-191.[Abstract]
    7. 7
  7. Martinez-Freijo, P., A. C. Fluit, F. J. Schmitz, V. S. Grek, J. Verhoef, and M. E. Jones. 1998. Class I integrons in Gram-negative isolates from different European hospitals and association with decreased susceptibility to multiple antibiotic compounds. J. Antimicrob. Chemother. 42:689-696.[Abstract/Free Full Text]
    8. 8
  8. Martinez-Martinez, L., A. Pascual, I. Garcia, J. Tran, and G. A. Jacoby. 2003. Interaction of plasmid and host quinolone resistance. J. Antimicrob. Chemother. 51:1037-1039.[Free Full Text]
    9. 9
  9. Martinez-Martinez, L., A. Pascual, and G. A. Jacoby. 1998. Quinolone resistance from a transferable plasmid. Lancet 351:797-799.[CrossRef][Medline]
    10. 10
  10. Meyer, E., D. Jonas, F. Schwab, H. Rueden, P. Gastmeier, and F. D. Daschner. 2003. Design of a surveillance system of antibiotic use and bacterial resistance in German intensive care units (SARI). Infection 31:208-215.[Medline]
    11. 11
  11. Minamisawa, K., K. Nishioka, T. Miyaki, B. Ye, T. Miyamoto, M. You, A. Saito, M. Saito, W. L. Barraquio, N. Teaumroong, T. Sein, and T. Sato. 2004. Anaerobic nitrogen-fixing consortia consisting of clostridia isolated from gramineous plants. Appl. Environ. Microbiol. 70:3096-3102.[Abstract/Free Full Text]
    12. 12
  12. National Committee for Clinical Laboratory Standards. 1997. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. Approved standard M7-A4. NCCLS, Wayne, Pa.
    13. 13
  13. Poole, K. 2000. Efflux-mediated resistance to fluoroquinolones in gram-negative bacteria. Antimicrob. Agents Chemother. 44:2233-2241.[Free Full Text]
    14. 14
  14. Relman, D. A., T. M. Schmidt, R. P. MacDermott, and S. Falkow. 1992. Identification of the uncultured bacillus of Whipple's disease. N. Engl. J. Med. 327:293-301.[Abstract]
    15. 15
  15. Rodriguez-Martinez, J. M., A. Pascual, I. Garcia, and L. Martinez-Martinez. 2003. Detection of the plasmid-mediated quinolone resistance determinant qnr among clinical isolates of Klebsiella pneumoniae producing AmpC-type beta-lactamase. J. Antimicrob. Chemother. 52:703-706.[Abstract/Free Full Text]
    16. 16
  16. Tran, J. H., and G. A. Jacoby. 2002. Mechanism of plasmid-mediated quinolone resistance. Proc. Natl. Acad. Sci. USA 99:5638-5642.[Abstract/Free Full Text]
    17. 17
  17. Valentine, C. R., M. J. Heinrich, S. L. Chissoe, and B. A. Roe. 1994. DNA sequence of direct repeats of the sulI gene of plasmid pSa. Plasmid 32:222-227.[CrossRef][Medline]
    18. 18
  18. Wang, M., D. F. Sahm, G. A. Jacoby, and D. C. Hooper. 2004. Emerging plasmid-mediated quinolone resistance associated with the qnr gene in Klebsiella pneumoniae clinical isolates in the United States. Antimicrob. Agents Chemother. 48:1295-1299.[Abstract/Free Full Text]
    19. 19
  19. Wang, M., J. H. Tran, G. A. Jacoby, Y. Zhang, F. Wang, and D. C. Hooper. 2003. Plasmid-mediated quinolone resistance in clinical isolates of Escherichia coli from Shanghai, China. Antimicrob. Agents Chemother. 47:2242-2248.[Abstract/Free Full Text]
    20. 20
  20. Wiegand, I., N. Khalaf, M. H. M. Al-Agamy, and B. Wiedemann. 2004. First detection of the transferable quinolone resistance determinant in clinical Providencia stuartii strains in Egypt. Clin. Microbiol. Infect. 10(Suppl. 3):64.

Antimicrobial Agents and Chemotherapy, February 2005, p. 773-775, Vol. 49, No. 2
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.2.773-775.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Strahilevitz, J., Jacoby, G. A., Hooper, D. C., Robicsek, A. (2009). Plasmid-Mediated Quinolone Resistance: a Multifaceted Threat. Clin. Microbiol. Rev. 22: 664-689 [Abstract] [Full Text]  
  • Pazhani, G. P., Niyogi, S. K., Singh, A. K., Sen, B., Taneja, N., Kundu, M., Yamasaki, S., Ramamurthy, T. (2008). Molecular characterization of multidrug-resistant Shigella species isolated from epidemic and endemic cases of shigellosis in India. J Med Microbiol 57: 856-863 [Abstract] [Full Text]  
  • Yamane, K., Wachino, J.-i., Suzuki, S., Kimura, K., Shibata, N., Kato, H., Shibayama, K., Konda, T., Arakawa, Y. (2007). New Plasmid-Mediated Fluoroquinolone Efflux Pump, QepA, Found in an Escherichia coli Clinical Isolate. Antimicrob. Agents Chemother. 51: 3354-3360 [Abstract] [Full Text]  
  • Wu, J.-J., Ko, W.-C., Tsai, S.-H., Yan, J.-J. (2007). Prevalence of Plasmid-Mediated Quinolone Resistance Determinants QnrA, QnrB, and QnrS among Clinical Isolates of Enterobacter cloacae in a Taiwanese Hospital. Antimicrob. Agents Chemother. 51: 1223-1227 [Abstract] [Full Text]  
  • Chen, Y.-T., Shu, H.-Y., Li, L.-H., Liao, T.-L., Wu, K.-M., Shiau, Y.-R., Yan, J.-J., Su, I.-J., Tsai, S.-F., Lauderdale, T.-L. (2006). Complete Nucleotide Sequence of pK245, a 98-Kilobase Plasmid Conferring Quinolone Resistance and Extended-Spectrum-{beta}-Lactamase Activity in a Clinical Klebsiella pneumoniae Isolate. Antimicrob. Agents Chemother. 50: 3861-3866 [Abstract] [Full Text]  
  • Toleman, M. A., Bennett, P. M., Walsh, T. R. (2006). ISCR Elements: Novel Gene-Capturing Systems of the 21st Century?. Microbiol. Mol. Biol. Rev. 70: 296-316 [Abstract] [Full Text]  
  • Leavis, H. L., Willems, R. J. L., Top, J., Bonten, M. J. M. (2006). High-Level Ciprofloxacin Resistance from Point Mutations in gyrA and parC Confined to Global Hospital-Adapted Clonal Lineage CC17 of Enterococcus faecium.. J. Clin. Microbiol. 44: 1059-1064 [Abstract] [Full Text]  
  • Corkill, J. E., Anson, J. J., Hart, C. A. (2005). High prevalence of the plasmid-mediated quinolone resistance determinant qnrA in multidrug-resistant Enterobacteriaceae from blood cultures in Liverpool, UK. J Antimicrob Chemother 56: 1115-1117 [Abstract] [Full Text]  
  • Nordmann, P., Poirel, L. (2005). Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae. J Antimicrob Chemother 56: 463-469 [Abstract] [Full Text]  
  • Poirel, L., Rodriguez-Martinez, J.-M., Mammeri, H., Liard, A., Nordmann, P. (2005). Origin of Plasmid-Mediated Quinolone Resistance Determinant QnrA. Antimicrob. Agents Chemother. 49: 3523-3525 [Abstract] [Full Text]  
  • Robicsek, A., Sahm, D. F., Strahilevitz, J., Jacoby, G. A., Hooper, D. C. (2005). Broader Distribution of Plasmid-Mediated Quinolone Resistance in the United States. Antimicrob. Agents Chemother. 49: 3001-3003 [Abstract] [Full Text]  
  • Poirel, L., Van De Loo, M., Mammeri, H., Nordmann, P. (2005). Association of Plasmid-Mediated Quinolone Resistance with Extended-Spectrum {beta}-Lactamase VEB-1. Antimicrob. Agents Chemother. 49: 3091-3094 [Abstract] [Full Text]  
  • Hegde, S. S., Vetting, M. W., Roderick, S. L., Mitchenall, L. A., Maxwell, A., Takiff, H. E., Blanchard, J. S. (2005). A Fluoroquinolone Resistance Protein from Mycobacterium tuberculosis That Mimics DNA. Science 308: 1480-1483 [Abstract] [Full Text]  
  • Saga, T., Kaku, M., Onodera, Y., Yamachika, S., Sato, K., Takase, H. (2005). Vibrio parahaemolyticus Chromosomal qnr Homologue VPA0095: Demonstration by Transformation with a Mutated Gene of Its Potential To Reduce Quinolone Susceptibility in Escherichia coli. Antimicrob. Agents Chemother. 49: 2144-2145 [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jonas, D.
Right arrow Articles by Daschner, F. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jonas, D.
Right arrow Articles by Daschner, F. D.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»