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.
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).
TABLE 1.
Properties of the clinical isolates examined in this study
| Isolate | Identity | Origin | ICU | Patient | Date of withdrawal (mo/day/yr) | MIC (μg/ml) ofa:
|
||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CIP | FOX | CAZ | CAZ + CLA | CTX | CTX + CLA | PIP | GEN | AMK | TET | SXT | TMP | SMZ | ||||||
| 37 | C. freundii | Tracheal | H1B | 9096 | 8/27/01 | 4 | >256 | 256 | 256 | 64 | 64 | 256 | >64 | 128 | >128 | 128 | >128 | >512 |
| 38 | C. freundii | Tracheal | H1B | 9702 | 9/3/01 | 2 | >256 | 256 | 128 | 64 | 32 | >256 | >64 | 32 | >128 | 128 | >128 | >512 |
| 39 | C. freundii | Tracheal | H1B | 9096 | 9/3/01 | 8 | >256 | 256 | 256 | 64 | 64 | >256 | >64 | 32 | >128 | 128 | >128 | >512 |
| 43 | C. freundii | Surgical suture | H1B | 9702 | 10/1/01 | 4 | >256 | 128 | 128 | 64 | 32 | >256 | >64 | 32 | >128 | 128 | >128 | >512 |
| 44 | C. freundii | Tracheal | H1B | 6485 | 9/27/01 | 2 | >256 | 256 | 128 | 64 | 32 | >256 | >64 | 32 | >128 | 128 | >128 | >512 |
| 458 | Enterobacter sp. | Swab | IME1 | 7410 | 5/11/03 | 8 | >256 | 128 | 128 | 64 | 128 | 256 | 4 | 4 | 16 | 128 | >128 | >512 |
CIP, ciprofloxacin; FOX, cefoxitin; CAZ, ceftazidime; CLA, clavulanic acid; CTX, cefotaxime; PIP, piperacillin; GEN, gentamicin; AMK, amikacin; TET, tetracycline; SXT, trimethoprim-sulfamethoxazole; TMP, trimethoprim; SMZ, sulfamethoxazole.
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.
TABLE 2.
Resistance profile of strains used in transconjugation experiments
| Group and strain | MIC (μg/ml) ofa:
|
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| CIP | FOX | CAZ | CTX | PIP | GEN | AMK | TET | SXT | TMP | SMZ | |
| Donor strain 39 | 8 | >256 | 256 | 64 | >256 | >64 | 32 | >128 | 128 | >128 | >512 |
| Recipients strain J53AzR | 0.03 | 8 | 2 | 0.125 | 1 | 0.25 | 4 | 1 | 0.5 | 0.5 | 16 |
| Transcon- jugants | |||||||||||
| 39-1 | 4 | >256 | 256 | 64 | >256 | >64 | 32 | >128 | 128 | >128 | >512 |
| 39-2 | 4 | >256 | 256 | 64 | >256 | >64 | 64 | >128 | 128 | >128 | >512 |
| 39-5 | 0.5 | 1 | 64 | 32 | >256 | >64 | 16 | >128 | 128 | >128 | >512 |
CIP, ciprofloxacin; FOX, cefoxitin; CAZ, ceftazidime; CTX, cefotaxime; PIP, piperacillin; GEN, gentamicin; AMK, amikacin; TET, tetracycline; SXT, trimethoprim-sulfamethoxazole; TMP, trimethoprim; SMZ, sulfamethoxazole.
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.
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).
REFERENCES
- 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. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Drlica, K., and X. Zhao. 1997. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. Mol. Biol. Rev. 61:377-392. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Jacoby, G. A., N. Chow, and K. B. Waites. 2003. Prevalence of plasmid-mediated quinolone resistance. Antimicrob. Agents Chemother. 47:559-562. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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. [DOI] [PubMed] [Google Scholar]
- 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. [DOI] [PubMed] [Google Scholar]
- 9.Martinez-Martinez, L., A. Pascual, and G. A. Jacoby. 1998. Quinolone resistance from a transferable plasmid. Lancet 351:797-799. [DOI] [PubMed] [Google Scholar]
- 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. [DOI] [PubMed] [Google Scholar]
- 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. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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.Poole, K. 2000. Efflux-mediated resistance to fluoroquinolones in gram-negative bacteria. Antimicrob. Agents Chemother. 44:2233-2241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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. [DOI] [PubMed] [Google Scholar]
- 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. [DOI] [PubMed] [Google Scholar]
- 16.Tran, J. H., and G. A. Jacoby. 2002. Mechanism of plasmid-mediated quinolone resistance. Proc. Natl. Acad. Sci. USA 99:5638-5642. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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. [DOI] [PubMed] [Google Scholar]
- 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. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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. [Google Scholar]