Skip Navigation


JAC Advance Access originally published online on July 26, 2006
Journal of Antimicrobial Chemotherapy 2006 58(3):673-677; doi:10.1093/jac/dkl297
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
58/3/673    most recent
dkl297v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Saito, R.
Right arrow Articles by Koike, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Saito, R.
Right arrow Articles by Koike, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Role of type II topoisomerase mutations and AcrAB efflux pump in fluoroquinolone-resistant clinical isolates of Proteus mirabilis

Ryoichi Saito1,2,*, Kenya Sato2, Wakako Kumita2, Natsuko Inami2, Hiroyuki Nishiyama2, Noboru Okamura2, Kyoji Moriya1 and Kazuhiko Koike1

1 Department of Infection Control and Prevention, The University of Tokyo Hospital Bunkyo-ku, Tokyo 113-8655, Japan 2 Department of Microbiology and Immunology, Graduate School of Allied Health Sciences, Tokyo Medical and Dental University Bunkyo-ku, Tokyo 113-8510, Japan

*Corresponding author. Department of Infection Control and Prevention, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Tel: +81-3-3815-5411; Fax: +81-3-5689-0495; E-mail: saito-lab{at}h.u-tokyo.ac.jp

Received 10 March 2006; returned 7 April 2006; revised 28 June 2006; accepted 2 July 2006


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
Objectives: We conducted a study to determine the role played by amino acid mutations in DNA gyrase and topoisomerase IV, and the AcrAB efflux pump in resistance to fluoroquinolones in clinical isolates of Proteus mirabilis.

Methods: Nine clinical isolates of P. mirabilis containing eight fluoroquinolone-resistant isolates and one fluoroquinolone-susceptible isolate as the causative pathogen were collected from different patients with urinary tract infections. Fluoroquinolone resistance was characterized by PCR and DNA sequencing. The role of the AcrAB efflux pump was investigated by semi-quantifying the transcriptional expression of the acrB gene.

Results: Double mutations were found in GyrA, at S83I and E87K, and single mutations in GyrB (S464F) and ParC (S80I) in four isolates with ciprofloxacin MICs of 16 to >128 mg/L. In three isolates (ciprofloxacin MICs of >128 mg/L), the level of acrB expression was 2.1- to 3.2-fold higher than that in the wild-type control strain (ciprofloxacin MIC of ≤0.12 mg/L) and these isolates also had increased MICs of minocycline (>64 versus 8–16 mg/L) and chloramphenicol (>256 versus 4–8 mg/L) compared with the five other fluoroquinolone-resistant isolates.

Conclusion: Our findings demonstrate that two mechanisms—mutations in GyrA (at S83I and E87K), GyrB and ParC, and overproduction of the AcrAB efflux pump—might synergistically contribute to a highest level of resistance to fluoroquinolones in clinical isolates of P. mirabilis.

Keywords: P. mirabilis , DNA gyrase , topoisomerase IV


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
Among Enterobacteriaceae, Proteus mirabilis is one of the most common causes of urinary tract infections (UTIs), which are often persistent and difficult to treat, and is also an important cause of nosocomial infections.1 Though wild-type strains of P. mirabilis are usually susceptible to fluoroquinolones, a progressive increase in fluoroquinolone resistance has been seen in clinical isolates of the bacterium.2,3

Two mechanisms that decrease susceptibility to fluoroquinolones have been identified so far in clinical isolates, which are alteration of the target proteins—DNA gyrase (encoded by gyrA and gyrB genes) and topoisomerase IV (encoded by parC and parE genes)—and reduced drug accumulation due to efflux pumps.4 In P. mirabilis, development of fluoroquinolone resistance requires a combination of two or more mutations in the quinolone resistance-determining region (QRDR) of the genes encoding DNA gyrase and topoisomerase IV and is mainly attributed to amino acid mutations at positions Ser-83 in GyrA, Ser-464 in GyrB and Ser-80 in ParC.5 In addition, there is growing evidence for the implication of the overexpression of multidrug efflux pumps in fluoroquinolone resistance in other bacteria, such as AcrAB in Escherichia coli6,7 and Salmonella enterica serovar Typhimurium8,9 or CmeABC in Campylobacter species.10 Moreover, the AcrAB efflux pump has been determined to be associated with reduced levels of susceptibility to tigecycline and minocycline in P. mirabilis11 but the role of this efflux pump in the fluoroquinolone resistance of P. mirabilis has been unclear.

Much data need to be collected from clinical isolates to assess risks from the increasing fluoroquinolone resistance of P. mirabilis. We therefore investigated the roles played by amino acid mutations in DNA gyrase and topoisomerase IV and the AcrAB efflux pump in the fluoroquinolone resistance of clinical isolates of P. mirabilis.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Transparency declarations
 References
 
Bacterial strains and susceptibility testing

The bacterial strains used in this study were those of P. mirabilis present in nine clinical isolates from different patients with acute, repeated or chronic UTIs at the University of Tokyo Hospital, collected from October 2003 through September 2005. All strains were identified by the conventional method and by the Vitek I system (bioMerieux Japan, Tokyo, Japan). Fluoroquinolones (ciprofloxacin, levofloxacin and sparfloxacin), minocycline, ampicillin, ceftazidime, gentamicin and imipenem were tested using panels manufactured by Eiken Chemical (Tokyo, Japan). Chloramphenicol (Sankyo, Tokyo, Japan), erythromycin (Shionogi Pharmaceutical, Osaka, Japan) and clarithromycin (Taishotoyama Pharmaceutical, Tokyo, Japan) were also used in this study. The MICs were determined by the broth microdilution method as described by the Clinical and Laboratory Standards Institute [CLSI, formerly known as the National Committee for Clinical Laboratory Standards (NCCLS)].12 Quality control for the MICs was performed using the following reference strains: Staphylococcus aureus ATCC 21293, E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853.

PFGE

Genomic DNA of the P. mirabilis strains was prepared in agarose plugs that had been treated with lysozyme and pronase K using a Gene Path reagent kit (Bio-Rad, Tokyo, Japan) according to the manufacturer's recommendations. DNA was digested with 25 U of the restriction endonuclease NotI (Roch Diagnostics, Tokyo, Japan). The DNA fragments generated were then separated in a 1% agarose gel and subjected to electrophoresis in Tris–borate–EDTA buffer at 14°C using a pulsed-field apparatus (CHEF-DR II, Bio-Rad) at 200 V for 19.7 h with pulse times of 5–35 s. BioNumerics software (version 3.0; Applied Maths, Kortrijk, Belgium) was used to analyse the DNA restriction patterns and determine their similarity, based on calculation of the Dice similarity coefficient and using the UPGMA algorithm (unweighted pair-group method using arithmetic averages).

PCR amplification and sequencing of QRDRs of gyrA, gyrB and parC genes

The DNA template for PCR amplification was obtained from the supernatant of a boiled extract of P. mirabilis cells harvested from Luria–Bertani (LB) broth. The QRDRs of the gyrA, gyrB and parC genes were amplified using primer sets according to a method described previously.5 PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Tokyo, Japan) in accordance with the manufacturer's recommendations. Purified PCR fragments were sequenced with an ABI PRISM 310 DNA sequencer (Applied Biosystems, Foster City, CA, USA). A similarity search for the deduced amino acid sequences against DDBJ/EMBL/GenBank sequence databases with the accession numbers AF397169 [GenBank] (gyrA), AF503506 [GenBank] (gyrB) and AF363611 [GenBank] (parC) from P. mirabilis ATCC 29906 was conducted using the BLAST program at the DNA Databank of Japan (Shizuoka, Japan).

Analysis of acrB gene expression by reverse transcription (RT)–PCR

Semi-quantitative RT–PCR was used to analyse the transcriptional expression of the acrB gene indicating expression of the AcrAB efflux pump. Overnight bacteria cultures were diluted 1:100 in LB broth and grown to the mid-logarithmic phase (OD600 = 0.5) at 37°C with shaking. Cultures were pelleted by centrifugation at 13 000 g for 10 min, and RNA was isolated using ISOGEN (Nippongen, Tokyo, Japan) according to the manufacturer's instructions. Total RNA (1 µg), 50 ng of random hexamers and 2 µL of a 10 mM deoxynucleoside triphosphates mixture (Invitrogen, Tokyo, Japan) were incubated for 5 min at 65°C, immediately cooled on ice and then reverse transcribed in a final volume of 20 µL—containing 2 µL of 10 mM dithiothreitol, 40 U of RNaseOUT ribonuclease inhibitor, First Strand Buffer 1x and 50 U of Superscript II reverse transcriptase (Invitrogen)—that was reacted for 50 min at 50°C. PCR amplification of cDNA was performed with an initial denaturation step of 5 min at 95°C, followed by 16 cycles of 30 s at 95°C, 30 s at 55°C and 1 min at 72°C, and finishing with one cycle of 7 min at 72°C, using primer sets for the acrB gene (Table 1). The number of PCR cycles used came within the linearity range for PCR amplification and constitutive expression of 16S rRNA assessed from the same cDNA preparation was used as a standard. Samples (10 µL) of each PCR product were separated by electrophoresis in 2.0% agarose and visualized by ethidium bromide staining. The bands of the acrB gene were semi-quantified using image scanning software (Scion Image) and results were standardized with the 16S rRNA band density.


View this table:
[in this window]
[in a new window]
  		Table 1. Primers used in the study

 
Statistics

The above gene expression experiment was repeated three times. Results were expressed as mean±SD. Statistical analysis was performed with a two-tailed Student's t-test. P values <0.05 were taken as significant.

Nucleotide sequence accession number

The partial DNA sequences of the gyrA gene in isolates PmNC and Pm506 have been assigned to the DDBJ/EMBL/GenBank database under accession numbers AB252193 [GenBank] (PmNC) and AB252194 [GenBank] (Pm506).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Transparency declarations
 References
 
PFGE

The genetic similarity of the nine isolates was evaluated using PFGE. Eight PFGE types were identified and among them isolates Pm506 and Pm609 were identical being of type E. Table 2 summarizes the results.


View this table:
[in this window]
[in a new window]
  		Table 2. QRDR amino acid mutations of GyrA, GyrB and ParC, and the antibiotic susceptibility in clinical isolates of P. mirabilis

 
Mutations in gyrA, gyrB and parC genes

The nucleotide sequences and derived amino acid sequences in the QRDRs of the gyrA, gyrB and parC genes for each P. mirabilis isolate were compared with those of the wild-type strain, ATCC 29906 (Table 2). One strain (PmNC) with a ciprofloxacin MIC of ≤0.12 mg/L showed no changes in GyrA, GyrB and ParC. Four isolates (Pm210, Pm405, Pm701 and Pm909) with ciprofloxacin MICs of 4–16 mg/L had a single mutation of Ser-83 to Arg or Ile in GyrA. Double mutations in GyrA, at Ser-83 and Glu-87, were found in four isolates (Pm311, Pm506, Pm609 and Pm805) having ciprofloxacin MICs of 16 to >128 mg/L. In these strains, the AGT codon for Ser-83 and the GAA codon for Glu-87 in the gyrA gene were replaced by ATT for Ile and AAA for Lys, respectively (Table 2). Further, all isolates showing resistance to ciprofloxacin (MIC of ≥4 mg/L) had one silent nucleotide substitution, a C to T transition for nucleotide position 148 of the gyrA gene, as compared with the P. mirabilis type strain, ATCC 29906. For GyrB, mutations such as Ser-464 to Tyr or Phe were detected in ciprofloxacin-resistant isolates (MICs of ≥8 mg/L), while no amino acid changes were detected at position 466 (Table 2). Furthermore, four isolates (Pm311, Pm506, Pm609 and Pm805) with a mutation of Ser-464 to Phe in GyrB showed a high level of resistance to ciprofloxacin (MICs of 16 to >128 mg/L). Except the isolate with a ciprofloxacin MIC of ≤0.12 mg/L, all isolates had amino acid changes affecting ParC: six isolates with Ser-80 to Ile and two isolates with Ser-80 to Arg (Table 2). No amino acid changes were detected at position 78 in ParC.

Expression of acrB gene

To determine the resistance to fluoroquinolones due to the AcrAB efflux pump, transcriptional expression of the acrB gene in the clinical isolates of P. mirabilis was analysed by semi-quantitative RT–PCR as mentioned above. The results are shown in Figure 1. Expression of the acrB gene in isolates Pm506, Pm609 and Pm805 (ciprofloxacin MICs of >128 mg/L) were 2.8, 2.1 and 3.2 times greater than in the wild-type control strain (PmNC), respectively. These were statistically significant differences (P < 0.05). For all of the other isolates, the expression of the acrB gene was of the same level as that of the wild-type control strain. The three isolates (Pm506, Pm609 and Pm805) which we hypothesized overexpress the acrB gene also had increased MICs of minocycline (>64 versus 8–16 mg/L) and chloramphenicol (>256 versus 4–8 mg/L) compared with the five other fluoroquinolone-resistant isolates (Table 2). A relationship between the other antibiotic susceptibilities (including susceptibilities to erythromycin, clarithromycin, ampicillin, ceftazidime, gentamicin and imipenem) and expression of the acrB gene was not recognized.


Figure 1
View larger version (30K):
[in this window]
[in a new window]
  		Figure 1. Analysis of expression of acrB gene and 16S rRNA in clinical isolates of P. mirabilis by semi-quantitative RT–PCR. (a) Representative electrophoresis of RT–PCR product of acrB gene and 16S rRNA. Lane 1, Pm210; lane 2, Pm311; lane 3, Pm405; lane 4, Pm506; lane 5, Pm609; lane 6, Pm701; lane 7, Pm805; lane 8, Pm909; lane 9, PmNC. (b) Semi-quantification of acrB gene expression—calculated using fluorescence ratio of 16S rRNA (n = 3 for each isolate). *P < 0.05 versus wild-type control strain (PmNC).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Transparency declarations
 References
 
The mechanisms by which bacteria develop resistance to fluoroquinolones include mutations in the target enzymes (DNA gyrase and topoisomerase IV) and overexpression of endogenous multidrug efflux pumps.4 The objective of the present study was to investigate the role of amino acid mutations in the target enzymes and the AcrAB efflux pump in clinical isolates of P. mirabilis showing resistance to fluoroquinolones.

Isolates Pm506 and Pm609 were both type E, suggesting that the infections caused by P. mirabilis in them shared a common origin. Since the seven other isolates were not clonally related there was unlikely to be a common source of infection for them.

Previous studies on amino acid mutations in DNA gyrase and topoisomerase IV associated with fluoroquinolone resistance in P. mirabilis have been limited.5 The study of Weigel et al.5 suggested that a double mutation affecting Ser-83 in GyrA and Ser-80 in ParC was not correlated with fluoroquinolone MICs and that mutation in GyrB is a frequent event in the acquisition of fluoroquinolone resistance. In the present study, mutations in GyrB and ParC occurred in the codons for Ser-464 and Ser-80, respectively, and this is similar to the previous findings.5 Four of our isolates (Pm311, Pm506, Pm609 and Pm805) having ciprofloxacin MICs of 16 to >128 mg/L had double mutations in GyrA, at Ser-83 and Glu-87, and this was the first time these mutations had been detected in P. mirabilis. Previous studies show that a high level of resistance to fluoroquinolones is associated with double mutations in GyrA and an additional mutation in ParC in E. coli.4,13,14 In the present study we did not find a clear relationship between the degree of fluoroquinolone resistance and the number of mutations in GyrA, GyrB and ParC in P. mirabilis. However, such a relationship may have been found if a larger number of isolates had been examined.

Recently, it has been demonstrated that DNA gyrase and topoisomerase IV mutations and the efflux pump have a multiplicative effect on the MICs of fluoroquinolones for E. coli6 and Campylobacter species.10 Also, in a previous study, a homologue of the E. coli AcrAB efflux pump was identified in P. mirabilis and this gene cluster appeared to be responsible for reducing susceptibility to tigecycline and minocycline,11 but it had yet to be shown that this efflux pump plays a role in the fluoroquinolone resistance of P. mirabilis. Thus, using clinical isolates of P. mirabilis, we investigated the role of the AcrAB efflux pump in fluoroquinolone resistance by semi-quantifying the transcriptional expression of the acrB gene. As indicated in Figure 1, in three isolates (Pm506, Pm609 and Pm805) with ciprofloxacin MICs of >128 mg/L, the degree of expression of the acrB gene was 2.8-, 2.1- and 3.2-fold higher, respectively, than that in the wild-type control strain (PmNC). Although we did not look at actual protein levels of the AcrAB efflux pump and did not confirm the role of this pump by inactivating the acrB gene via genetic means, overproduction of this pump was consistent with the increased MICs of minocycline and chloramphenicol compared with the other five isolates. Therefore, these results suggest that overproduction of this pump may play a role in fluoroquinolone resistance in clinical isolates of P. mirabilis. These three isolates had double mutations in GyrA (corresponding to Ser-83 and Glu-87) in addition to mutations in GyrB and ParC. Thus, overall, our results indicate that two mechanisms—mutations in DNA gyrase and topoisomerase IV and overproduction of the AcrAB efflux pump—might synergistically contribute to the acquisition of a highest level of resistance to fluoroquinolones in clinical isolates of P. mirabilis. To our knowledge, this is the first study that has analysed the role of the AcrAB efflux pump in fluoroquinolone-resistant clinical isolates of P. mirabilis.


   Transparency declarations
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
References
 
None to declare.


   Acknowledgements
 
We thank Toshio Chida for useful discussions. We did not receive any financial support from third parties.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
1 Rozalski A, Sidorczyk Z, Kotelko K. (1997) Potential virulence factors of Proteus bacilli. Microbiol Mol Biol Rev 61:65–89.[Abstract]

2 de Champs C, Bonnet R, Sirot D, et al. (2000) Clinical relevance of Proteus mirabilis in hospital patients: a two year survey. J Antimicrob Chemother 45:537–9.[Abstract/Free Full Text]

3 Hernandez JR, Martinez-Martinez L, Pascual A, et al. (2000) Trends in the susceptibilities of Proteus mirabilis isolates to quinolones. J Antimicrob Chemother 45:407–8.[Free Full Text]

4 Hooper DC. (1999) Mechanisms of fluoroquinolone resistance. Drug Resist Updat 2:38–55.[CrossRef][Web of Science][Medline]

5 Weigel LM, Anderson GJ, Tenover FC. (2002) DNA gyrase and topoisomerase IV mutations associated with fluoroquinolone resistance in Proteus mirabilis. Antimicrob Agents Chemother 46:2582–7.[Abstract/Free Full Text]

6 Oethinger M, Kern WV, Jellen-Ritter AS, et al. (2000) Ineffectiveness of topoisomerase mutations in mediating clinically significant fluoroquinolone resistance in Escherichia coli in the absence of the AcrAB efflux pump. Antimicrob Agents Chemother 44:10–3.[Abstract/Free Full Text]

7 Jellen-Ritter AS and Kern WV. (2001) Enhanced expression of the multidrug efflux pumps AcrAB and AcrEF associated with insertion element transposition in Escherichia coli mutants selected with a fluoroquinolone. Antimicrob Agents Chemother 45:1467–72.[Abstract/Free Full Text]

8 Baucheron S, Imberechts H, Chaslus-Dancla E, et al. (2002) The AcrB multidrug transporter plays a major role in high-level fluoroquinolone resistance in Salmonella enterica serovar Typhimurium phage type DT204. Microb Drug Resist 8:281–9.[CrossRef][Web of Science][Medline]

9 Olliver A, Valle M, Chaslus-Dancla E, et al. (2004) Role of an acrR mutation in multidrug resistance of in vitro-selected fluoroquinolone-resistant mutants of Salmonella enterica serovar Typhimurium. FEMS Microbiol Lett 238:267–72.[Web of Science][Medline]

10 Ge B, McDermott PF, White DG, et al. (2005) Role of efflux pumps and topoisomerase mutations in fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli. Antimicrob Agents Chemother 49:3347–54.[Abstract/Free Full Text]

11 Visalli MA, Murphy E, Projan SJ, et al. (2003) AcrAB multidrug efflux pump is associated with reduced levels of susceptibility to tigecycline (GAR-936) in Proteus mirabilis. Antimicrob Agents Chemother 47:665–9.[Abstract/Free Full Text]

12 National Committee for Clinical Laboratory Standards. (2003) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically: Approved Standard M7-A6 (NCCLS, Wayne, PA, USA).

13 Vila J, Ruiz J, Marco F, et al. (1994) Association between double mutation in gyrA gene of ciprofloxacin-resistant clinical isolates of Escherichia coli and MICs. Antimicrob Agents Chemother 38:2477–9.[Abstract/Free Full Text]

14 Vila J, Ruiz J, Goni P, et al. (1996) Detection of mutations in parC in quinolone-resistant clinical isolates of Escherichia coli. Antimicrob Agents Chemother 40:491–3.[Abstract]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
58/3/673    most recent
dkl297v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Saito, R.
Right arrow Articles by Koike, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Saito, R.
Right arrow Articles by Koike, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?