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 Ricci, V.
Right arrow Articles by Piddock, L. J. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ricci, V.
Right arrow Articles by Piddock, L. J. V.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, April 2004, p. 1344-1346, Vol. 48, No. 4
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.4.1344-1346.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Role of Topoisomerase Mutations and Efflux in Fluoroquinolone Resistance of Bacteroides fragilis Clinical Isolates and Laboratory Mutants

Vito Ricci,1 Marnie L. Peterson,1,2 John C. Rotschafer,2 Hannah Wexler,3 and Laura J. V. Piddock1*

Antimicrobial Agents Research Group, Division of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, United Kingdom,1 University of Minnesota, Minneapolis, Minnesota 55455,2 Department of Medicine, University of California at Los Angeles School of Medicine, Los Angeles, California 900243

Received 21 May 2003/ Returned for modification 22 November 2003/ Accepted 19 December 2003


arrow
ABSTRACT
 
Twelve laboratory mutants and 32 ciprofloxacin-resistant isolates of Bacteroides fragilis were examined for the mechanism(s) of fluoroquinolone resistance. Five mutants had mutations in gyrA. One mutant and two clinical isolates contained a mutation in gyrB. Eight mutants and five clinical isolates accumulated significantly less ciprofloxacin than did wild-type isolates; the mutants and clinical isolates were restored to wild-type characteristics when carbonyl cyanide m-chlorophenylhydrazone was used.


arrow
TEXT
 
DNA gyrase is the primary target of fluoroquinolones for most gram-negative bacteria (3, 4, 6). DNA topoisomerase IV is also a primary target for specific fluoroquinolones in certain gram-positive bacteria (15, 16). Studies to explore mechanisms of fluoroquinolone resistance in Bacteroides fragilis have identified mutations in gyrA as well as active efflux of norfloxacin (2, 7, 9, 10). However, analysis of the literature suggests that gyrA is a secondary (not primary) target of fluoroquinolones in B. fragilis (2, 10). The aim of this study was to identify the mechanism(s) of resistance in fluoroquinolone-resistant B. fragilis selected in vitro from agar and a pharmacodynamic (PD) model compared with those of 32 ciprofloxacin-resistant clinical isolates from the United States.

The MIC of each antibiotic was determined for each strain with the agar doubling-dilution method described by the British Society for Antimicrobial Chemotherapy (1). Mutants were selected as described previously (5) from B. fragilis NCTC9343/ATCC25285 (Z14) on Wilkins and Chalgren agar in a single step at 2x MIC of trovafloxacin, moxifloxacin, and ciprofloxacin at mutation frequencies of 2.6 x 10-10, 6.6 x 10-9, and 4.5 x 10-9, respectively. In vitro mutants were also selected in a PD model (11, 12) from Z14, a clinical isolate (Z54), and ATCC 23745 (Z55). From Z14, three mutants (Z50, Z72, and Z73) were selected with trovafloxacin and two mutants (Z69 and Z70) were selected with moxifloxacin. From Z54, one mutant (Z51) was selected with levofloxacin and one mutant (Z52) was selected with trovafloxacin. From Z55, two mutants (Z48 and Z49) were selected with sparfloxacin.

Except for garenoxacin and isolate Z72, the MICs of all agents were 2- to 16-fold higher for all mutants than those seen with their parent strain (Table 1). Mutants selected with trovafloxacin on agar (isolate Z56) had a smaller decrease in susceptibility to garenoxacin than mutants selected in the PD model (Z50 and Z72). To study any role of efflux in fluoroquinolone resistance, the efflux inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used. For all parent strains and mutants (except Z69 and Z70), CCCP lowered the MIC of ciprofloxacin two- to fourfold. A total of 32 ciprofloxacin-resistant strains isolated in 1999 from a tertiary hospital in the United States were also investigated. Two isolates were inhibited by >=8 µg of ciprofloxacin/ml, 15 were inhibited by >=16 µg of ciprofloxacin/ml, 10 were inhibited by >=32 µg of ciprofloxacin/ml, and 5 were inhibited by >=64 µg of ciprofloxacin/ml (Table 1). Compared with the laboratory mutants, CCCP had a smaller effect on the ciprofloxacin MICs for these isolates. For comparison, six ciprofloxacin-sensitive isolates collected in the United Kingdom in 2002 were examined; all required 4 µg of ciprofloxacin/ml for inhibition.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Phenotype and genotype of B. fragilis laboratory mutants and clinical isolates

Amplification of a 267-bp product of gyrA was achieved using primers BFA1 (5'-ACTACTCCATGTCGGTCATC-3') and BFA2 (5'-CAGAACCGAAGTTACCTTGC-3'). Amplification of a 260-bp product of gyrB was achieved using primers BFB1 (5'-CTATGTCAGGTGGCGGTCTT-3') and BFB2 (5'-GTCTTCTTCCGTTCCGATAG-3'). The PCR parameters were initial denaturation at 95°C for 5 min followed by 30 cycles of 95°C for 30 s, 52°C for 30 s (gyrA) or 53°C for 30 s (gyrB), and extension at 72°C for 30 s, with a final extension at 72°C for 10 min. DNA sequences of amplimers were determined commercially on an LI-COR 4200 automatic DNA sequencer (MWG Biotech, Ebersberg, Germany).

The only strains of B. fragilis in the present study to have mutations in gyrA were the 12 mutants selected in the PD model. A total of 5 of the 12 mutants had a substitution in GyrA (Ser82 to Phe; equivalent to Ser83 of Escherichia coli) and these strains (Z49, Z50, Z51, Z52, and Z72) were the strains most resistant to fluoroquinolones. Two mutants (Z69 and Z70) had different substitutions in GyrA at position 81 (Asp to Asn and Asp to Gly, respectively). The MICs of fluoroquinolones for mutants with substitutions at Asp81 were lower than those for mutants with substitutions at Ser82. Only one laboratory mutant selected in the PD model, Z73, contained a mutation in gyrB, replacing Gly478 with Lys. Five of the clinical isolates contained a mutation in gyrB, replacing Leu415 with Val (Table 1). The increases in fluoroquinolone MICs for mutants with mutations in gyrB were only increased two- to eightfold compared to the results seen with the controls. It is hypothesized that as so few ciprofloxacin-resistant mutants and clinical isolates contained a mutation in the quinolone resistance-determining regions (QRDR) of gyrA or gyrB, all mutants from the PD model and clinical isolates with mutations in these genes are at least second-step mutants. Previously reported studies focused upon identifying mutations in the QRDR of gyrA in either laboratory mutants or clinical isolates of B. fragilis (2, 9, 10), with only one of these studies exploring the QRDR of both gyrA and gyrB (10). Analyses of data from all studies indicate that mutations in gyrA or gyrB are usually not the first step to resistance.

Topoisomerase IV has been shown to be a secondary target for fluoroquinolone action in E. coli, in which high-level resistance (ciprofloxacin MICs >= 32 µg/ml) is commonly associated with at least one mutation in gyrA and another mutation in the gene encoding the A subunit of topoisomerase IV (parC) (3, 4, 6). In Staphylococcus aureus, however, mutations in the A subunit gene (grlA) can be selected prior to those in gyrA (16). Varon et al. (15) also suggested that parC and gyrA of S. pneumoniae might be interchangeable initial targets of certain fluoroquinolones, such as ciprofloxacin, sparfloxacin, and moxifloxacin. Due to the overwhelming amount of data supporting the idea of a role of topoisomerase IV (ParC/ParE) in fluoroquinolone resistance in other species, the role of parC and parE in B. fragilis was investigated. A parC/parE homologue was identified, but as yet no role in fluoroquinolone resistance has been established (M. L. Peterson and L. J. V. Piddock, unpublished data).

Both Miyamae et al. and Ricci and Piddock have demonstrated that B. fragilis actively causes the efflux of norfloxacin (7, 13). More recently Miyamae et al. described bexA from B. thetaiotaomicron (8). The BexA transporter has been shown to be responsible for the efflux of norfloxacin, ciprofloxacin, and ethidium bromide and is a member of the multidrug and toxic compound extrusion family. The concentrations of ciprofloxacin accumulated by all strains in the present study were measured by a fluorescence assay as previously described (14). All experiments were performed in duplicate on three separate days to obtain the mean value, from which the standard deviation was calculated. Differences in accumulation values between different experiments were analyzed by Student's t test. A P value of <0.05 was considered significant.

All mutants selected from isolate Z14 accumulated significantly less ciprofloxacin than Z14 (Table 1). Addition of 100 µM CCCP significantly increased the steady-state concentrations (SSC) of ciprofloxacin accumulated by all strains, including Z14; Z14 accumulated approximately 120 ng of ciprofloxacin/mg (dry weight) of cells. The concentrations of ciprofloxacin accumulated by mutants of Z54 and Z55 were similar to the results seen with the respective parent strains (data not shown). Six ciprofloxacin-sensitive clinical isolates of ATCC 23745 (strain Z55) and M97-117 (Z54; ciprofloxacin-sensitive clinical isolates from the United States) accumulated significantly less ciprofloxacin (40 to 50 ng of ciprofloxacin/µg [dry weight] of cells) than did the type strain for the species NCTC 9343/ATCC 25285 (Z14). Compared with the results seen with respect to the mean concentration of ciprofloxacin accumulated by the sensitive isolates, only five ciprofloxacin-resistant isolates (Z20, Z35, Z23, Z28, and Z31) accumulated significantly lower concentrations. CCCP increased the SSC for all isolates (irrespective of susceptibility) to 80 to 100 ng of ciprofloxacin/mg (dry weight) of cells.

In summary, mutations were observed in gyrA and gyrB but for 22 clinical isolates no mutations were revealed. Further studies are in progress to identify the fluoroquinolone resistance mechanisms in these isolates. Eight laboratory mutants and five isolates accumulated significantly less ciprofloxacin than the parent strain. Addition of CCCP significantly increased these concentrations, suggesting that enhanced efflux might play a role in fluoroquinolone resistance in some strains of B. fragilis.

In conclusion, mutations were observed in gyrA and gyrB; for some mutants and isolates, the accumulation data suggest either enhanced efflux or decreased uptake. Current work is characterizing these strains further.


arrow
ACKNOWLEDGMENTS
 
We are grateful to Bayer AG for supporting Vito Ricci in the early stages of this work and Health Partners Research Foundation for supporting Marnie Peterson.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: University of Birmingham, Div. of Immunity and Infection, Birmingham B15 2TT, United Kingdom. Phone: 44-121-414-6969. Fax: 44-121-414-3599. E-mail: l.j.v.piddock{at}bham.ac.uk. Back


arrow
REFERENCES
 
    1
  1. Andrews, J. M. 2001. Determination of minimum inhibitory concentrations. J. Antimicrob. Chemother. 48(Suppl. S1):5-16.[Abstract]
    2. 2
  2. Bachoual, R., L. Dubreuil, C. J. Soussy, and J. Tankovic. 2000. Roles of gyrA mutations in resistance of clinical isolates and in vitro mutants of Bacteroides fragilis to the new fluoroquinolone trovafloxacin. Antimicrob. Agents Chemother. 44:1842-1845.[Abstract/Free Full Text]
    3. 3
  3. Bagel, S., V. Hullen, B. Weidemann, and P. Heisig. 1999. Impact of gyrA and parC mutations on quinolone resistance, doubling time, and supercoiling degree of Escherichia coli. Antimicrob. Agents Chemother. 43:868-875.[Abstract/Free Full Text]
    4. 4
  4. Everett, M. J., Y. F., Jin, V. Ricci, and L. J. V. Piddock. 1996. Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals. Antimicrob. Agents Chemother. 40:2380-2386.[Abstract]
    5. 5
  5. Griggs, D. J., H. Marona, and L. J. V. Piddock. 2003. Selection of moxifloxacin-resistant Staphylococcus aureus compared with five other fluoroquinolones. J. Antimicrob. Chemother. 51:1403-1407.[Abstract/Free Full Text]
    6. 6
  6. Heisig, P. 1996. Genetic evidence for a role of parC mutations in development of high-level fluoroquinolone resistance in Escherichia coli. Antimicrob. Agents Chemother. 40:879-885.[Abstract]
    7. 7
  7. Miyamae, S., H. Nikaido, Y. Tanaka, and F. Yoshimura. 1998. Active efflux of norfloxacin by Bacteroides fragilis. Antimicrob. Agents Chemother. 42:2119-2121.[Abstract/Free Full Text]
    8. 8
  8. Miyamae, S., O. Ueda, F. Yoshimura, J. Hwang, Y. Tanaka, and H. Nikaido. 2001. A MATE family multidrug efflux transporter pumps out fluoroquinolones in Bacteroides thetaiotaomicron. Antimicrob. Agents Chemother. 45:3341-3346.[Abstract/Free Full Text]
    9. 9
  9. Oh, H. C. E. Nord, L. Barkholt, M. Hedberg, and C. Edlund. 2000. Ecological disturbances in intestinal microflora caused by clinafloxacin, an extended-spectrum quinolone. Infection 28:272-277.[CrossRef][Medline]
    10. 10
  10. Onodera, Y., and K. Sato. 1999. Molecular cloning of the gyrA and gyrB genes of Bacteroides fragilis encoding DNA gyrase. Antimicrob. Agents Chemother. 43:2423-2429.[Abstract/Free Full Text]
    11. 11
  11. Peterson, M. L., L. B. Hovde, D. H. Wright, A. D. Hoang, J. K. Raddatz, P. J. Boysen, and J. C. Rotschafer. 1999. Fluoroquinolone resistance in Bacteroides fragilis following sparfloxacin exposure. Antimicrob. Agents Chemother. 43:2251-2255.[Abstract/Free Full Text]
    12. 12
  12. Peterson, M. L., L. B. Hovde, D. H. Wright, G. H. Brown, A. D. Hoang, and J. C. Rotschafer. 2002. Pharmacodynamics of trovafloxacin and levofloxacin against Bacteroides fragilis in an in vitro pharmacodynamic model. Antimicrob. Agents Chemother. 46:203-210.[Abstract/Free Full Text]
    13. 13
  13. Ricci, V., and L. J. V. Piddock. 2000. Accumulation of norfloxacin by Bacteroides fragilis. Antimicrob. Agents Chemother. 44:2361-2366.[Abstract/Free Full Text]
    14. 14
  14. Ricci, V., and L. Piddock. 2003. Accumulation of garenoxacin by Bacteroides fragilis compared with that of five fluoroquinolones. J. Antimicrob. Chemother. 52:605-609.[Abstract/Free Full Text]
    15. 15
  15. Varon, E., C. Janoir, M. D. Kitzis, and L. Gutmann. 1999. ParC and GyrA may be interchangeable initial targets of some fluoroquinolones in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 43:302-306.[Abstract/Free Full Text]
    16. 16
  16. Yamagishi, J., T. Kojima, Y. Oyamada, K. Fujimoto, H. Hattori, S. Nakamura, and M. Inoue. 1996. Alterations in the DNA topoisomerase IV grlA gene responsible for quinolone resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 40:1157-1163.[Abstract]

Antimicrobial Agents and Chemotherapy, April 2004, p. 1344-1346, Vol. 48, No. 4
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.4.1344-1346.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Wexler, H. M. (2007). Bacteroides: the Good, the Bad, and the Nitty-Gritty. Clin. Microbiol. Rev. 20: 593-621 [Abstract] [Full Text]  
  • Pumbwe, L., Glass, D., Wexler, H. M. (2006). Efflux Pump Overexpression in Multiple-Antibiotic-Resistant Mutants of Bacteroides fragilis.. Antimicrob. Agents Chemother. 50: 3150-3153 [Abstract] [Full Text]  
  • Pumbwe, L., Chang, A., Smith, R. L., Wexler, H. M. (2006). Clinical significance of overexpression of multiple RND-family efflux pumps in Bacteroides fragilis isolates. J Antimicrob Chemother 58: 543-548 [Abstract] [Full Text]  
  • Pumbwe, L., Ueda, O., Yoshimura, F., Chang, A., Smith, R. L., Wexler, H. M. (2006). Bacteroides fragilis BmeABC efflux systems additively confer intrinsic antimicrobial resistance. J Antimicrob Chemother 58: 37-46 [Abstract] [Full Text]  
  • Poole, K. (2005). Efflux-mediated antimicrobial resistance. J Antimicrob Chemother 56: 20-51 [Abstract] [Full Text]  
  • Gruger, T., Nitiss, J. L., Maxwell, A., Zechiedrich, E. L., Heisig, P., Seeber, S., Pommier, Y., Strumberg, D. (2004). A Mutation in Escherichia coli DNA Gyrase Conferring Quinolone Resistance Results in Sensitivity to Drugs Targeting Eukaryotic Topoisomerase II. Antimicrob. Agents Chemother. 48: 4495-4504 [Abstract] [Full Text]  
  • Peric, M., Jacobs, M. R., Appelbaum, P. C. (2004). Antianaerobic Activity of a Novel Fluoroquinolone, WCK 771, Compared to Those of Nine Other Agents. Antimicrob. Agents Chemother. 48: 3188-3192 [Abstract] [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 Ricci, V.
Right arrow Articles by Piddock, L. J. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ricci, V.
Right arrow Articles by Piddock, L. J. V.

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