Plasmid-mediated complementation of gyrA and gyrB in fluoroquinolone-resistant Bacteroides fragilis

M. L. Peterson1,2, J. C. Rotschafer1 and L. J. V. Piddock2,*

1 University of Minnesota, Minneapolis, MN, USA; 2 Antimicrobial Agents Research Group, Division of Immunity and Infection, School of Medicine, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

Received 4 April 2003; returned 1 May 2003; revised 12 June 2003; accepted 12 June 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To identify whether mutations in gyrA and gyrB confer fluoroquinolone resistance in Bacteroides fragilis.

Methods: Eight fluoroquinolone-resistant (FQR) strains were complemented with plasmid-mediated B. fragilis wild-type gyrA (pMP1) and gyrB (pMP2), and MICs determined. Sequence analysis of the gyrA and gyrB quinolone resistance determining region (QRDR) was performed for all strains.

Results: MICs of fluoroquinolones were two- to 32-fold higher than wild-type for all mutants. Five mutants had a substitution in GyrA (Ser-82->Phe), one mutant had a substitution in GyrA (Asp-81->Gly), one mutant had a substitution in GyrB (Glu-478->Lys), and one resistant strain did not contain mutations in the QRDR of gyrA or gyrB. Following complementation with pMP1 or pMP2, the MICs of fluoroquinolones were reduced two- to 32-fold for the mutants.

Conclusion: These studies verify that substitutions in GyrA and GyrB confer resistance in B. fragilis. Other mechanisms are also responsible for resistance since not all resistant strains fully complemented to the wild-type phenotype.

Keywords: anaerobe, quinolone, antibiotic resistance


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteroides fragilis, the most recognized anaerobic pathogen, is often isolated from anaerobic bacteraemia and intra-abdominal infections.1,2 In addition to possessing in vitro antimicrobial activity against aerobic organisms, newer fluoroquinolones have in vitro activity against anaerobes, including B. fragilis.1,3,4 However, Snydman et al.2 recently reported increasing fluoroquinolone resistance in B. fragilis. In previous studies, mutations in the quinolone resistance determining region (QRDR) of gyrA were identified, suggesting that GyrA contributes to resistance in B. fragilis.57 Despite the identification of mutations in gyrA, many first-step laboratory mutants and fluoroquinolone-resistant (FQR) clinical isolates lacked mutations in the QRDR of this gene, suggesting other mechanisms of resistance. No mutations in gyrB have been reported, and no topoisomerase IV genes have been described.

Plasmid-mediated wild-type gyrA is known to confer fluoroquinolone susceptibility to aerobic Gram-negative bacteria with mutations in gyrA.8 In light of the latter, and published data suggesting that mutations in the QRDR of gyrA of B. fragilis may not be the primary mechanism of fluoroquinolone resistance, we sought to construct plasmids containing wild-type B. fragilis gyrA or gyrB genes, to analyse FQR laboratory mutants, and identify mutations in these genes outside the QRDR or other novel mechanisms of resistance.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and mutant selection

Fluoroquinolone-susceptible B. fragilis NCTC 9342/ATCC 28285, ATCC 23745 and clinical isolate M97-117 served as controls. Eight FQR mutants were selected from controls following exposure to trovafloxacin, moxifloxacin, levofloxacin or sparfloxacin in an in vitro pharmacodynamic model.3,4 B. fragilis strains were grown at 37°C in Wilkens–Chalgren (WC) media within a Bactron IV anaerobic chamber (Anaerobe Systems, Morgan Hill, CA, USA). Escherichia coli, DH5{alpha}mcr, was used in transformations, and was grown in Luria–Bertani (LB) medium.

Antibiotics and susceptibility determination

Antibiotics were supplied and used according to the manufacturers’ instructions: ampicillin, trimethoprim, gentamicin, tetracycline (Sigma, St. Louis, MO, USA); levofloxacin (R. W. Johnson Pharmaceutical Research Institute, Spring House, PA, USA); trovafloxacin (Pfizer Inc., Groton, CT, USA); ciprofloxacin and moxifloxacin (Bayer Pharmaceuticals, West Haven, CT, USA); sparfloxacin (Rhone-Poulenc Rorer, Collegeville, PA, USA). The MIC of ciprofloxacin, trovafloxacin, levofloxacin and moxifloxacin were determined in accordance with NCCLS methods. Tetracycline (3 mg/L) was used for plasmid maintenance during MIC determinations of B. fragilis transformants.

PCR and sequencing of the QRDR of gyrA and gyrB

A 267 bp gyrA fragment and a 260 bp gyrB fragment were amplified by PCR. For gyrA, the forward primer was 5'-ACTACTCCATGTCGGTCATC-3' (positions 65 to 84) and the reverse primer 5'-CAGAACCGAACTTACCTTGC-3' (312 to 331). For gyrB, the forward primer was 5'-CTATGTCAGGTGGCGTCTT-3' (positions 1229 to 1248) and the reverse primer 5'-GTCTTCTTCCGTTCCGATAG-3' (1469 to 1488). PCR was carried out in a volume of 0.05 mL containing 100 pmol each primer, 1 x PCR buffer, 0.2 mM dNTPs, 1.5 mM MgCl2, 1.25 U of Taq polymerase (ABgene, Surrey, UK) and 100 ng of DNA template, isolated by DNase Spin Cell culture kit (Bioline, Randolph, MA, USA). PCR conditions were as follows: one cycle of 95°C for 5 min; 30 cycles of 95°C for 30 s, 52°C (gyrA) or 53°C (gyrB) for 30 s and 72°C for 30 s; one cycle of 72°C for 10 min. PCR products were purified using a Qiaquick PCR purification kit (Qiagen, Valencia, CA, USA) and sequenced.

Construction of pMP1 (gyrA) and pMP2 (gyrB)

gyrA and gyrB plus promoter regions were amplified by PCR with template DNA from B. fragilis ATCC 25285/NCTC 9343. For gyrA (3053 bp), the forward primer was 5'-GGTGGATCCTACGCATTTCATATTGTTTGCATGC-3' (BamHI; gyrA positions –351 to –327); and the reverse primer was 5'-AGTTGTTAAGCTTTTGCGAAGTCAGG-3' (HindIII; 2677 to 2702).6 For gyrB (2471 bp), the forward primer was 5'-GATGGTACCAGATAAGTCGAGATGGCAAATC-3' (KpnI; gyrB positions –447 to –426); and the reverse primer was 5'-ATACTGCAGATTTTCCTTCAGCGCCG-3' (PstI; 2008 to 2024). PCR was carried out similarly except that 1 x cloned Pfu DNA polymerase reaction buffer and 2.5 U of Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA, USA) were used. PCR conditions were as follows: one cycle of 95°C for 5 min; 35 cycles of 95°C for 30 s, 61°C (gyrA) or 58°C (gyrB) for 30 s and 72°C for 9 min; one cycle of 72°C for 10 min.

Plasmids containing gyrA (pMP1; 11152 bp) and gyrB (pMP2; 10524 bp) were constructed by cloning wild-type gyrA and gyrB of B. fragilis ATCC 25285/NCTC9343 into shuttle vector pLYL01.9 The PCR products, gyrA and gyrB, were purified, digested with restriction enzymes (BamHI and HindIII: gyrA) and (KpnI and PstI: gyrB), ligated into pLYL01 and transformed into E. coli DH5{alpha}mcr. Transformants were selected on LB/ampicillin (100 mg/L)/IPTG /X-gal agar plates. The presence of the plasmids was verified by extracting plasmid DNA (Qiagen), performing restriction digestions (gyrA:BamHI and HindIII or gyrB:KpnI and PstI) and DNA sequencing.

Conjugation and transformation of B. fragilis strains with pLYL01, pMP1 and pMP2

pLYL01, pMP1 and pMP2 were mobilized by a co-resident IncPß plasmid, R751, from E. coli into B. fragilis by conjugation via filter mating. Transconjugants were selected on WC agar plates containing tetracycline (3 mg/mL), gentamicin (200 mg/mL) and 5% lysed horse blood. Colonies appeared after 48 h–1 week. The presence of the plasmid in the transconjugants was verified as above. Following identification and isolation of plasmids from B. fragilis, electroporation experiments were conducted by preparing and transforming competent cells of B. fragilis strains with pLYL01, pMP1 and pMP2. Transformants were selected on agar plates containing tetracycline (3 mg/L) and gentamicin (200 mg/L).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Susceptibility and characterization of FQR B. fragilis

Eight FQR strains were obtained from in vitro pharmacodynamic studies (Table 1). Four strains (Z50, Z52, Z72, Z73) were selected following trovafloxacin exposure (2–3.5 x MIC), one strain (Z70) following moxifloxacin exposure (1 x MIC), two strains (Z48 and Z49) following sparfloxacin exposure (1 x MIC) and one strain (Z51) following exposure to levofloxacin (4 x MIC). The MIC of the tested fluoroquinolones for the mutants were two- to 32-fold higher than for the parent strains (Table 1). DNA sequencing of the QRDR of gyrA and gyrB revealed that five mutants had a substitution in GyrA, from Ser-82->Phe, one had a substitution in GyrA from Asp-81->Gly and one had a substitution in GyrB from Glu-478->Lys. One resistant strain did not contain mutations in the QRDR of gyrA or gyrB.


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Table 1.  Bacterial strains, descriptions and susceptibilities pre- and post-transformed
 
Contribution of mutations in gyrA and gyrB to resistant phenotype

All strains were transformed with plasmids pMP1, pMP2 and pLYL01. The transformation frequency obtained for pLYL01 was 1 x 103 transformants/µg compared with 1 x 102 transformants/µg obtained for pMP1 and pMP2. Transformation of control strains B. fragilis ATCC 25285, ATCC 23745 and clinical isolate M97-117 with pLYL01, pMP1 and pMP2 had no effect on antimicrobial susceptibility. Following transformation of the FQR mutants with pMP1 and pMP2, the MICs of the fluoroquinolones decreased (Table 1).

Those strains with mutations in gyrA became more susceptible to fluoroquinolones in the presence of pMP1 (gyrA), whereas introduction of the vector control pLYL01 or pMP2 (gyrB) had no effect (Z49, Z50, Z51, Z52, Z70, Z72). Only two strains (Z70 and Z72) became as susceptible as their parent strain (ATCC 25285) when complemented with (pMP1). Z73, which contained a mutation in gyrB, also became as susceptible, or within one dilution of the MIC, to the fluoroquinolones as ATCC 25285 when complemented with (pMP2). No increase in susceptibility was apparent for Z48, the FQR strain with no mutation in the QRDR of gyrA or gyrB.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fluoroquinolone resistance in B. fragilis has been attributed to mutations in gyrA.57 However, despite these mutations in gyrA, many first-step laboratory mutants and resistant clinical isolates of B. fragilis lacked mutations in the QRDR of this gene, suggesting other resistance mechanisms. To identify whether mutations in gyrA and gyrB can confer fluoroquinolone resistance alone, plasmids were constructed containing wild-type B. fragilis gyrA or gyrB and used to complement mutants from three different strains.

Utilizing this approach, our study confirms that a single substitution in the QRDR of GyrA, Ser-82->Phe or Asp-81->Gly, confers fluoroquinolone resistance. We report for the first time that a single substitution in GyrB (Glu-478->Lys) alone confers a four- to eight-fold increase in MIC to fluoroquinolones. However, these data also suggest that other mechanisms, independent of GyrA or GyrB, contribute to resistance, as complementation did not always confer reversal to full wild-type susceptibility.

Previously, other researchers have utilized complementation with cloned wild-type gyrA or gyrB plasmids to discriminate between mutations in gyrA or gyrB and other resistance mechanisms. Nakamura et al.10 reported that E. coli gyrA and gyrB mutants became fully susceptible to nalidixic acid when transformed with a corresponding wild-type gyrA or gyrB gene, whereas a transport mutant did not. The existence of topoisomerase IV in B. fragilis has not been described, so its potential role in quinolone resistance cannot be determined.

These studies confirm that fluoroquinolone resistance in B. fragilis can be conferred by mutation in gyrA or gyrB. However, studies are warranted to rule out fluoroquinolone transport as an additional mechanism of fluoroquinolone resistance in B. fragilis, as well as the role of topoisomerase IV.


    Acknowledgements
 
This study was funded by HealthPartners Research Foundation (Minneapolis, MN, USA) and the British Society for Antimicrobial Chemotherapy (BSAC) (Birmingham, UK). The authors would also like to thank Dr Abigail Salyers and Nadja Shoemaker, Department of Microbiology, University of Illinois, Urbana, IL, USA, for their assistance and contribution of several bacterial strains containing necessary plasmids, including pLYL01. In addition, M.L.P. is grateful to Dr Patrick Schlievert for his continued support throughout this work.


    Footnotes
 
* Corresponding author. Tel: +44-121-414 6966; Fax: +44-121-414 3454; E-mail: l.j.v.piddock{at}bham.ac.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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