1 Toronto Centre for Antimicrobial Research and Evaluation (ToCARE), Room 1483, Department of Microbiology, Mount Sinai Hospital, 600 University Avenue, Toronto; 2 Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada; 3 Triangle Research and Development Center, Kfar Quara, Israel; 4 The Infectious Diseases Unit, Sheba Medical Center, Tel Aviv University School of Medicine, Tel Hashomer, Israel; 5 GR Micro Ltd., London, UK
Received 20 January 2004; returned 9 March 2004; revised 19 April 2004; accepted 21 April 2004
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Abstract |
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Methods: Fluoroquinolone-resistant isolates of the Sterne and Russian Anthrax Vaccine STi strains were obtained following serial passage in the presence of increasing concentrations of four different fluoroquinolones. The quinolone-resistance-determining regions of the type II topoisomerase genes from the resistant strains were amplified by PCR and characterized by DNA sequence analysis. The MICs in the presence and absence of reserpine were determined using broth microdilution as a means of detecting active efflux.
Results: Single and double amino acid substitutions in the GyrA (Ser-85-Leu; Glu-89-Arg/Gly/Lys) and GrlA (Ser-81-Tyr; Val-96-Ala; Asn-70-Lys) were most common. A single amino acid substitution in GyrB (Asp-430-Asn) was also identified. Efflux only applied to isolates selected for by either levofloxacin or ofloxacin.
Conclusions: Specific amino acid substitutions in the type II topoisomerase enzymes significantly contributed to the development of high-level fluoroquinolone resistance in B. anthracis. However, notable differences between the strains and the drugs tested were identified including the role of efflux and the numbers and types of mutations identified.
Keywords: anthrax , DNA gyrase , topoisomerase IV
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Introduction |
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Currently recommended antibiotics remain effective in the treatment of anthrax, however, there is a fear that future attacks might incorporate drug-resistant strains which would be problematic to treat.9 To prepare for this possibility, the mechanisms of resistance utilized by B. anthracis need to be fully investigated and understood, particularly the potential for development of fluoroquinolone resistance.
Fluoroquinolones are broad-spectrum antimicrobial agents that act by inhibiting the bacterial type II topoisomerases, DNA gyrase and topoisomerase IV.10 In Bacillus, these heterotetrameric enzymes are of the form GyrA2GyrB2 and GrlA2GrlB2 for DNA gyrase and topoisomerase IV, respectively.11 Resistance is driven primarily by the principles of natural selection, whereby specific chromosomal mutations occur in discrete regions of the topoisomerase genes, designated as the quinolone-resistance-determining-region (QRDR).10 These nucleotide changes occur in a stepwise fashion, with each additional mutation adding to a further increase in the MIC of the drug to which the bacteria were exposed. Moreover, an increase in the MIC of one fluoroquinolone leads to MIC increases in other members of the class although this is not always the case for newer fluoroquinolones as multiple nucleotide changes are often required for resistance. More recently, resistance caused by enhanced expression of endogenous efflux systems that pump drug out of the cell has been described in respiratory pathogens, however their role in high-level resistance remains unclear.12
Despite the obvious global importance of B. anthracis, it was not until recently that a mechanism for fluoroquinolone resistance in this organism was reported.13 Price and co-workers13 used the attenuated plasmid-cured Ames strain and showed that a substitution at Ser-85 or Glu-89 in GyrA was associated with low-level ciprofloxacin resistance (MIC 0.5 mg/L) and that an additional substitution at either Ser-81 or Glu-85 in GrlA and/or a substitution at Asn-475 or Asp-437 in GrlB was associated with higher levels of resistance (MIC 864 mg/L). As the newer respiratory fluoroquinolones like levofloxacin and moxifloxacin are likely to be more efficacious than older agents such as ciprofloxacin and ofloxacin in the treatment of inhalational anthrax,14 we explored the mechanisms of high-level resistance to all four of these agents.
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Materials and methods |
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Fluoroquinolone resistance was selected for in the B. anthracis Sterne (a gift from the Colorado Serum Institute, Denver, CO, USA) and the Russian Anthrax Vaccine STi (purchased commercially in Moscow, Russia) strains by serial passage on Brain Heart Infusion (BHI) agar (Difco Laboratories, Detroit, MI, USA) supplemented with increasing concentrations of drugs. The fluoroquinolones used included ofloxacin (Aventis, Israel), levofloxacin (Aventis, France), ciprofloxacin and moxifloxacin (Bayer, Germany). All drugs were prepared according to the manufacturer's instructions. Bacteria were first grown to become vegetative forms for 1824 h at 37°C in BHI broth and subsequently transferred to a non-selective BHI agar plate. Ten colonies were transferred using sterile toothpicks from the original non-selective BHI agar plate, to a plate containing the initial MIC of each quinolone and thereafter onto a plate containing the next two-fold higher concentration. This procedure was repeated 30 times at which point isolates were chosen and characterized for mechanisms of resistance.
Susceptibility testing
MICs were determined using the broth microdilution method according to the NCCLS standards for Staphylococcus aureus.9,15 Susceptibility testing in the presence of 10 mg/L of reserpine (Sigma Chemical Co., St. Louis, MO, USA) was carried out to examine for evidence of active efflux. The efflux-positive Escherichia coli Yoni strain was used as the positive control. Strains for which there was a four-fold or greater decrease in the MIC of the selecting fluoroquinolone in the presence of reserpine were considered to be positive for reserpine-inhibited efflux.
PCR and DNA analysis
Genomic DNA from 109 bacterial cells was harvested and purified using the DNeasy purification system according the manufacturer's instructions (Qiagen Inc.). The QRDR for each isolate was amplified by PCR and sequenced in both directions. PCR and DNA sequencing primers are listed in Table 1 and were designed using genomic DNA sequence data from GenBank16 for B. anthracis A201217 [Accession No. NC_003995], B. subtilis11 [Accession No. NC_000964] and Streptococcus pneumoniae R618 [Accession No. NC_003098]. For amplification, a 50 µL total reaction mixture was used containing 5 µL of genomic DNA extract, 0.1 µM of each primer, 0.2 mM deoxynucleoside triphosphates, 1 x PCR buffer, 2.5 U Taq DNA polymerase (Invitrogen Life Technologies, Burlington, Ontario, Canada), 1.5 mM MgCl2 (for grlA), 4 mM (grlB), 1.5 mM (gyrA) and 2 mM (gyrB), and deionized water. Amplification was carried out in a Perkin-Elmer 9600 thermocycler: initial denaturation at 94°C for 4 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 53.8°C (for grlA), 50.2°C (grlB), 51.6°C (gyrA) or 52.5°C (gyrB) for 30 s and extension at 72°C for 30 s. A final extension of 5 min at 72°C was carried out for each amplicon. Amplicons were purified with the QIAquick PCR purification kit (Qiagen Inc., Mississauga, Ontario, Canada). DNA sequencing was carried out by ABI prism Big Dye terminator cycle sequencing with the ABI 377 automated sequencer (PE Applied Biosystems, Mississauga, Ontario, Canada). Primer design and sequence comparisons were carried out with the primer tool analysis and multiple-alignment sequence functions of the Vector NTI Suite software, respectively (InforMax Inc., Bethesda, MD, USA).
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Results |
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All of the fluoroquinolones tested selected for resistance with MIC increases ranging from 16-fold to 2048-fold (Table 2). However, the MIC of the selecting agent was always greater than, or equal to, the MIC of the other fluoroquinolones. For example, a moxifloxacin-selected Sterne isolate had a moxifloxacin MIC of 32 mg/L but an ofloxacin, ciprofloxacin and levofloxacin MIC of only 4 mg/L. Likewise, an ofloxacin-selected STi isolate had an ofloxacin MIC of 32 mg/L but a ciprofloxacin, levofloxacin and moxifloxacin MIC of 1632 mg/L. Only those isolates selected for by levofloxacin and ofloxacin showed evidence of active efflux as measured by a reserpine inhibition assay (Table 2).
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The sequential selection of B. anthracis on increasing concentrations of drug resulted in the accumulation of QRDR mutations, however, the numbers and types of mutations selected varied, depending on the drug and strain tested (Table 2). All isolates had at least one amino acid substitution in GyrA, but only half had an additional substitution in GrlA; only one had an additional substitution in GrlB, and none had a substitution in GyrB. Whereas double substitutions in GyrA were noted in both Sterne and STi isolates, double substitutions in GrlA were only noted in STi isolates. Amino acid substitutions of Glu-89-Lys/Gly in GyrA were the most common as was the substitution of Ser-85-Leu (Table 2). A Glu-89-Arg substitution was also noted but only once. In GrlA, a substitution of Ser-81-Tyr was observed more frequently than substitutions of Val-96-Ala and Asn-70-Lys. The only identified GrlB substitution was Asp-430-Asn.
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Discussion |
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Although similarities between the amino acid substitutions identified in B. anthracis and those found in other Gram-positive bacteria are evident, significant differences are apparent. In S. pneumoniae and S. aureus, ciprofloxacin, ofloxacin and levofloxacin preferentially target and bind to topoisomerase IV, i.e. the ParC and GrlA subunits, respectively. By convention, these agents select for a first-step mutation in a gene that encodes topoisomerase IV, followed by a second-step mutation in a gene that encodes DNA gyrase. Third- and even fourth-step mutations have been reported in genes encoding topoisomerase IV and DNA gyrase, respectively.10 The reverse is however thought to be true for moxifloxacin and S. pneumoniae whereby the preferential target is DNA gyrase and the secondary target is topoisomerase IV. Notwithstanding that which is seen in other Gram-positive bacteria, the fact remains that all our mutants as well as those reported by Price et al. harboured a single, and in some instances a double substitution in GyrA. By comparison, not all harboured a substitution in GrlA. Taken together, this would suggest that DNA gyrase is the preferential target in B. anthracis. However, since moxifloxacin selected two GrlA substitutions but only one substitution in GyrA in the STi strain, it is possible that preferentially targeting occurs at the strain level and that moxifloxacin preferentially targets GrlA in this particular strain. Also noteworthy was our observation that the amino acid substitutions in GrlA were highly variable (Val-96-Ala, Ser-81-Tyr and Asn-70-Lys) in comparison to the rather conserved substitutions identified in GyrA. Third-step mutations also appeared to be unpredictable and strain-dependent as moxifloxacin selected a third-step GyrA substitution at either Glu-89 or Ser-85 in the Sterne strain but a third-step GrlA mutation at either Asn-70 or Ser-81 in the STi strain. Although overall, our work and that of Price et al.13 support DNA gyrase and topoisomerase IV as preferential and secondary targets, respectively, more studies are needed to determine whether certain mutations are indeed drug and/or strain specific as identified with moxifloxacin and STi.
The roles of the GyrB and GrlB subunits in resistance are also unclear as has been previously reported in other Gram-positive bacteria. Under no circumstances did we select a substitution in GyrB although Price et al.13 reported two when testing the Ames strain with ciprofloxacin. By comparison, we report a novel substitution in GrlB (Asp-430-Asn).
In our study, a direct correlation between presence of QRDR mutations and increasing MIC was not always evident as has been demonstrated with other bacteria. Price and co-workers13 showed that a substitution of Ser-85-Leu or Glu-89-Lys in GyrA was associated with low-level ciprofloxacin resistance (MIC of 0.5 mg/L), whereas an additional substitution of Ser-81-Phe/Tyr or Glu-85-Lys in GrlA was associated with higher MICs (832 mg/L). In their study, isolates with a ciprofloxacin MIC of 64 mg/L had either an additional substitution in GrlA (Ser-81-Phe) or GyrB (Asn-475-Asp or Asp-437-Asn), with or without a double substitution in GyrA (Ser-85-Leu and Glu-89-Lys/Ala). In contrast, we have shown that some isolates with ciprofloxacin MICs of 864 mg/L had either a single or double substitution in GyrA but no additional QRDR substitutions in any of the other three subunits suggesting that substitutions outside the QRDR of either the GrlA, GrlB or GyrB subunits may be involved in high-level ciprofloxacin resistance in the Sterne and STi strains. This observation contradicts the conventional stepwise selection of QRDR mutations in other Gram-positive bacteria and indicates the need for a better definition of the QRDR region in B. anthracis.
Efflux mediated by the proton-dependent membrane protein, NorA in S. aureus and PmrA in S. pneumoniae has been previously shown to contribute to fluoroquinolone resistance.12 However, this particular pump has been shown to be less efficient in effecting efflux of newer agents with bulky C-7 substituents, such as moxifloxacin.19 Similar pumps in B. subtilis (BmrA)20 and in B. anthracis17 have also been identified but their role in high-level fluoroquinolone resistance remains unclear. Notwithstanding this uncertainty, we showed evidence for proton-dependent efflux in isolates selected for by levofloxacin and ofloxacin but not with ciprofloxacin as reported by Price et al.13 As expected, those isolates selected for by moxifloxacin showed no evidence of active efflux as tested for by reserpine and thereby support the notion that fluoroquinolones with bulky substituents are not affected by such pumps. Although it has yet to be determined whether the BmrA homologue in B. anthracis17 contributes to resistance, there is reason to believe that it or other drug efflux systems play a significant role and may help explain the discrepancies in resistance profiles between the different strains.
Our findings indicate the potential and relative ease for the emergence of resistance in B. anthracis to antibiotics used for treatment of anthrax. Our identification of differences between strains poses additional problems and in terms of national security it is vital we explore further the mechanisms of resistance not only to the fluoroquinolones but also to other commonly used antibiotics in the event of another bioterrorist attack.
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Footnotes |
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References |
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