JAC Advance Access originally published online on October 24, 2006
Journal of Antimicrobial Chemotherapy 2006 58(6):1268-1273; doi:10.1093/jac/dkl421
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In vitro development of resistance to DX-619 and other quinolones in enterococci
Department of Medical Microbiology and Immunology, Center for Research in Anti-Infectives and Biotechnology Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA
*Corresponding author. Tel: +1-402-280-2921; Fax: +1-402-280-1875; E-mail: pwickman{at}creighton.edu
Received 26 June 2006; returned 31 July 2006; revised 25 August 2006; accepted 25 September 2006
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Abstract |
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Objectives: To investigate the molecular events involved in the development of quinolone resistance in enterococci.
Methods: Clinical isolates of Enterococcus faecium and Enterococcus faecalis were exposed to inhibitory and subinhibitory concentrations of DX-619, ciprofloxacin, levofloxacin, gatifloxacin and moxifloxacin. Mutational frequencies were calculated and susceptibility changes were determined. The quinolone resistance determining regions (QRDRs) of gyrA and parC in less-susceptible mutants were amplified by PCR and sequenced.
Results: Single-step mutants of E. faecalis and E. faecium were selected with all drugs. There were no differences in the frequencies of mutant selection among drugs, with frequencies ranging from 10–5 to 10–8. All single-step mutants were inhibited by 0.03–1 mg/L DX-619, 0.25–8 mg/L moxifloxacin, 0.5–8 mg/L gatifloxacin, 1–16 mg/L levofloxacin and 1–32 mg/L ciprofloxacin. No QRDR changes were observed in single-step mutants. Less-susceptible mutants selected after five passages on agar containing subinhibitory quinolone concentrations were inhibited by 0.12–8 mg/L DX-619, 1–64 mg/L moxifloxacin, 2–64 mg/L gatifloxacin and 2–128 mg/L levofloxacin and ciprofloxacin. QRDR changes were detected in only 9 of the 20 fifth-passage mutants, suggesting that mutations outside the purported QRDRs and/or other resistance mechanisms were also involved.
Conclusions: The relatively high frequencies at which single-step mutants were selected with all drugs indicate that caution is necessary if quinolones are to be considered for monotherapy of serious enterococcal infections. DX-619, the most potent quinolone, may have potential as an anti-enterococcal agent if sufficient concentrations can be safely attained in vivo.
Keywords: moxifloxacin , ciprofloxacin , levofloxacin , mutational frequency , gatifloxacin
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Introduction |
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Enterococci are a leading cause of serious infections in hospitalized patients.1,2 DX-619 is a novel des-fluoro (6) quinolone with potent anti-Gram-positive activity.3 With the aim of investigating its anti-enterococcal potential, a study was designed to compare it and several other quinolones for their activity and propensity to select less susceptible mutants. We also aimed to investigate the molecular events responsible for reduced quinolone susceptibility in Enterococcus faecalis and Enterococcus faecium. Previous studies have identified amino acid changes within the quinolone resistance determining regions (QRDRs) of the GyrA subunit of DNA gyrase (encoded by gyrA) and the ParC subunit of DNA topoisomerase IV (encoded by parC) in clinical isolates.4–6 To date, there has only been one report investigating the in vitro development of quinolone resistance in enterococci.7 That study only examined mutants selected with levofloxacin from a single strain of E. faecalis. Therefore, we felt a more extensive study that included strains of E. faecium, more quinolones and a greater number of mutants was warranted.
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Materials and methods |
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Bacterial strains and growth conditions
Mutants were selected from two clinical isolates of E. faecalis (one vancomycin-susceptible, one vancomycin-resistant) and five clinical isolates of E. faecium (one vancomycin-susceptible, four vancomycin-resistant). E. faecalis 178 and E. faecium 169 and 171 were provided by Daniel Sahm, Barnes Jewish Hospital, St Louis MO, USA. E. faecalis 114 and E. faecium 137 were provided by Dennis Schaberg, University of Michigan Medical Center, Ann Arbor, MI, USA, and Gail Woods, Medical College Hospitals, Elkins Park, PA, USA, respectively. E. faecium 127 and 144 were acquired from the Methodist Hospital, Omaha, NE, USA, and the Creighton University Medical Center, Omaha, NE, USA, respectively. E. faecalis ATCC 29212, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27583 were used as control strains for susceptibility testing.
Antibiotic susceptibility testing
Antibiotic susceptibility to DX-619 (Daiichi Pharmaceutical Co. Ltd, Tokyo, Japan), ciprofloxacin and moxifloxacin (Bayer AG, Pharma Research Center, Elberfeld, Germany), gatifloxacin (Bristol-Myers Squibb Co., Princeton, NJ, USA) and levofloxacin (The R. W. Johnson Pharmaceutical Research Institute, Spring House, PA, USA) was determined by CLSI agar dilution methodology using Mueller–Hinton agar (MHA) (Oxoid, Basingstoke, UK).8 Mutants selected from E. faecium 127 with levofloxacin, moxifloxacin and DX-619 grew poorly or not at all on MHA and were retested on brain heart infusion agar (BHIA) (Becton Dickinson, Sparks, MD, USA). Plates were inoculated with a Steers replicator to produce an inoculum of 104 cfu per spot and incubated at 37°C for 24 h.
Single-step mutant selection
Enterococci were grown in brain heart infusion broth (BHIB) (Becton Dickinson) to an OD540 of 0.5. Inocula of 
107–109 cfu were added to BHIA containing inhibitory concentrations (1x, 2x and 4x the MIC) of quinolone. The actual inoculum was determined with viable counts. After 48–72 h of incubation at 37°C, mutant colonies were counted to calculate spontaneous mutational frequencies.
Gradient plate mutant selection
Inocula of approximately 108 cfu were plated as a lawn culture onto BHIA plates containing a quinolone concentration gradient ranging from sub-MIC levels to a concentration of two times the MIC. After overnight incubation at 37°C, bacterial growth on the portion of the plate containing a subinhibitory concentration of quinolone was pushed with a sterile bacteriological loop across the growth-free portion of the plate containing inhibitory concentrations. Growth from the previous antibiotic gradient plate was used to seed a plate containing a higher gradient concentration. The procedure was repeated four times. Mutants were frozen after one and five passages and used for susceptibility testing and sequencing.
Mutational analysis by PCR amplification and DNA sequencing
The nucleotide sequences representing the QRDRs of gyrA and parC in selected mutants were amplified by PCR and compared with the corresponding sequences of the parental strains. QRDRs from E. faecalis were amplified with previously described primers: the forward primer Pr-EFGA1 and reverse primer Pr-EFGA4 for gyrA, and the forward primer Pr-EFPC1 and the reverse primer Pr-EFPC4 for parC.7 For E. faecium, a 196 bp fragment from gyrA was amplified with the forward primer 5'-CGGCGGCACCGTCACCGTCAACAG-3' and the reverse primer 5'-GAATTGGGTGTGACACCGGATAAAG-3'. For parC, a 151 bp fragment was amplified with the forward primer 5'-TTCCCGTGCATTTCGATCAGTACTTC-3' and the reverse primer 5'-CGTATGACAAAGGATTCCGTAAATC-3'. PCR experiments were conducted in a total volume of 50 µL containing 0.25 mM of each dNTP, 2 mM MgCl2, 0.5 µM of the appropriate primer, 1.25 U of Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) and 2 µL of genomic DNA that was isolated using a previously described method.9 PCR reactions were conducted for 25 cycles with the following parameters: 94°C for 30s, 50°C for 15s and 68°C for 2 min. PCR products were sequenced by automated PCR cycle-sequencing with dye-terminator chemistry using a DNA stretch sequencer (Applied Biosystems, Foster City, CA, USA). The DNA sequences of E. faecalis were BLAST searched against GenBank accession numbers AB059405 [GenBank] and AB059406 [GenBank] for gyrA and parC, respectively. For E. faecium, sequences were BLAST searched against accession numbers AF060881 [GenBank] and AB017811 [GenBank] for gyrA and parC, respectively.
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Results and discussion |
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DX-619 was the most potent agent against single-step and gradient plate mutants of E. faecalis and E. faecium. All single-step mutants were inhibited by 1 mg/L DX-619, 8 mg/L gatifloxacin and moxifloxacin, 16 mg/L levofloxacin and 32 mg/L ciprofloxacin (Tables 1 and 2). All fifth-passage gradient plate mutants were inhibited by 8 mg/L DX-619, 64 mg/L gatifloxacin and moxifloxacin, and 128 mg/L levofloxacin and ciprofloxacin (Tables 3 and 4).
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In single-step mutant selection experiments, spontaneous mutants of E. faecalis and E. faecium were selected at relatively high frequencies with each quinolone, ranging from 10–5 to 10–7 following exposure to DX-619, moxifloxacin and gatifloxacin, and 10–5 to 10–8 with ciprofloxacin and levofloxacin (Tables 1 and 2). Although the increases in quinolone MICs were generally small for the single step mutants (2- to 4-fold), the mutations did not affect all quinolones equally, with DX-619 being least affected. Of the 49 single-step mutants, only 19 (39%) exhibited
4-fold reductions in susceptibility to DX-619 and levofloxacin. However, the activity of levofloxacin was more compromised than DX-619 in that 8 of the 19 mutants (42%) exhibited 8-fold decreases in susceptibility to it while only 1 mutant (5.3%) exhibited a similar reduction in susceptibility to DX-619. In comparison, 26 (53%), 32 (65%) and 34 (69%) mutants demonstrated 4-fold or greater reductions in susceptibility to ciprofloxacin, moxifloxacin and gatifloxacin, respectively. The QRDRs of gyrA and parC were sequenced. Mutations were not detected in either the single-step mutants or the first-passage gradient plate mutants, suggesting that mechanisms other than target mutations in GyrA and ParC often contributed to decreased quinolone susceptibility. These findings are consistent with those of Onodera et al.7 who did not detect QRDR mutations in single-step mutants of E. faecalis selected with levofloxacin. Because we did not sequence gyrB or parE, the possibility of resistance-conferring mutations in these genes cannot be excluded.
In contrast to the first-passage mutants, seven of the ten fifth-passage mutants selected from E. faecalis contained QRDR alterations (Table 3). Their presence and location differed with the selecting agent. While ciprofloxacin selected mutants from both E. faecalis 114 and E. faecalis 178 with single alterations in ParC (serine-80), mutants selected with moxifloxacin and gatifloxacin from E. faecalis 114 contained amino acid changes in GyrA (serine-83). In addition, the fifth-passage mutant selected from E. faecalis 178 with moxifloxacin contained mutations in both GyrA and ParC. To our knowledge, these are the first mutations linked to the in vitro development of resistance to ciprofloxacin, moxifloxacin and gatifloxacin in enterococci. In contrast to the effects of the other quinolones, no QRDR mutations were observed in either E. faecalis fifth-passage mutant after exposure to DX-619.
Most fifth-passage mutant strains of E. faecium did not harbour QRDR changes (Table 4). However, two novel mutations associated with quinolone resistance were observed in mutants of E. faecium 137: a serine to arginine mutation at position 80 of ParC and a glutamate to glycine mutation at position 87 of GyrA. el Amin et al.4 previously characterized alterations involved in quinolone resistance in clinical isolates of E. faecium, reporting serine to isoleucine mutations at position 80 of ParC, serine to arginine, isoleucine and tyrosine mutations at position 83 of GyrA, and glutamate to lysine mutations at position 87 of GyrA. Taken together, these results indicate that the serine-80 position of ParC, as well as the serine-83 and glutamate-87 positions of GyrA, play important roles in quinolone resistance in E. faecium.
In conclusion, DX-619 was the most potent anti-enterococcal quinolone and was least affected by the resistance mechanisms of the mutants. If appropriate in vivo concentrations can be safely attained, DX-619 may be a useful anti-enterococcal agent. However, because single-step mutants were selected at relatively high frequencies with all of the quinolones, caution is necessary if any of these agents is considered for monotherapy of serious enterococcal infections.
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None to declare.
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Acknowledgements |
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This research was supported by a grant from Daiichi Pharmaceutical Co. Ltd, Tokyo, Japan.
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References |
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1 Edmond MB, Wallace SE, McClish DK, et al. (1999) Nosocomial bloodstream infections in United States hospitals: a three-year analysis. Clin Infect Dis 29:239–44.[Web of Science][Medline]
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Fujikawa K, Chiba M, Tanaka M, et al. (2005) In vitro antibacterial activity of DX-619, a novel des-fluoro(6) quinolone. Antimicrob Agents Chemother 49:3040–5.
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el Amin N, Jalal S, Wretlind B. (1999) Alterations in GyrA and ParC associated with fluoroquinolone resistance in Enterococcus faecium. Antimicrob Agents Chemother 43:947–9.
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Grohs P, Houssaye S, Aubert A, et al. (2003) In vitro activities of garenoxacin (BMS-284756) against Streptococcus pneumoniae, viridans group streptococci, and Enterococcus faecalis compared to those of six other quinolones. Antimicrob Agents Chemother 47:3542–7.
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Kanematsu E, Deguchi T, Yasuda M, et al. (1998) Alterations in the GyrA subunit of DNA gyrase and the ParC subunit of DNA topoisomerase IV associated with quinolone resistance in Enterococcus faecalis. Antimicrob Agents Chemother 42:433–5.
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Onodera Y, Okuda J, Tanaka M, et al. (2002) Inhibitory activities of quinolones against DNA gyrase and topoisomerase IV of Enterococcus faecalis. Antimicrob Agents Chemother 46:1800–4.
8 Clinical and Laboratory Standard Institute. Performance Standards for Antimicrobial Susceptibility Testing: Fifteenth Informational Supplement M100-S15. CLSI, Wayne, PA, USA, 2005.
9 Pan XS, Ambler J, Mehtar S, et al. (1996) Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae. Antimicrob Agents Chemother 40:2321–6.[Abstract]
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