Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Road, Mason, OH 45040, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recently, a series of 8-methoxy, non-fluorinated quinolones (NFQs; Figure 1) has been developed and shown to have broad-spectrum antibacterial activity9 with relatively high potency against quinolone-resistant staphylococci.10 In this report, we describe: (i) the enzyme inhibitory activity of these NFQs against E. coli DNA gyrase; and (ii) the antibacterial activity of the NFQs against clinical isolates of E. coli and S. pneumoniae, including strains with specific point mutations in gyrA and parC.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Benchmark and test quinolones were synthesized in-house. For the MIC analysis, clinical isolates of E. coli and penicillin-resistant S. pneumoniae (PRSP) were obtained from Procter & Gamble Pharmaceuticals' internal culture collection and major hospitals in the greater Cincinnati area (Health Alliance Laboratories) during 19981999. These isolates were collected at random without regard for their susceptibility to specific quinolones. For studies with gyrA and parC mutants, E. coli strain WT, its in vitro-selected mutant MI, and the clinical isolate U12987 were obtained from Dr Peter Heisig.11,12 Additional E. coli clinical isolates C-4, 1363, 1331 and 1334 were obtained from Dr Jordi Vila.13 S. pneumoniae strains 02J1016, 02J1056 and their in vitro-selected mutants 1016-27, 1016-27-23, 1056-13 and 1056-13-18 were obtained from Dr Thomas D. Gootz.14 Clinical isolates of S. pneumoniae with mutations in parC and gyrA were obtained from Dr Patrice Courvalin.15,16 BM4203 and BM4204 were ciprofloxacin-susceptible strains isolated from patients before antibiotic therapy, while BM4203-R and BM4204-R were the corresponding ciprofloxacin-resistant strains isolated after therapy. In both cases, pre- and post-treatment isolates were indistinguishable based on pulsed-field gel electrophoresis patterns of genomic DNA digests, suggesting that the mutants were selected in vivo as a result of quinolone therapy.15 BM4205 was a clinical isolate of S. pneumoniae, while BM4205-R3 was its in vitro-selected mutant.15 Additional clinical isolates of ciprofloxacin-resistant S. pneumoniae, strains 1048, 1055 and 1056 were obtained from Dr Peter C. Appelbaum (Hershey Medical Center, PA, USA).
Experimental procedures
DNA gyrase was purified as subunits A and B from E. coli JM109 containing plasmids pPH3 and pAG111 to overexpress the gyrA and gyrB genes, respectively, from the tac promoter.17 The mutated form of the A subunit (Ser-83Trp) was purified from E. coli JM109 containing the overexpression plasmid pPH483.18 The subunits were purified via anion-exchange column chromatography, following a previously described procedure with minor modifications.17 The active, multimeric (A2B2) form of DNA gyrase was prepared by incubating equimolar amounts of the A and B subunits at room temperature in 50 mM Tris (pH 7.5)/100 mM KCl/1 mM EDTA/5 mM dithiothreitol/10% glycerol.17 Inhibition of DNA gyrase supercoiling activity was ascertained by separating supercoiled and relaxed DNA via agarose gel electrophoresis, following an established procedure.19 The IC50 was determined as the compound concentration at which 50% of the DNA gyrase supercoiling activity was inhibited.
The MIC of the test compounds was determined by incubating bacterial cultures (~5 x 105 cfu/mL) at 37°C overnight (1824 h) in brainheart infusion (BHI) broth in the presence of test compounds in a two-fold, broth microdilution series in duplicate.20 S. pneumoniae strains were tested in the presence of 3% lysed horse blood in BHI broth and incubated in a 5% CO2-enriched environment. In analysing multiple isolates of E. coli and penicillin-resistant S. pneumoniae, the MIC50 and MIC90 values were estimated as the minimum compound concentration at which growth of 50% and
90% of the strains were inhibited.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The supercoiling activity of E. coli DNA gyrase was inhibited by the NFQs with IC50s in the 1.63.2 mg/L range, while the IC50s of ciprofloxacin, trovafloxacin, gatifloxacin and clinafloxacin were 1.6, 1.6, 0.8 and 0.2 mg/L, respectively (Figure 2). Using the same assay, IC50s of additional benchmark compounds for the wild-type gyrase were as follows: nalidixic acid 50 mg/L; ofloxacin 1 mg/L; lomefloxacin 1.75 mg/L; sparfloxacin 1 mg/L and tosufloxacin 0.5 mg/L. When tested against the mutant gyrase [GyrA (Ser-83
Trp)], the IC50s of the NFQs rose by factors of two to four, to 6.4 mg/L, while those of ciprofloxacin, trovafloxacin, gatifloxacin and clinafloxacin rose by factors of 64, 8, 4 and 5, respectively, to 102, 13, 3 and 1 mg/L (Figure 2
).
|
The NFQs and benchmark quinolones were tested against 20 clinical isolates collected at random from patients with urinary tract infections for in vitro susceptibility. The MIC data are presented in Table I. Based on the MIC90s against these strains, two NFQs, PGE9262932 and PGE9509924, were as potent as trovafloxacin and eight-, four- and two-fold less potent than clinafloxacin, ciprofloxacin and gatifloxacin, respectively.
|
|
In vitro antibacterial activity of the NFQs along with other quinolone and non-quinolone compounds was tested against 23 clinical isolates of PRSP. The results, summarized in Table III, show that the MICs of many classes of antibacterials including ß-lactams (e.g. ampicillin) and macrolides (e.g. azithromycin) were relatively high (MIC90 4 >16 mg/L) for these strains, although they appeared susceptible to vancomycin (MIC90 1 mg/L). Based on the MIC90s, the NFQs were more potent than other quinolones, such as ciprofloxacin, trovafloxacin and gatifloxacin, and at least as potent as clinafloxacin. Among the NFQs, PGE9262932 was 64-, 16-, eight- and four-fold more potent than ciprofloxacin, gatifloxacin, trovafloxacin and clinafloxacin, respectively, based on the MIC90 data. The cumulative susceptibility data, shown in Figure 3
, also indicated that PGE9262932 was the most potent of all the quinolones tested in this study.
|
|
S. pneumoniae strains with mutations in the target genes (parC and gyrA) were tested against the NFQs and other quinolones in this study. The results from three sets of mutants are presented in Table IV. When tested against the laboratory-generated ParC mutants (Ser-79
Phe in strains 101627 and 105613), the MICs of the NFQs remained unchanged relative to those for the parent, while those of ciprofloxacin, trovafloxacin, gatifloxacin and clinafloxacin were elevated two- to four-fold (Table IV
). When tested against the laboratory-generated, second-step mutant strains 1016-27-23 [ParC (Ser-79
Phe); GyrA (Ser-81
Phe)] and 1056-13-18 [ParC (Ser-79
Phe); GyrA (Glu-85
Lys)], the MICs of the NFQs were in the 0.1251.0 mg/L range and were elevated two- to eight-fold (relative to the MICs for the parent), while the MICs of the other quinolones were in the range 0.532 mg/L and were elevated eight- to 32-fold (relative to the MICs for the parent). For a laboratory-generated GyrA mutant (Ser-81
Phe in strain BM4205-R3, Table IV
), the NFQ MICs were elevated two- to four-fold, while those of the other quinolones were elevated four- to 16-fold (relative to the MICs for the parent).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In E. coli, the GyrA subunit of DNA gyrase is believed to be a primary target for quinolones, while the ParC subunit of topoisomerase IV is considered a secondary target. This is based on the association of mutations in gyrA with low levels of ciprofloxacin resistance, and that of mutations in gyrA and parC with high levels of resistance to ciprofloxacin.13 It appears that in E. coli, once GyrA is mutated, quinolones such as ciprofloxacin inhibit ParC with higher potency and thereby exert their antibacterial activity via topoisomerase IV as the effective molecular target. However, since newer quinolones, such as the NFQs, have higher potency toward the Ser-83 mutant of GyrA, these compounds could potentially exert their antibacterial activity, at least in part, by inhibiting the mutated form of DNA gyrase, instead of utilizing the second target, topoisomerase IV, as the sole target. This scenario is consistent with our finding that the potency difference between the NFQs and ciprofloxacin was less against the GyrA (Ser-83Leu) mutant MI than against the parent WT. A similar phenomenon was apparent against the clinical isolate C-4. While potency differences against various clinical isolates with similar mutations could be attributable to mechanisms other than mutations in gyrA and parC, the overall higher potency of the NFQs, relative to ciprofloxacin and trovafloxacin, against clinical isolates with gyrA and parC mutations, suggests that the NFQs retain higher potency against mutated forms of GyrA and/or ParC.
The in vitro potency data on the PRSP isolates indicated that the NFQs were extremely potent against this clinically important pathogen, which is widely resistant to penicillins and macrolides. PGE9262932 was exceptionally potent, with 100% of the isolates being susceptible at 0.031 mg/L. The high antibacterial potency of the NFQs could be attributable to their high potency against DNA gyrase and/or topoisomerase IV in S. pneumoniae. Unlike E. coli, previous studies showed that depending on the quinolone, the first-step mutations in S. pneumoniae could map to either parC or gyrA.2123 This suggests that in S. pneumoniae, certain quinolones, such as ciprofloxacin and trovafloxacin, have higher potency toward ParC (topoisomerase IV) than GyrA (DNA gyrase), while the reverse is true in the case of other quinolones, such as sparfloxacin and gatifloxacin.21,22 As shown in Table IV
, the MICs of the NFQs remained unchanged in the ParC mutants (Ser-79
Phe in strains 101627 and 105613), and were elevated twoto four-fold in a GyrA mutant (Ser-81
Phe in strain BM4205-R3) relative to the corresponding parents. These data suggest that in S. pneumoniae, wild-type GyrA is the target with higher effective potency for the NFQs.
Against S. pneumoniae strain 1016-27-23, with mutations in the serine hot spots of both ParC and GyrA, the NFQ MICs increased two-fold (PGE9262932 and 4175997) or four-fold (PGE9509924) compared with eight- to 32-fold for the other quinolones. Against strain 1056-13-18, with mutations in the glutamate and serine hot spots of GyrA and ParC, respectively, the NFQ MICs increased eight-fold, compared with 16- to 64-fold for the other quinolones. These data suggest that while the NFQs have higher potency against the Ser-81Phe mutant form of GyrA than the Glu-85
Lys form, they are more potent than ciprofloxacin, trovafloxacin and gatifloxacin and at least as potent as clinafloxacin against the serine/glutamate hot spot mutants of GyrA.
Data obtained with clinical isolates of S. pneumoniae with GyrA and ParC mutations were consistent with those obtained with laboratory-derived mutants. In vivo-selected mutations in either the Ser-79 or the Asp-83 residues of ParC (strains BM4203-R and BM4204-R) failed to show a consistent increase in the NFQ MICs relative to the parent strain, suggesting that in S. pneumoniae GyrA is the higher potency target for these compounds. In addition, the relatively high NFQ potency against clinical isolates with ParC and GyrA mutations (strains 1048 and 1055) suggest that these compounds are more potent than ciprofloxacin, trovafloxacin and gatifloxacin, and at least as potent as clinafloxacin against the mutated targets. Our data suggest that, like clinafloxacin, the NFQs are highly potent against PRSP, and are less affected in vitro by pre-existing and characterized target mutations that reduce quinolone potency against S. pneumoniae.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Lister, P. D. (2000). Emerging resistance problems among respiratory tract pathogens. American Journal of Managed Care 6, Suppl. 8, S40918.[ISI][Medline]
3
.
Chen, D. K., McGeer, A., de Azavedo, J. C. & Low, D. E. (1999). Decreased susceptibility of Streptococcus pneumoniae to fluoroquinolones in Canada. Canadian Bacterial Surveillance Network. New England Journal of Medicine 341, 2339.
4
.
Kahlmeter, G. (2000). The ECO.SENS project: a prospective, multinational, multicentre epidemiological survey of the prevalence and antimicrobial susceptibility of urinary tract pathogensinterim report. Journal of Antimicrobial Chemotherapy 46, Suppl. S1, 1522.
5
.
Thornsberry, C., Jones, M. E., Hickey, M. L., Mauriz, Y., Kahn, J. & Sahm, D. F. (1999). Resistance surveillance of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis isolated in the United States, 19971998. Journal of Antimicrobial Chemotherapy 44, 74959.
6 . Oram, M. & Fisher, L. M. (1991). 4-Quinolone resistance mutations in the DNA gyrase of Escherichia coli clinical isolates identified by using the polymerase chain reaction. Antimicrobial Agents and Chemotherapy 35, 3879.[ISI][Medline]
7
.
Tavio, M., Vila, J., Ruiz, J., Martin-Sanchez, A. M. & de Anta M. T. (1999). Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli isolates. Journal of Antimicrobial Chemotherapy 44, 73542.
8
.
Bast, D. J., Low, D. E., Duncan, C. A., Kilburn, L., Mandell, L. A., Davidson, R. J. et al. (2000). Fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae: contributions of type II topoisomerase mutations and efflux to levels of resistance. Antimicrobial Agents and Chemotherapy 44, 304954.
9 . Brown, S. D., Fuchs, P. C & Barry, A. L. (1999). In vitro activities of 3 non-fluorinated quinolones and 3 fluoroquinolones against consecutive clinical isolates from eleven U. S. medical centers. In Program and Abstracts of the Fortieth Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 2000. Abstract F-1510, p. 210. American Society for Microbiology, Washington, DC.
10
.
Roychoudhury, S., Catrenich, C. E., McIntosh, E. J., McKeever, H. D., Makin, K. M., Koenigs, P. M. et al. (2001). Quinolone resistance in staphylococci: activities of new nonfluorinated quinolones against molecular targets in whole cells and clinical isolates. Antimicrobial Agents and Chemotherapy 45, 111520.
11 . Heisig, P. & Tschomy, R. (1994). Characterization of fluoroquinolone-resistant mutants of Escherichia coli selected in vitro. Antimicrobial Agents and Chemotherapy 38, 128491.[Abstract]
12 . López-Brea, M. & Alarcón, T. (1990). Isolation of fluoroquinolone-resistant Escherichia coli and Klebsiella pneumoniae from an infected Hickman catheter. European Journal of Clinical Microbiology and Infectious Diseases 9, 3457.[ISI][Medline]
13 . Vila, J., Ruiz, J., Gonñi & de Anta, M. T. (1996). Detection of mutations on parC in quinolone-resistant clinical isolates of Escherichia coli. Antimicrobial Agents and Chemotherapy 40, 4913.[Abstract]
14 . Gootz, T. D., Zaniewski, R., Haskell, S., Schmieder, B., Tankovic, J., Girard, D. et al. (1996). Activity of the new fluoroquinolone trovafloxacin (CP-99,219) against DNA gyrase and topoisomerase IV mutants of Streptococcus pneumoniae selected in vitro. Antimicrobial Agents and Chemotherapy 40, 26917.[Abstract]
15 . Tankovic, J., Perichon, B., Duval, J. & Courvalin, P. (1996). Contribution of mutations in gyrA and parC genes to fluoroquinolone resistance of mutants of Streptococcus pneumoniae obtained in vivo and in vitro. Antimicrobial Agents and Chemotherapy 40, 250510.[Abstract]
16 . Perez-Trallero, E., Garcia-Arenzana, J. M., Jimenez, J. A. & Peris, A. (1990). Therapeutic failure and selection of resistance to quinolones in a case of pneumococcal pneumonia treated with ciprofloxacin. European Journal of Clinical Microbiology and Infectious Diseases 9, 9056.[ISI][Medline]
17 . Hallett, P., Grimshaw, A. J., Wigley, D. B. & Maxwell, A. (1990). Cloning of the DNA gyrase genes under tac promoter control: overproduction of the gyrase A and B proteins. Gene 93, 13942.[ISI][Medline]
18 . Willmott, C. J. R. & Maxwell, A. (1993). A single point mutation in the DNA gyrase protein A greatly reduces binding of fluoroquinolones to the gyraseDNA complex. Antimicrobial Agents and Chemotherapy 37, 1267.[Abstract]
19 . Barrett, J. F., Bernstein, J. I., Krause, H. M., Hillard, J. J. & Ohemeng, K. A. (1993). Testing potential gyrase inhibitors of bacterial DNA gyrase: a comparison of the supercoiling inhibition assay and "cleavable complex" assay. Analytical Biochemistry 214, 3137.[ISI][Medline]
20 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA.
21 . Pan, X. S. & Fisher, L. M. (1997). Targeting of DNA gyrase in Streptococcus pneumoniae by sparfloxacin: selective targeting of gyrase or topoisomerase IV by quinolones. Antimicrobial Agents and Chemotherapy 41, 4714.[Abstract]
22
.
Fukuda, H. & Hiramatsu, K. (1999). Primary targets of fluoroquinolones in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 43, 4102.
23
.
Pan, X. S. & Fisher, L. M. (1998). DNA gyrase and topoisomerase IV are dual targets of clinafloxacin action in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 42, 28106.
Received 21 December 2000; returned 23 February 2001; revised 26 March 2001; accepted 2 April 2001