Departments of Pathology and Medicine, Northwestern University Medical School, Divisions of Microbiology and Infectious Diseases, Northwestern Memorial Hospital, Chicago, IL 60611, USA
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Abstract |
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Introduction |
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Fluoroquinolones act by inhibiting homologous type II topoisomerases, DNA gyrase and DNA topoisomerase IV, enzymes that control DNA topology and are vital for chromosome function and replication. Each of these enzymes is a tetramer composed of two subunits: GyrA and GyrB forming an A2B2 complex in DNA gyrase; and ParC and ParE forming a C2E2 complex in DNA topoisomerase IV. Amino acid substitutions in any of the subunits of either gyrase or topoisomerase IV have the potential for associated fluoroquinolone resistance in S. pneumoniae. The ParC subunit of DNA topoisomerase IV is the initial, or primary, target of the older fluoroquinolones, such as ciprofloxacin.24 Changes in this enzyme are commonly associated with ciprofloxacin resistance in clinical isolates of S. pneumoniae.5
It was recently demonstrated, however, that fluoroquinolones with a different molecular structure could have other primary targets in S. pneumoniae. For example, the primary target of sparfloxacin is now believed to be GyrA, possibly as a result of a substituent change made at position C-8 (Figure 1).3 Furthermore, energy-dependent active efflux is increasingly seen as important in the development of bacterial resistance to many drugs, including fluoroquinolone antimicrobials.6,7 Our initial investigations on trovafloxacin suggested that the bulky substituent at position C-7 on this molecule may hinder an organism's ability to export that agent.8,9 Moxifloxacin has the bulkiest C-7 substituent of the currently available fluoroquinolone agents, leading to our hypothesis that it, too, will be poorly exported from the bacterial cell.
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Materials and methods |
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Clinical isolates of S. pneumoniae with varying fluoroquinolone sensitivity were obtained from the collection of strains at Northwestern Memorial Hospital in Chicago, IL, USA. A highly susceptible laboratory strain, CP1000, used in this study was described previously.11
This isolate was recovered before the introduction of fluoroquinolones, and we used it as a susceptible control and as the strain for selection of resistant mutants. Organisms were grown at 35°C in ToddHewitt broth (Difco Laboratories, Detroit, MI, USA) supplemented with 0.5% yeast extract (THBY), or on Tryptic Soy agar plates (Difco) supplemented with 5% sheep's blood. A casein hydrolysateyeast extracttryptone medium (CAT) was used for mutant selection.12 Archived strains were preserved at 80°C with 12% (v/v) glycerol.
Testing susceptibility to antimicrobial agents
The susceptibility of strains to antimicrobial agents was determined by a microdilution method, using Mueller Hinton broth (Difco) supplemented with 5% lysed horse blood.13 The following agents were used: levofloxacin (Ortho-McNeil Pharmaceuticals, Raritan, NJ, USA), ciprofloxacin (Bayer Corporation, West Haven, CT, USA), sparfloxacin (Rhône-Poulenc Rorer R-D, Vitry-sur-Seine, France) and moxifloxacin (Bayer Corporation). Before testing, individual strains were incubated overnight at 35°C on Tryptic Soy agar plates (Difco) supplemented with 5% sheep's blood. These cultures were used to prepare an inoculum of 15 x 105 cfu/mL. Inoculated panels were incubated at 35°C for 24 h. All testing was in duplicate.
Selection of mutants
First-step mutants were obtained by exposing S. pneumoniae CP1000 to the MIC of each agent: ciprofloxacin, 0.5 mg/L; levofloxacin, 1.0 mg/L; sparfloxacin, 0.25 mg/L; moxifloxacin, 0.125 mg/L. Between 108 and 109 cells from an S. pneumoniae CP1000 culture grown in THBY were plated on to the top layer of CAT agar on a two-layer plate, with double the concentration of corresponding antimicrobial agent in the bottom layer of the agar. In each experiment, a total of 1 x 1010 cells (using multiple plates) were used for mutant selection. Individual clones were harvested after 48 h incubation at 35°C. Second-step mutants were obtained essentially by the same procedure, this time exposing first-step mutants to 2 x MIC of the respective selection agent for each strain. Fluoroquinolone-resistant mutants were tested for stability of the acquired resistance by two passages on drug-free blood agar plates (DiMed Inc., St Paul, MN, USA), incubated at 35°C for a total of 48 h. Organisms were then transferred on to blood agar plates containing a concentration of drug equivalent to half the new MIC in order to determine that resistance was stable after 48 h growth in a drug-free environment.
PCR amplification and DNA sequence analysis
To investigate whether the tested strains had amino acid substitutions in ParC, ParE, GyrA or GyrB, the nucleotide sequences of parC, parE, gyrA and gyrB gene fragments that include regions corresponding to quinolone resistance determining regions (QRDRs) of the respective proteins (amino acids 43121 in GyrA, 361511 in GyrB, 55167 in ParC and 392529 in ParE) were determined and compared with the corresponding sequences from the reference strain CP1000. The sequences of gyrA, gyrB, parC and parE were published previously and are available in the NCBI database. The QRDR regions that we assessed correspond to amino acids 47125 in GyrA, 352502 in GyrB, 59171 in ParC and 383520 in ParE of Escherichia coli.3 A 253 bp fragment of gyrA (bp 129363), a 453 bp fragment of gyrB (bp 10801533), a 337 bp fragment of parC (bp 164501) and a 413 bp fragment of parE (bp 11751587) were amplified using the following pairs of primers: GyrA1 (5'-CGTCGCATTCTCTACGGA-3') and GyrA2 (5'-CGTCGCATTCTCTACGGA-3'); GyrB1 (5'-CTCTTCAGTGAAGCCTTCTCC-3') and GyrB2 (5'-CTCCATCGACATCGGCATC-3'); ParC1 (5'- TGACAAGAGCTACCGTAAGTCG-3') and ParC2 (5-TCGAACCATTGACCAAGAGG-3'); and ParE1 (5'-ACGTAAGGCGCGTGATGAG-3') and ParE2 (5'-CTAGCGGACGCATGTAACG-3'). PCR amplifications were performed with AmpliTaq DNA polymerase (PerkinElmer Cetus, Foster City, CA, USA) on an MJ Research Peltier Thermal Cycler PTC-100. A 1 µL sample of the bacterial culture at a density of 1 x 108 cells/mL was used as a template in standard 50 µL PCR reactions. Sequencing was carried out on the amplified PCR products using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Cetus) and an ABI PRISM 310 Genetic Analyzer according to the protocol of the manufacturer.
Growth inhibition studies: evaluation of the effect of reserpine on susceptibility of S. pneumoniae CP1000 to fluoroquinolones
S. pneumoniae cultures were started at an optical density (OD550) of 0.001 (approximately 1 x 106 cells/mL) in THBY broth containing either ciprofloxacin 0.12 mg/L, levofloxacin 0.25 mg/L, sparfloxacin 0.06 mg/L or moxifloxacin 0.03 mg/L, or a combination of each of these fluoroquinolones with 10 mg/L of reserpine.8 After a 7 h incubation at 35°C, the OD550 reached by each culture was determined. A drug concentration of 0.25 x MIC was used as this inhibited growth of S. pneumoniae CP1000 cultures by two- to four-fold relative to a logarithmically growing culture without added antimicrobial agent, yet allowed enough growth over 7 h to observe the action of the drug, as well as the reserpine-mediated inhibition of growth.8 The extent of growth inhibition by each fluoroquinolone with or without reserpine was determined by comparing the optical density of cultures containing the corresponding fluoroquinolone, or the combination of this fluoroquinolone with reserpine, with the optical density of control cultures grown in the absence of antimicrobial agents, with reserpine added to one of the two controls.
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Results |
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The activity of moxifloxacin against clinical isolates and laboratory mutants bearing changes in major fluoroquinolone target enzymes, DNA gyrase and topoisomerase IV, is shown in Table I. Strain SP30 is a recent clinical isolate susceptible to all fluoroquinolones tested. Clinical isolates 6406 and 6678 bear changes in the ParC subunit of DNA topoisomerase IV. Each of these strains has an additional substitution, Ile-460
Val, in ParE, which was found not to contribute to fluoroquinolone resistance using genetic transformation of the corresponding mutation into strain CP1000.9 Strains 1C1 and 1L1 are first-step mutants selected with ciprofloxacin (C) and levofloxacin (L) using laboratory strain CP1000.9,14 As is evident from Table I
, moxifloxacin was the most active of the fluoroquinolones tested, against both the wild-type isolates and those with various mutations. It consistently had the highest activity against clinical isolates and first-step laboratory mutants bearing a Ser-79
Phe/Tyr substitution in ParC. These strains demonstrated only a two-fold increase in the MIC of moxifloxacin (to 0.25 mg/L), suggesting that an important target for moxifloxacin differs from that of ciprofloxacin and levofloxacin.
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Selection of resistance with moxifloxacin and sparfloxacin
To identify the molecular targets of moxifloxacin and to investigate whether the primary target of this antimicrobial agent differs from that of older fluoroquinolones, we performed stepwise selection of resistant mutants using moxifloxacin. For comparison, we also selected first-step mutants with sparfloxacin. The frequencies of direct mutant selection using moxifloxacin and sparfloxacin are shown in Table II. Interestingly, moxifloxacin-resistant first-step mutants appeared with a frequency of 1.3 x 107 on plates containing 0.125 mg/L (the MIC) of this agent, while no mutant colonies were detected on those containing a higher concentration of moxifloxacin. In contrast, sparfloxacin-resistant mutants could be selected at concentrations up to twice the agent's MIC. However, using a first-step mutant (1M8) for selection of higher levels of moxifloxacin resistance, it was possible to obtain second-step mutants at 2 x MIC with a frequency of 1.8 x 107. Thus, once first-step resistance develops, the emergence of high-level, second-step resistance appears to be facilitated.
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Table III presents the results of sequence analysis of first-step (1M8, 1M15) and second-step (2M1, 2M2, 2M3, 2M4) mutants selected using moxifloxacin. We also analysed two first-step mutants (1S1, 1S2) selected by the same method with sparfloxacin. First-step mutants selected with either moxifloxacin or sparfloxacin bore changes in the gyrA gene that resulted in a deduced Ser
Phe substitution of the GyrA subunit. The acquisition of this amino acid substitution leads to a four-fold increase in the level of resistance to sparfloxacin and moxifloxacin for mutant strains, but only to a two-fold increase in the resistance to ciprofloxacin. No change in levofloxacin resistance was detected. The second-step mutants selected from strain 1M8 using 1 mg/L of moxifloxacin accumulated amino acid substitutions in both the GyrA subunit of DNA gyrase and the ParC subunit of DNA topoisomerase IV (Table III
). A combination of these two substitutions raised the MIC of sparfloxacin, ciprofloxacin and levofloxacin to
8.0 mg/L, while the MIC of moxifloxacin rose only to 2.0 mg/L, again suggesting its enhanced activity against either these altered sites, or the remaining, non-mutated enzymes GyrB or ParE.
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To assess the role of active efflux as a cellular response to moxifloxacin, we studied the effect of the plant alkaloid reserpine combined with this fluoroquinolone on the growth of S. pneumoniae CP1000, and compared the results with those obtained with sparfloxacin, levofloxacin and ciprofloxacin. Our experiments were based on a known observation that several fluoroquinolones that are substrates of active efflux exhibit an increased antibacterial activity in the presence of reserpine. Being a specific inhibitor of an active multidrug efflux mechanism, reserpine potentiates the action of these agents by increasing quinolone intracellular accumulation.15,16 The extent of growth inhibition by each fluoroquinolone with and without reserpine was compared in 7 h S. pneumoniae cultures (Figure 2). As evident from this figure, growth with ciprofloxacin was inhibited nearly three-fold when combined with reserpine, and growth with levofloxacin and sparfloxacin in the presence of reserpine was reduced to approximately two-thirds of that without reserpine. Importantly, reserpine had little effect on the growth of S. pneumoniae CP1000 in the presence of moxifloxacin, suggesting that this agent is a poor substrate for active efflux by S. pneumoniae. Thus, in addition to the likelihood of multiple homologous type II topoisomerase targets for moxifloxacin, these data imply moxifloxacin's possibility of avoiding other bacterial resistance mechanisms, such as active efflux, in the cellular response after drug exposure.
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Discussion |
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The low level of resistance in ParC mutants to moxifloxacin suggests a different primary target of this fluoroquinolone in S. pneumoniae. Stepwise in vitro mutant selection demonstrated that, unlike older fluoroquinolone agents, moxifloxacin targets the GyrA subunit of DNA gyrase first. Similar target preference was demonstrated by an 8-fluoro-substituted quinolone, sparfloxacin, as shown here and reported in earlier studies.5 Recently this effect was noted for another fluoroquinolone, gatifloxacin.19 The C-8-substituted quinolones also exhibit enhanced bacteriostatic and lethal activities against GyrA mutants in Escherichia coli and Mycobacterium spp.20,21 Furthermore, in another Gram-positive bacterium, Staphylococcus aureus, the substitution was the most lethal for both wild-type cells and those with a pre-existing topoisomerase IV mutation.22 As shown in Figure 1, moxifloxacin and sparfloxacin carry a methoxyl group and fluorine, respectively, at position C-8, with levofloxacin (and ofloxacin) having a benzoxazine bridge between C-8 and N-1, while ciprofloxacin carries no C-8 substituent. Taken together, our results show that C-8-substituted quinolones appear to have an enhanced affinity within the Gram-positive bacterial cell for GyrA rather then ParC (compared with C-8-H agents), with increased lethality of the C-8-substituted agents persisting even against strains resistant to older quinolones. Of note is the observation that the fused C-8 substituent of ofloxacin and levofloxacin does not provide the same advantage, suggesting the need for a free substituent at C-8.
In this study, we also assessed the role of active efflux as a bacterial response to moxifloxacin. The involvement of active efflux as a key mechanism in the avoidance of S. pneumoniae resistance to moxifloxacin was suggested by the low frequency of resistance selection at drug concentrations exceeding the agent's MIC. With reduced active efflux, this drug appears to accumulate within the S. pneumoniae cell, leading to early death of the entire bacterial population and markedly reduced survival of first-step mutants. The involvement of active efflux as a mechanism of resistance to certain fluoroquinolone agents in S. pneumoniae was indicated in earlier studies.68,15 Reports now suggest that at least half of resistant clinical pneumococcal isolates express an active efflux phenotype.6,7 It was shown earlier that bulkiness at the C-7 substituent and the overall hydrophobicity of the fluoroquinolone molecule contribute to the reduction of the active efflux, therefore increasing their cellular accumulation in S. aureus.23 As further evidence, we recently demonstrated this trait for another quinolone with a bulky C-7 substituent, trovafloxacin, in S. pneumoniae8,9 Since moxifloxacin carries a bulky substituent, a bicyclic fused ring composed of a pyrrolidine and piperazine ring (Figure 2), we anticipated that the efflux of this compound would be impaired in S. pneumoniae. Our hypothesis was confirmed by the results of the growth inhibition experiments showing poor efflux of this fluoroquinolone agent. Of the fluoroquinolones tested in this study, ciprofloxacin has the least bulky C-7 substituent, a piperazine moiety, with levofloxacin and sparfloxacin each carrying slightly larger piperazine derivatives, a 4-methyl piperazine and 3,5-dimethyl piperazine, respectively, while moxifloxacin has the bulkiest C-7 substituent. In our study, moxifloxacin appeared to be least susceptible to efflux, while ciprofloxacin was the most sensitive of all fluoroquinolones tested.
In conclusion, the C-8-methoxyl substituent and a bulky bicyclic fused ring carried by moxifloxacin provide an advantageous increase in activity of this agent against the Gram-positive pathogen S. pneumoniae compared with older fluoroquinolones. This is probably the consequence of a combination of factors, including the increased lethality of moxifloxacin against parC mutants, as well as the decrease in the efficiency of its active efflux from the pneumococcal cell. The benefit of these structural innovations is most pronounced on wild-type pneumococcal cells with no pre-existing mutations in any QRDR. Here, the entire bacterial population appears to be killed at drug concentrations exceeding the MIC. This is in contrast to older compounds (ciprofloxacin and levofloxacin) where some resistant mutants can survive at even 8 x MIC.8 However, once a first-step resistant mutant is selected, whether by moxifloxacin or another fluoroquinolone, the development of more-resistant bacterial cells proceeds more readily. Thus fluoroquinolones with specific novel substitutions, like moxifloxacin, may be potent tools for maintaining long-term quinolone activity against Gram-positive organisms, but only if they are used preferentially over the older agents that appear more prone to selecting drug-resistant bacteria.
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Acknowledgments |
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Notes |
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References |
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Received 21 May 1999; returned 23 September 1999; revised 29 October 1999; accepted 1 December 1999