K Sato

Type II Topoisomerase Mutations in Fluoroquinolone-Resistant Clinical Strains of Pseudomonas aeruginosa Isolated in 1998 and 1999: Role of Target Enzyme in Mechanism of Fluoroquinolone Resistance

ABSTRACT The major mechanism of resistance to fluoroquinolones forPseudomonas aeruginosa is the modification of type II topoisomerases (DNA gyrase and topoisomerase IV). We examined the mutations in quinolone-resistance-determining regions (QRDR) ofgyrA, gyrB, parC, and parE genes of recent clinical isolates. There were 150 isolates with reduced susceptibilities to levofloxacin and 127 with reduced susceptibilities to ciprofloxacin among 513 isolates collected during 1998 and 1999 in Japan. Sequencing results predicted replacement of an amino acid in the QRDR of DNA gyrase (GyrA or GyrB) for 124 of the 150 strains (82.7%); among these, 89 isolates possessed mutations in parC orparE which lead to amino acid changes. Substitutions of both Ile for Thr-83 in GyrA and Leu for Ser-87 in ParC were the principal changes, being detected in 48 strains. These replacements were obviously associated with reduced susceptibilities to levofloxacin, ciprofloxacin, and sparfloxacin; however, sitafloxacin showed high activity against isolates with these replacements. We purified GyrA (The-83 to Ile) and ParC (Ser-87 to Leu) by site-directed mutagenesis and compared the inhibitory activities of the fluoroquinolones. Sitafloxacin showed the most potent inhibitory activities against both altered topoisomerases among the fluoroquinolones tested. These results indicated that, compared with other available quinolones, sitafloxacin maintained higher activity against recent clinical isolates with multiple mutations ingyrA and parC, which can be explained by the high inhibitory activities of sitafloxacin against both mutated enzymes.

Antimicrobial activity of DU-6859, a new potent fluoroquinolone, against clinical isolates.

DU-6859, (-)-7-[(7S)-amino-5-azaspiro(2,4)heptan-5-yl]-8-chloro-6- fluoro-1-[(1R,2R)-cis-2-fluoro-1-cyclopropyl]-1,4-dihydro-4-oxoquinol one-3- carboxylic acid, is a new fluoroquinolone with antibacterial activity which is significantly better than those of currently available quinolones. The MICs for 90% of methicillin-susceptible and -resistant Staphylococcus aureus and Staphylococcus epidermidis clinical isolates (MIC90s) were 0.1, 3.13, 0.1, and 0.39 microgram/ml, respectively. MIC50s of DU-6859 against quinolone-resistant, methicillin-resistant S. aureus were 8-, 32-, 64-, and 128-fold lower than those of tosufloxacin and sparfloxacin, ofloxacin and fleroxacin, ciprofloxacin, and lomefloxacin, respectively. DU-6859 inhibited the growth of all strains of Streptococcus pneumoniae and Streptococcus pyogenes at 0.1 and 0.2 microgram/ml, respectively, and was more active against enterococci than the other quinolones tested. Although the activity of DU-6859 against Pseudomonas aeruginosa was roughly comparable to that of ciprofloxacin at the MIC50 level, it was fourfold more active than ciprofloxacin at the MIC90 level. DU-6859 was also more active against other glucose-nonfermenting bacteria, Haemophilus influenzae, Moraxella catarrhalis, and Neisseria gonorrhoeae, than the other drugs tested. Strains of Bacteroides fragilis and Peptostreptococcus spp. were susceptible to DU-6859; MIC90s were 0.39 and 0.2 microgram/ml, respectively. DU-6859 generally showed activities twofold or greater than those of ciprofloxacin and the other drugs against almost all members of the family Enterobacteriaceae. The action of DU-6859 against the clinical isolates was bactericidal at concentrations near the MICs. DU-6859 activity was not affected by different media, pH, inoculum size, or human serum but was decreased in human urine.

In vitro and in vivo antifungal activities of DU-6859a, a fluoroquinolone, in combination with amphotericin B and fluconazole against pathogenic fungi.

DU-6859a is an investigational fluoroquinolone agent with potent bactericidal activity, but by itself it has no antifungal activity. When combined with amphotericin B (AmB), however, DU-6859a clearly enhanced the in vitro antifungal activity of AmB against Candida albicans, Candida tropicalis, Candida krusei, Candida glabrata, and Cryptococcus neoformans in microdilution checkerboard studies. Positive interactions of DU-6859a with AmB against Aspergillus fumigatus were dependent on the medium used; yeast nitrogen base supplemented with amino acids, ammonium sulfate, and 1% glucose was better for demonstrating synergism, while in RPMI 1640 medium, unexpected antagonism between the drugs occurred against three of the strains tested. In combination with fluconazole (Flu), DU-6859a increased the activity of Flu against C. albicans both in synthetic amino acid medium fungal and in supplemented yeast nitrogen base. An in vitro time-kill study revealed that DU-6859a combined with AmB significantly suppressed the regrowth of C. albicans compared with the suppression brought about by AmB used alone in a concentration-dependent fashion. Furthermore, in a model of C. albicans infection in mice, the fungal load in infected kidneys was significantly less in mice given the combination treatment of DU-6859a plus either AmB or Flu, and thus, the combination treatment resulted in prolonged survival of infected mice compared with treatment with either antifungal alone. The prolonged survival in mice given the combined treatment was also observed in mice with A. fumigatus infection, indicating that DU-6859a potentiated the actions of the antifungal agents in vivo as well as in vitro.

Inhibitory effects of quinolones on DNA gyrase of Escherichia coli and topoisomerase II of fetal calf thymus.

The in vitro inhibitory effects of quinolones on the bacterial DNA gyrase of Escherichia coli KL-16 and topoisomerase II of fetal calf thymus were compared. All the quinolones tested required higher concentrations to inhibit the topoisomerase II than to inhibit the DNA gyrase, and no correlation existed among their inhibitory activities against both enzymes. However, there was a large difference among the quinolones in their selectivities between the bacterial enzyme and its eucaryotic counterpart. The selectivity of ofloxacin was highest, and the selectivities of CI-934 and nalidixic acid were lowest.

Contribution of the C-8 substituent of DU-6859a, a new potent fluoroquinolone, to its activity against DNA gyrase mutants of Pseudomonas aeruginosa.

Inhibitory effects of five quinolones against DNA gyrases purified from four quinolone-resistant clinical isolates of Pseudomonas aeruginosa and the quinolone-susceptible strain PAO1 were examined. All of the quinolone-resistant strains tested were found to be DNA gyrase mutants. The 50% inhibitory concentrations (IC50s) of the quinolones for these DNA gyrases roughly correlated with their MICs. Interestingly, gyrase inhibition by DU-6859a was found to be significantly less affected by these mutations that inhibition by other currently available quinolones. To assess the enhanced activity shown by DU-6859a, the effects of quinolones with altered substituents at the N-1, C-7, and C-8 positions of the quinolone ring of DU-6859a were tested. Measurement of MICs for four DNA gyrase mutants and IC50s for their purified DNA gyrases showed that removal of the C-8 chlorine of DU-6859a significantly increased MICs and IC50s for DNA gyrase mutants. However, no deleterious effects were observed when either the fluorine on the cyclopropyl substituent at the N-1 position or the cyclopropyl ring at the C-7 substituent was removed. Moreover, removal of the C-8 chlorine also increased the MIC for 19 of 20 quinolone-resistant clinical isolates. Our results led to the conclusion that DU-6859a is much more active against quinolone-resistant clinical isolates of P. aeruginosa than other currently available quinolones, probably because of its strong inhibitory effects against mutant quinolone-resistant DNA gyrases, and that the C-8 chlorine is necessary for these potent effects.

Significance of the methyl group on the oxazine ring of ofloxacin derivatives in the inhibition of bacterial and mammalian type II topoisomerases.

A study was made of the correlation between the in vitro inhibitory effects of several quinolones, including four ofloxacin derivatives, on bacterial DNA gyrase from Escherichia coli KL-16 and on topoisomerase II from fetal calf thymus. No correlation was observed between the inhibitions of DNA gyrase activity and topoisomerase II activity. On the other hand, the inhibitory effects of these quinolones against topoisomerase II were closely correlated with their inhibition of cell growth. Furthermore, among the oxazine derivatives tested, the derivative with a methyl group at position 3 in an S configuration showed the highest activity against DNA gyrase and derivatives without a methyl group on the oxazine ring were more potent against topoisomerase II than those with a methyl group. Among these derivatives, DR-3355, the S isomer of ofloxacin, showed the highest activity against DNA gyrase and low activity against topoisomerase II. These results indicate that the methyl group on the oxazine ring plays an important role in the inhibitory activities of ofloxacin derivatives for these enzymes.

Inhibition by quinolones of DNA gyrase from Staphylococcus aureus.

In order to clarify the mechanism of action of quinolones against Staphylococcus aureus, the subunit A and B proteins of DNA gyrase were separately purified from a crude extract of S. aureus FDA 209-P. The reconstituted enzyme exhibited ATP-dependent DNA supercoiling activity. The inhibitory effects of quinolones on the supercoiling activity of the purified enzyme were measured by the quantitative electrophoresis method (17), using plasmid DNA, pBR322 or pUB110, as substrates and expressed as the 50% inhibitory concentrations (IC50s). The IC50s of ofloxacin, DR-3355 (l-ofloxacin), ciprofloxacin, tosufloxacin, sparfloxacin, and DS-4524, a new quinolone derivative, for pBR322 were 63.0, 37.8, 30.5, 46.0, 28.5, and 3.2 micrograms/ml, respectively. These values were closely correlated with antibacterial activity (MIC), with correlation coefficients of 0.953 for pBR322 and 0.938 for pUB110. These results indicate that, in S. aureus, as in gram-negative bacteria, DNA gyrase is likely to be a major target enzyme of quinolones.

Antimicrobial activity of DV-7751a, a new fluoroquinolone.

We compared the in vitro antibacterial activity of DV-7751a against gram-positive and -negative bacteria with those of quinolones currently available. MICs for 90% of the strains tested (MIC90s) against clinical isolates of methicillin-susceptible and -resistant Staphylococcus aureus and Staphylococcus epidermidis were 0.20, 0.39, 0.20, and 0.78 micrograms/ml, respectively. Moreover, MIC50s for DV-7751a against ofloxacin-resistant methicillin-resistant S. aureus were 4-, 8-, 16-, 32-, and 64-fold lower than those for tosufloxacin and sparfloxacin, levofloxacin, ofloxacin and fleroxacin, ciprofloxacin, and lomefloxacin, respectively. DV-7751a inhibited the growth of all strains of Streptococcus pneumoniae, Streptococcus pyogenes, and Peptostreptococcus spp. at 0.39, 0.39, and 0.78 micrograms/ml, respectively, and was 4- to > 16-fold more active against enterococci at the MIC90 level than the other quinolones tested. The activity of DV-7751a against Pseudomonas aeruginosa was roughly comparable to those of levofloxacin and sparfloxacin at the MIC90 level and was two- to fourfold less than that of ciprofloxacin. DV-7751a showed activity comparable to those of levofloxacin and ciprofloxacin against the other glucose-nonfermenting bacteria Haemophilus influenzae, Neisseria gonorrhoeae, and Moraxella catarrhalis (MIC90s of 0.025, 0.20, and 0.10 micrograms/ml, respectively). DV-7751a activity was not affected by medium, inoculum size, or the addition of human serum but was decreased under acidic conditions and in human urine, as were the other quinolones tested. Time-kill curve studies demonstrated the rapid bactericidal action of DV-7751a against S. aureus, S. pneumoniae, Escherichia coli, and P. aeruginosa. The frequency of spontaneous resistance to DV-7751a was less than or equal to those of the reference drugs. DV-7751a inhibited the supercoiling activity of DNA gyrases from S. aureus, E. coli, and P. aeruginosa at concentrations comparable to those of levofloxacin and sparfloxacin.

Quinolone Resistance Mechanisms in Klebsiella pneumoniae

K. pneumoniae 900129 was a quinolonesusceptible clinical isolate; spontaneous quinoloneresistant mutants of this strain were selected by plating. K. pneumoniae G1, a first step mutant of 900129, was selected with nalidixic acid at a concentration of 12.5 (2 x the MIC against 900129). K. pneumoniae G2, a second-step mutant of strain G1, was selected with ciprofloxacin at a concentration of 1.56 (2 x the MIC against G1). MICs of nalidixic acid, ciprofloxacin, ofloxacin, tetracycline, chloramphenicol, imipenem and cephaloridine against the 3 strains mentioned above were determined by the agar dilution method. The MICs of quinolones against G1 were 16to 32-fold higher than those against 900129. Compared with G1, the MICs of quinolones to G2 were further increased by 2to 4-fold. Both Gl and G2 had 4to 32-fold lower susceptibilities to tetracycline and chloramphenicol than the parent strain. The susceptibility of G2 to ~-lactams was 2to 4-fold lower than both G1 and the parent strain. DNA gyrases from the 3 strains were purified. DNA supercoiling activity and doses required to inhibit supercoiling by 50% (IDsos) of quinolones were determined by methods described by Sato et al.[l] Gyrases from G1 and G2 were equally resistant to quinolones; IDsos were 3to II-fold higher than those for gyrase from 900129. The IDsos of quinolones for the cross-reconstituted gyrase containing subunit A from G1 or G2 were the same as those for gyrases from G1 and G2. Exchange of subunit B of the gyrases did not affect the 1Dsos (table I). This indicates that modification of subunit A is one of the resistance mechanisms of K. pneumoniae. Outer membrane proteins were prepared by the method of Sawai et alP] and analysed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of outer membrane protein profiles with those of strain 900129 showed that G1 and G2 lacked a 42kD protein. Additionally, G2 lacked a protein of 34kD and 2 lower molecular weight proteins of 24.7 and 21kD. Accumulation of ciprofloxacin by the bacterial cell was determined by the fluorometric method

Resistance Mechanism of MRSA to Quinolones

Methicillin-resistant Staphylococcus aureus (MRSA) is a common nosocomial pathogen accounting for 5 to 24% of all staphylococci isolated in Hua Shan HospitaL It has recently emerged as a major clinical and epidemiological problem_ Most strains of MRS A are resistant to all commonly used antimicrobial agents except vancomycin. Some newer quinolones demonstrate good antibacterial activity against MRSA, and have been used in the treatment of MRSA infections. However, it has been reported that resistance may rapidly develop. In this study, MIC values for 101 clinical strains of MRSA were determined by the agar dilution method, by use of Mueller-Hinton agar with an inoculum of 105 cfu/spot. From) 985 to 1987 the rates of inhibition with norflox~cin, ofloxacin and ciprofloxacin at a concentration i of 2 mg/L against MRSA were 80, 88 and 88%, ~espectively, much higher than in 1991 when rates had decreased to 9.8%, 19.6% and 19.6%, respectively. DNA gyrases from 3 strains of MRSA and S. aureus FDA 209P were purified. DNA supercoiling activity and 50% inhibitory doses (IC50S) of quinolones were examined by methods described in previous reports, with some slight modification. The IC50 values of 5 quinolones tested against DNA gyrases from quinolone-resistant MRSA 89-33 and 90-37 were much higher than those tested against quinolone-susceptible strains. The IC50 values closely correlated with MIC values (table I). DNA gyrase was reconstructed by combining heterologous gyrase subunits. The ICso values of ofloxacin and ciprofloxacin against subunit A (from MRSA 89-33) plus subunit B (from MRSA 87-53) were the same as that against subunit A (from MRSA 89-33) plus subunit B (from MRSA 89-33). The ICso values against subunit A (from MRSA 87-53) plus subunit B (from MRSA 87-53) were similar to those against subunit A (from MRSA 8753) plus subunit B (from MRSA 89-33). This showed that modification of subunit A is one of the resistance mechanisms of MRSA. The following conclusions were reached: 1) resistance of MRSA to norfloxacin, ofloxacin and ciprofloxacin appeared soon after the drugs were widely used in Hua Shan Hospital, Shanghai; 2) DNA gyrase is the main site of action of qui nolones, and the IC50 values reflect the ability of quinolones to inhibit DNA supercoiling and bacterial growth; and 3) alteration of gyrase subunit A is the major mechanism of quinolone resistance.