MICs Of Sparfloxacin Research Articles

Combination of known and unknown mechanisms confers high-level resistance to fluoroquinolones in Enterococcus faecium

In order to elucidate the mechanisms of fluoroquinolone resistance in Enterococcus faecium, spontaneous mutants isolated from Ent. faecium ATCC 19434 by stepwise selection with sparfloxacin (SPX) or norfloxacin (NOR) and 13 clinical isolates of Ent. faecium were characterized by analysing quinolone-resistance-determining regions (QRDRs) of the gyrA, gyrB, parC and parE genes and examining changes in MICs of SPX and NOR in the presence of efflux pump inhibitors. The SPX-selected first-step mutant had a point mutation only in gyrA, and the mutants QR7-18 and QR7-39, and clinical isolates that had point mutations in parC, showed NOR resistance. These results indicate that the primary targets of SPX and NOR are DNA gyrase and topoisomerase IV, respectively, and therefore that the primary target of fluoroquinolones in Ent. faecium differs depending on the structure of the compound used. The characterization of the spontaneous mutants and the clinical isolates demonstrates that in addition to the previously reported alterations in GyrA and ParC, an alteration in GyrB, a NorA-like pump, an unknown efflux pump, which excretes both SPX and NOR from bacterial cells, and probably other unknown mechanism(s) all contribute to fluoroquinolone resistance in Ent. faecium.

In vitro, ex-vivo and in vivo activities of ethambutol and sparfloxacin alone and in combination against mycobacteria

The MIC of ethambutol and sparfloxacin for Mycobacterium smegmatis and M. avium was determined using a broth dilution method. The MICs of sparfloxacin and ethambutol were lower for M. smegmatis than M. avium. The combination of ethambutol and sparfloxacin was additive against M. smegmatis but synergistic for M. avium. The effect of these drugs alone and in combination used at C max levels was screened against M. avium and M. tuberculosis H 37Rv growing intracellularly in murine macrophage cell lines. In vivo studies using this combination given at 100 mg/kg of body weight once weekly showed greater activity than the drugs used singly. These results suggest that combination of ethambutol and sparfloxacin has significant activity intracellularly and in animal models against M. avium and M. tuberculosis.

Activities of new fluoroquinolones against fluoroquinolone-resistant pathogens of the lower respiratory tract.

The activities of six new fluoroquinolones (moxifloxacin, grepafloxacin, gatifloxacin, trovafloxacin, clinafloxacin, and levofloxacin) compared with those of sparfloxacin and ciprofloxacin with or without reserpine (20 microg/ml) were determined for 19 Streptococcus pneumoniae isolates, 5 Haemophilus sp. isolates, and 10 Pseudomonas aeruginosa isolates with decreased susceptibility to ciprofloxacin from patients with clinically confirmed lower respiratory tract infections. Based upon the MICs at which 50% of isolates were inhibited (MIC50s) and MIC90s, the most active agent was clinafloxacin, followed by (in order of decreasing activity) trovafloxacin, moxifloxacin, gatifloxacin, sparfloxacin, and grepafloxacin. Except for clinafloxacin (and gatifloxacin and trovafloxacin for H. influenzae), none of the new agents had improved activities compared with that of ciprofloxacin for P. aeruginosa and H. influenzae. A variable reserpine effect was observed for ciprofloxacin and S. pneumoniae; however, for 9 of 19 (47%) isolates the MIC of ciprofloxacin was decreased by at least fourfold, suggesting the presence of an efflux pump contributing to the resistance phenotype. The laboratory parC (Ser79) mutant strain of S. pneumoniae required eightfold more ciprofloxacin for inhibition than the wild-type strain, but there was no change in the MIC of sparfloxacin and only a 1-dilution increase in the MICs of the other agents. For efflux pump mutant S. pneumoniae the activities of all the newer agents, except for levofloxacin, were reduced. Except for clinafloxacin, all second-step laboratory mutants required at least 2 microg of all fluoroquinolones per ml for inhibition.

Sparfloxacin resistance in clinical isolates of Streptococcus pneumoniae: involvement of multiple mutations in gyrA and parC genes.

Antimicrobial susceptibility testing revealed among 150 clinical isolates of Streptococcus pneumoniae 4 pneumococcal isolates with resistance to fluoroquinolones (MIC of ciprofloxacin, >/=32 microgram/ml; MIC of sparfloxacin, >/=16 microgram/ml). Gene amplification and sequencing analysis of gyrA and parC revealed nucleotide changes leading to amino acid substitutions in both GyrA and ParC of all four fluoroquinolone-resistant isolates. In the case of strains 182 and 674 for which sparfloxacin MICs were 16 and 64 microgram/ml, respectively, nucleotide changes were detected at codon 81 in gyrA and codon 79 in parC; these changes led to an Ser-->Phe substitution in GyrA and an Ser-->Phe substitution in ParC. Strains 354 and 252, for which sparfloxacin MICs were 128 microgram/ml, revealed multiple mutations in both gyrA and parC. These strains exhibited nucleotide changes at codon 85 leading to a Glu-->Lys substitution in GyrA, in addition to Ser-79-->Tyr and Lys-137-->Asn substitutions in ParC. Moreover, strain 252 showed additional nucleotide changes at codon 93, which led to a Trp-->Arg substitution in GyrA. These results suggest that sparfloxacin resistance could be due to the multiple mutations in GyrA and ParC. However, it is possible that other yet unidentified mutations may also be involved in the high-level resistance to fluoroquinolones in S. pneumoniae.

High-level fluoroquinolone resistance in Streptococcus pneumoniae requires mutations in parC and gyrA.

The mechanism of high-level fluoroquinolone resistance was studied in strains of Streptococcus pneumoniae, either selected in vitro or isolated from clinical samples. By using DNA from these high-level-resistant strains, low-level-resistant transformants (MIC of pefloxacin, > or = 32 micrograms/ml; MIC of ciprofloxacin, 4 micrograms/ml; MIC of sparfloxacin, 0.50 micrograms/ml) were obtained at high frequencies (ca.10(-2)), while high-level-resistant transformants (MIC of pefloxacin, > or = 64 micrograms/ml; MIC of ciprofloxacin, 16 to 64 micrograms/ml; MIC of sparfloxacin, > or = 8 micrograms/ml) were obtained only at low frequencies (ca.10(-4)). This suggested that mutations in at least two unlinked genes were necessary to obtain high-level resistance. Low-level resistance was associated with ParC mutations (change from Ser to Tyr at position 79 [Ser79Tyr], Ser79Phe, or Asp83Gly). ParC mutations were associated, in high-level-resistant strains and transformants, with alterations in the quinolone resistance-determining region of GyrA (Ser84Tyr, Ser84Phe, and/or Glu88Lys). Low-level resistance was shown to be necessary for expression of the gyrA mutations. No mutation in the region corresponding to the quinolone resistance-determining region of GyrB and no alteration of drug accumulation were found.

Gyrase mutations in laboratory-selected, fluoroquinolone-resistant mutants of Mycobacterium tuberculosis H37Ra.

To characterize mechanisms of resistance to fluoroquinolones by Mycobacterium tuberculosis, mutants of strain H37Ra were selected in vitro with ofloxacin. Their quinolone resistance-determining regions for gyrA and gyrB were amplified and sequenced to identify mutations in gyrase A or B. Three types of mutants were obtained: (i) one mutant (TKp1) had no mutations in gyrA or gyrB; (ii) mutants that had single missense mutations in gyrA, and (iii) mutants that had two missense mutations resulting in either two altered gyrase A residues or an altered residue in both gyrases A and B. The TKp1 mutant had slightly reduced levels of uptake of [14C]norfloxacin, which was associated with two- to fourfold increases in the MICs of ofloxacin, ciprofloxacin, and sparfloxacin. Gyrase mutations caused a much greater increase in the MICs of fluoroquinolones. For mutants with single gyrA mutations, the increases in the MICs were 4- to 16-fold, and for mutants with double gyrase mutations, the MICs were increased 32-fold or more compared with those for the parent. A gyrA mutation in TKp1 secondary mutants was associated with 32- to 128-fold increases in the MICs of ofloxacin and ciprofloxacin compared with the MICs for H37Ra and an eight-fold increase in the MIC of sparfloxacin. Sparfloxacin was the most active fluoroquinolone tested. No sparfloxacin-resistant single-step mutants were selected at concentrations of > 2.5 micrograms/ml, and high-level resistance (i.e., MIC, > and = 5 micrograms/ml) was associated with two gyrase mutations. Mutations in gyrB and possibly altered levels of intracellular accumulation of drug are two additional mechanisms that may be used by M. tuberculosis in the development of fluoroquinolone resistance. Because sparfloxacin is more active in vitro and selection of resistance appears to be less likely to occur, it may have important advantage over ofloxacin or ciprofloxacin for the treatment of tuberculosis.

In vitro susceptibility of pneumococci to trovafloxacin, penicillin G, and other antimicrobial agents in the Czech Republic and Slovakia.

The in vitro activity of the new naphthyridone trovafloxacin (CP 99,219) was compared with those of penicillin G and six other agents (cefpodoxime, erythromycin, azithromycin, clindamycin, ciprofloxacin, and sparfloxacin) against 316 penicillin-susceptible and -resistant pneumococci isolated in the former Czechoslovakia. Trovafloxacin was very active against strains of Streptococcus pneumoniae (MIC50 and MIC90 0.25 microgram/ml). Ciprofloxacin was less active (MIC50 1.0 microgram/ml, MIC90 2.0 micrograms/ml), and MICs of sparfloxacin were between those of trovafloxacin and ciprofloxacin (MIC50 and MIC90 both 0.5 microgram/ml). MICs of cefpodoxime, erythromycin, azithromycin, and clindamycin were higher for strains intermediately resistant or resistant to penicillin than for penicillin-susceptible strains.

Inhibitory effects of ciprofloxacin and sparfloxacin on DNA gyrase purified from fluoroquinolone-resistant strains of methicillin-resistant Staphylococcus aureus.

The activities of new quinolones against 140 strains of methicillin-resistant Staphylococcus aureus were determined. From the relationship between the MICs of sparfloxacin and ciprofloxacin, fluoroquinolone-resistant S. aureus 6171 (MIC of sparfloxacin, 200 micrograms/ml; MIC of ciprofloxacin, 100 micrograms/ml) and fluoroquinolone-susceptible S. aureus FDA 209-P were selected for purification of the subunit A and B proteins of their DNA gyrases. The supercoiling activities of reconstituted ArBr (r, resistant) (from strain 6171) and ArBs (s, susceptible) gyrases were 40-fold more resistant to new quinolones than those of AsBs from FDA 209-P and AsBr gyrases. The 50% inhibitory doses of ciprofloxacin and sparfloxacin for AmBm (from mutant 19) and AmBs (m, moderately resistant) gyrases were 15- to 27-fold higher than those for AsBs and AsBm gyrases. These findings indicate that one of the resistance mechanisms of S. aureus against fluoroquinolones is a modification of the gyrase subunit A protein.

In vitro studies with five quinolones: evidence for changes in relative potency as quinolone resistance rises

A panel of 203 staphylococci, Enterobacteriaceae, Pseudomonas aeruginosa, and miscellaneous nonfermentative gram-negative bacilli were chosen for their various susceptibilities to ciprofloxacin. On the basis of agar dilution susceptibilities, each of the four taxonomic groups was divided into ciprofloxacin-susceptible, moderately resistant, and highly resistant subgroups, and each subgroup was then further analyzed for its susceptibility to the fluoroquinolones CI-960, CI-990, sparfloxacin, and ofloxacin. Although the MICs of each quinolone increased as ciprofloxacin resistance increased, the potency of CI-960 appeared to increase relative to the potencies of the other quinolones. Similarly, the MICs of sparfloxacin and ofloxacin appeared to be less affected by ciprofloxacin resistance than were the MICs of ciprofloxacin or CI-990. Single-step mutants of representative clinical isolates with different levels of ciprofloxacin resistance were selected to determine whether the study quinolones differed in their propensity to select resistant mutants and whether the presence of preexisting ciprofloxacin resistance influenced the subsequent development of resistance. Each of the five fluoroquinolones and nalidixic acid selected mutants that exhibited generally modest decreases in quinolone susceptibility (4- to 16-fold). However, CI-960 inhibited significantly more mutants (80%) than did the other quinolones (39 to 59%) at a concentration of 1 microgram/ml. The presence of preexisting ciprofloxacin resistance appeared to be associated with higher mutational frequencies in coagulase-negative staphylococci exposed to each of the fluoroquinolones and in Serratia marcescens exposed to nalidixic acid. Preexisting ciprofloxacin resistance did not influence the development of resistance in the strains of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, or Pseudomonas aeruginosa that were studied. The results of this study suggest that quinolones are not affected equally by all resistance mechanisms, and although each one can select mutants, some quinolones may be active against these mutants at clinically achievable concentrations.

In vitro susceptibility of Mycobacterium avium complex to the new fluoroquinolone sparfloxacin (CI-978; AT-4140) and comparison with ciprofloxacin

We tested the activity of the new fluoroquinolone sparfloxacin (CI-978; AT 4140) against 30 strains of Mycobacterium avium complex (MAC) isolated from patients with acquired immune deficiency syndrome. MICs of sparfloxacin (range, less than or equal to 0.06 to 4 micrograms/ml) were lower than MICs of ciprofloxacin for all 30 strains, and MBCs for acid-fast bacteria were lower for 28 of the 30 strains. In synergism experiments using 10 strains of MAC, fractional inhibitory concentration indices revealed that the combination of sparfloxacin plus ethambutol was synergistic against 9 strains, and the three-drug combination of sparfloxacin plus ethambutol plus rifampin was synergistic against all strains. In the absence of ethambutol, the combination of sparfloxacin plus rifampin appeared to be antagonistic against three of the MAC strains.