Isolation and characterization of a dibenzofuran-degrading yeast: identification of oxidation and ring cleavage products

PURPOSE: To investigate the development of fluoroquinolone resistance among Neisseria gonorrhoeae isolates in Japan and the frequency and patterns of mutations involving the GyrA and ParC proteins, which confer quinolone resistance to the bacteria, in isolates. MATERIALS AND METHODS: Antimicrobial susceptibilities of 145 gonococcal isolates, including 79 isolated from February 1992 through February 1993 and 66 isolated from February 1995 through February 1996, to six fluoroquinolones and several other antibiotics were compared with those of 27 isolates obtained from 1981 through 1984. To identify mutations in gyrA and parC genes of the isolates, the quinolone resistance-determining regions of the gyrA and parC genes were PCR-amplified and the PCR products were directly sequenced. RESULTS: The minimum inhibitory concentration for 90% of strains (MIC90) values of norfloxacin for the isolates from 1992 to 93 (4 microg./ml.) and 1995 to 96 (8 microg./ml.) were 16- and 32-fold, respectively, higher than those for isolates from 1981 to 84 (0.25 microg./ml.). The MIC90 values of ciprofloxacin for isolates from 1992 to 93 (0.5 microg./ml.) and 1995 to 96 (1 microg./ml.) showed increase of 8- and 16-fold, respectively, in comparison with those from 1981 to 84 (0.063 microg./ml.). The isolates from 1992 to 93 and 1995 to 96 were also less susceptible to newer fluoroquinolones including levofloxacin, sparfloxacin, DU-6859a and AM-1155, as compared with those from 1981 to 84. In 46 (67.6%) and 16 (23.5%) of the 68 gonococcal strains sequenced, GyrA and ParC mutations were identified, respectively. No ParC substitutions were identified in any isolates without co-existence of the GyrA mutation. A Ser-91 to Phe mutation, which was detected in 30 (65.2%) of the 46 isolates with GyrA mutations, was the most common GyrA mutation. Mutants with the single Ser-91 to Phe substitution in GyrA were 12-fold and at least 13-fold, respectively, less susceptible to norfloxacin and ciprofloxacin than the wild type. CONCLUSIONS: The results obtained in this study suggest that a high prevalence of gonococcal isolates with the Ser-91 to Phe mutation in GyrA has reduced the susceptibility of this organism to fluoroquinolones in Japan.     

Expression patterns of proliferating cell nuclear antigen in the small intestine of mice infected with Metagonimus yokogawai and Metagonimus Miyata type

The in vitro antimicrobial activities of AM-1155, a new fluoroquinolone, tosufloxacin and fleroxacin were tested against 55 clinical isolates of Neisseria gonorrhoeae using the agar dilution method. In our previous study, all the strains had been examined for mutations in the region corresponding to the quinolone-resistance determining region of the Escherichia coli gyrA gene and the analogous region of the parC gene, and tested for susceptibility to ciprofloxacin. In this study, the 55 isolates of N. gonorrhoeae were assigned to one of three categories based on the presence or absence of alterations in GyrA and ParC. In each category, the antimicrobial activity of AM-1155 against the isolates was compared with those of tosufloxacin and fleroxacin. The MICs of AM-1155 for 11 highly fluoroquinolone-resistant isolates with alterations in both GyrA and ParC ranged from 0.06 to 1.0 microgram/ml. The MICs inhibiting 50% (MIC50) and 90% (MIC90) of these isolates were 0.125 and 1.0 microgram/ml, respectively. The MICs of AM-1155 for 20 moderately fluoroquinolone-resistant isolates with alterations only in GyrA ranged from 0.03 to 0.25 microgram/ml (MIC50, 0.06 microgram/ml; MIC90m, 0.125 microgram/ml). The MICs of AM-1155 for 24 of the quinolone-susceptible isolates without alterations in either GyrA or ParC ranged from 0.004 to 0.03 microgram/ml (MIC50, 0.008 microgram/ml. MIC90, 0.015 microgram/ml). There were significant differences between the MIC distribution of AM-1155 and each corresponding MIC distribution of tosufloxacin and fleroxacin in these three categories to which the 55 isolates were assigned (p < 0.05). Based on the MIC90S of the tested fluoroquinolones, AM-1155 was two- and eightfold more active against the highly fluoroquinolone-resistant isolates than tosufloxacin and fleroxacin, respectively. Against the moderately fluoroquinolone-resistant isolates, AM-1155 was four- and sixteenfold more active than tosufloxacin and fleroxacin, respectively. Against the quinolone-susceptible strains, AM-1155 was also two- to fourfold more active than the other fluoroquinolones. Overall, AM-1155 exhibited more potent in vitro activity against both quinolone-resistant and quinolone-susceptible isolates of N. gonorrhoeae than tosufloxacin and fleroxacin. In ciprofloxacin treatment failures of gonorrhea at single doses of 500 mg. MICs for the causative organisms have ranged from 1.0 to 16.0 micrograms/ml. The MICs of AM-1155 for the isolates harboring quinolone resistance-associated genetic alterations, including strains exhibiting ciprofloxacin MICs of 2.0 and 8.0 micrograms/ml, still ranged from 0.03 to 1.0 microgram/mL A single-dose study in humans has demonstrated higher peak serum concentrations and longer half-lives of AM-1155, resulting in the AUC0-00 values of AM-1155, which are threefold greater than those of ciprofloxacin at the single doses of 400 and 600 mg. Because of its potent in vitro antimicrobial activity and advantageous pharmacokinetic behavior, AM-1155 may be a clinically useful agent for treating gonorrhea including that caused by quinolone-resistant strains.     

Interferons alpha/beta inhibit IL-7-induced proliferation of CD4- CD8- CD3- CD44+ CD25+ thymocytes, but do not inhibit that of CD4- CD8- CD3- CD44- CD25- thymocytes

We have determined partial sequences of the gyrA and parC genes of Enterobacter cloacae type strain including the regions analogous to the quinolone resistance-determining region of the Escherichia coli gyrA gene. The deduced 65- and 49-amino acid sequences of the determined regions of the E. cloacae gyrA and parC genes were identical to the corresponding regions of the E. coli GyrA and ParC proteins, respectively. We examined 40 clinical strains of E. cloacae isolated from patients with urinary tract infection for susceptibilities to nalidixic acid and ciprofloxacin. Based on the nalidixic acid and ciprofloxacin MICs, these isolates were divided into 19 quinolone-susceptible strains (MICs of nalidixic acid, 3.13-25 mg/L; MICs of ciprofloxacin, < or = 0.025 mg/L) and 21 quinolone-resistant strains (MICs of nalidixic acid, 400 to > 800 mg/L; MICs of ciprofloxacin, 0.39-100 mg/L). We analysed five quinolone-susceptible and 21 quinolone-resistant strains for alterations in GyrA and ParC. The five quinolone-susceptible strains had amino acid sequences in GyrA and ParC identical to those of type strain. Of the 21 quinolone-resistant isolates, three (MICs of nalidixic acid, 400 to > 800 mg/L; MICs of ciprofloxacin, 0.39-3.13 mg/L) had a single amino acid change at the position equivalent to Ser-83 in the E. coli GyrA protein and no alterations in ParC; one (MIC of nalidixic acid, > 800 mg/L; MIC of ciprofloxacin, 3.13 mg/L) had a single amino acid change at Ser-83 in GyrA and a single amino acid change at the position equivalent to Glu-84 in the E. coli ParC protein; two (MIC of nalidixic acid, > 800 mg/L; MIC of ciprofloxacin, 25 mg/L) had double amino acid changes at Ser-83 and Asp-87 in GyrA and no alterations in ParC; and 15 (MICs of nalidixic acid, > 800 mg/L; MICs of ciprofloxacin, 25-100 mg/L) had double amino acid changes at Ser-83 and Asp-87 in GyrA and a single amino acid change at Ser-80 or Glu-84 in ParC. This study suggests, that in clinical isolates of E. cloacae, DNA gyrase is a primary target of quinolones, that only a single amino acid change at Ser-83 in GyrA is sufficient to generate high-level resistance to nalidixic acid and to decrease susceptibility to ciprofloxacin, and that the accumulation of amino acid changes in GyrA and the simultaneous presence of the ParC alterations play a central role in developing high-level resistance to ciprofloxacin.     

Contribution of mutations in gyrA and parC genes to fluoroquinolone resistance of mutants of Streptococcus pneumoniae obtained in vivo and in vitro

We have analyzed by gene amplification and sequencing mutations in the quinolone resistance-determining regions of the gyrA, gyrB, and parC genes of fluoroquinolone-resistant Streptococcus pneumoniae mutants obtained during therapy or in vitro. Mutations leading to substitutions in ParC were detected in the two mutants obtained in vivo, BM4203-R (substitution of a histidine for an aspartate at position 84 [Asp-84-->His]; Staphylococcus aureus coordinates) and BM4204-R (Ser-80-->Phe), and in two mutants obtained in vitro (Ser-80-->Tyr). An additional mutant obtained in vitro, BM4205-R3, displayed a higher level of fluoroquinolone resistance and had a mutation in gyrA leading to a Ser-84-->Phe change. We could not detect any mutation in the three remaining mutants obtained in vitro. Total DNA from BM4203-R, BM4204-R, and BM4205-R3 was used to transform S. pneumoniae CP1000 by selection on fluoroquinolones. For the parC mutants, transformants with phenotypes indistinguishable from those of the donors were obtained at frequencies (5 x 10(-3) to 8 x 10(-3)) compatible with monogenic transformation. By contrast, transformants were obtained at a low frequency (4 x 10(-5)), compatible with the transformation of two independent genes, for the gyrA mutant. Resistant transformants of CP1000 were also obtained with an amplified fragment of parC from BM4203-R and BM4204-R but not with a gyrA fragment from BM4205-R3. All transformants had mutations identical to those in the donors. These data strongly suggest that ParC is the primary target for fluoroquinolones in S. pneumoniae and that BM4205-R3 is resistant to higher levels of the drugs following the acquisition of two mutations, including one in gyrA.     

ParC and GyrA may be interchangeable initial targets of some fluoroquinolones in Streptococcus pneumoniae

To evaluate the role of known topoisomerase IV and gyrase mutations in the fluoroquinolone (FQ) resistance of Streptococcus pneumoniae, we transformed susceptible strain R6 with PCR-generated fragments encompassing the quinolone resistance-determining regions (QRDRs) of parC or gyrA from different recently characterized FQ-resistant mutants. Considering the MICs of FQs and the GyrA and/or ParC mutations of the individual transformants, we found three levels of resistance. The first level was obtained when a single target, ParC or GyrA, depending on the FQ, was modified. An additional mutation(s) in a second target, GyrA or ParC, led to the second level. The highest increases in resistance levels were seen for Bay y3118 and moxifloxacin with the transformant harboring a double mutation in both ParC and GyrA. When a single modified target was considered, only the ParC mutation(s) led to an increase in the MICs of pefloxacin and trovafloxacin. In contrast, the GyrA or ParC mutation(s) could lead to increases in the MICs of ciprofloxacin, sparfloxacin, grepafloxacin, Bay y3118, and moxifloxacin. These results suggest that the preferential target of trovafloxacin and pefloxacin is ParC, whereas either ParC or GyrA may both be initial targets for the remaining FQs tested. The contribution of the ParC and GyrA mutations to efflux-mediated FQ resistance was also examined. Active efflux was responsible for two- to fourfold increases in the MICs of ciprofloxacin for the transformants, regardless of the initial FQ resistance levels of the recipients.     

Analysis of quinolone resistance mechanisms in a sparfloxacin-resistant clinical isolate of Neisseria gonorrhoeae

BACKGROUND AND OBJECTIVES: Recently, a reduction in the susceptibility of clinical isolates of Neisseria gonorrhoeae to newer fluoroquinolones including sparfloxacin in vitro has been recognized in Japan. The quinolone resistance mechanisms in gonococcal isolates from a patient with clinical failure of sparfloxacin treatment was investigated. GOAL: To report a man with gonococcal urethritis in whom clinical failure of sparfloxacin treatment occurred and to examine the quinolone resistance mechanisms in gonococcal isolates from the patient. STUDY DESIGN: A man with gonococcal urethritis was treated with oral 100 mg sparfloxacin three times daily for 5 days. However, clinical failure of the sparfloxacin treatment was observed. The antimicrobial susceptibilities of pretreatment and posttreatment isolates to sparfloxacin and other agents were measured. To analyze quinolone resistance mechanisms in the set of isolates, DNA sequencing of the genes corresponding to the quinolone resistance-determining regions within the GyrA and ParC proteins was performed. We also assayed the intracellular sparfloxacin accumulation level in these gonococcal cells. Moreover, we performed pulsed-field gel electrophoresis analysis to determine whether the pretreatment and posttreatment isolates were isogenic. RESULTS: The minimum inhibitory concentration of sparfloxacin for the posttreatment isolate (4 micrograms/ml) was 16 times higher than that for the pretreatment isolate (0.25 microgram/ml). The pretreatment isolate contained three mutations, including a Ser-91 to Phe mutation and an Asp-95 to Asn mutation in GyrA and a Ser-88 to Pro mutation in ParC. The posttreatment isolate had four mutations, including the same three mutations and an additional Glu-91 to Gly mutation in ParC. The sparfloxacin accumulation level within 30 minutes in the posttreatment isolate was four times less than that in the pretreatment isolate. There were no differences in the pulsed-field gel electrophoresis patterns between the pretreatment and posttreatment isolates from the patient. CONCLUSIONS: The emergence of a fluoroquinolone-resistant N. gonorrhoeae isolate with multiple mutations involving GyrA and ParC reduced the response to sparfloxacin treatment. Multiple dosing and long-term treatment with sparfloxacin seems to induce a mutation in ParC and an alteration leading to reduced drug accumulation that contribute to increasing the fluoroquinolone resistance level.     

Detection of mutations in parC in quinolone-resistant clinical isolates of Escherichia coli

The gene parC encodes the A subunit of topoisomerase IV of Escherichia coli. Mutations in the parC region analogous to those in the quinolone resistance-determining region of gyrA were investigated in 27 clinical isolates of E. coli for which ciprofloxacin MICs were 0.0007 to 128 micrograms/ml. Of 15 isolates for which ciprofloxacin MICs were > or = 1 microgram/ml, 8 showed a change in the serine residue at position 80 (Ser-80), 4 showed a change in Glu-84, and 3 showed changes in both amino acids. No mutations were detected in 12 clinical isolates for which ciprofloxacin MICs were < or = 0.25 micrograms/ml. These findings suggest that ParC from E. coli may be another target for quinolones and that mutations at residues Ser-80 and Glu-84 may contribute to decreased fluoroquinolone susceptibility.     

Targeting of DNA gyrase in Streptococcus pneumoniae by sparfloxacin: selective targeting of gyrase or topoisomerase IV by quinolones

gyrA and parC mutations have been identified inn Streptococcus pneumoniae mutants stepwise selected for resistance to sparfloxacin, an antipneumococcal fluoroquinolone. GyrA mutations (at the position equivalent to resistance hot spot Ser-83 in Escherichia coli GyrA) were found in all 17 first-step mutants examined and preceded DNA topoisomerase IV parC mutations (at Ser-79 or Glu-83), which appeared only in second-step mutants. The targeting of gyrase by sparfloxacin in S. pneumoniae but of topoisomerase IV by ciprofloxacin indicates that target preference can be altered by changes in quinolone structure.     

Alterations in topoisomerase IV and DNA gyrase in quinolone-resistant mutants of Mycoplasma hominis obtained in vitro

Mycoplasma hominis mutants were selected stepwise for resistance to ofloxacin and sparfloxacin, and their gyrA, gyrB, parC, and parE quinolone resistance-determining regions were characterized. For ofloxacin, four rounds of selection yielded six first-, six second-, five third-, and two fourth-step mutants. The first-step mutants harbored a single Asp426-->Asn substitution in ParE. GyrA changes (Ser83-->Leu or Trp) were found only from the third round of selection. With sparfloxacin, three rounds of selection generated 4 first-, 7 second-, and 10 third-step mutants. In contrast to ofloxacin resistance, GyrA mutations (Ser83-->Leu or Ser84-->Trp) were detected in the first-step mutants prior to ParC changes (Glu84-->Lys), which appeared only after the second round of selection. Further analysis of eight multistep-selected mutants of M. hominis that were previously described (2) revealed that they carried mutations in ParE (Asp426-->Asn), GyrA (Ser83-->Leu) and ParE (Asp426-->Asn), GyrA (Ser83-->Leu) and ParC (Ser80-->Ile), or ParC (Ser80-->Ile) alone, depending on the fluoroquinolone used for selection, i.e., ciprofloxacin, norfloxacin, ofloxacin, or pefloxacin, respectively. These data indicate that in M. hominis DNA gyrase is the primary target of sparfloxacin whereas topoisomerase IV is the primary target of pefloxacin, ofloxacin, and ciprofloxacin.     

Role of mutations in DNA gyrase genes in ciprofloxacin resistance of Pseudomonas aeruginosa susceptible or resistant to imipenem

In Pseudomonas aeruginosa, resistance to imipenem is mainly related to a lack of protein OprD and resistance to fluoroquinolones is mainly related to alterations in DNA gyrase. However, strains cross resistant to fluoroquinolones and imipenem have been selected in vitro and in vivo with fluoroquinolones. We investigated the mechanisms of resistance to fluoroquinolones in 30 clinical strains of P. aeruginosa resistant to ciprofloxacin (mean MIC, >8 micrograms/ml), 20 of which were also resistant to imipenem (mean MIC, >16 micrograms/ml). By immunoblotting, OprD levels were markedly decreased in all of the imipenem-resistant strains. Plasmids carrying the wild-type gyrA gene (pPAW207) or gyrB gene (pPBW801) of Escherichia coli were introduced into each strain by transformation. MICs of imipenem did not change after transformation, whereas those of ciprofloxacin and sparfloxacin dramatically decreased (25- to 70-fold) for all of the strains. For 28 of them (8 susceptible and 20 resistant to imipenem), complementation was obtained with pPAW207 but not with pPBW801. After complementation, the geometric mean MICs of ciprofloxacin and sparfloxacin (MICs of 0.3 microgram/ml and 0.5 microgram/ml, respectively) were as low as those for wild-type strains. Complementation was obtained only with pPBW801 for one strain and with pPAW207 and pPBW801 for one strain highly resistant to fluoroquinolones. These results demonstrate that in clinical practice, gyrA mutations are the major mechanism of resistance to fluoroquinolones even in the strains of P. aeruginosa resistant to imipenem and lacking OprD, concomitant resistance to these drugs being the result of the addition of at least two independent mechanisms.     

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.     

Autoimmune thyroid disease and insulin-dependent diabetes mellitus during pregnancy and post partum

The in-vitro antimicrobial activity of HSR-903, a new fluoroquinolone, was tested against 51 clinical Neisseria gonorrhoeae isolates in comparison with ciprofloxacin, levofloxacin and sparfloxacin. The MICs of HSR-903 for 11 isolates with alterations in both GyrA and ParC, for 19 isolates with alterations only in GyrA and for 21 isolates without alterations in either GyrA or ParC ranged from 0.03 mg/L to 1.0 mg/L (MIC90 = 0.25 mg/L), from 0.03 mg/L to 0.5 mg/L (MIC90 = 0.125 mg/L) and from < or = 0.001 mg/L to 0.008 mg/L (MIC90 = 0.004 mg/L), respectively. Levofloxacin and ciprofloxacin were the least active of the four quinolones tested, particularly against the mutant strains. Sparfloxacin was more active, but HSR-903 exhibited the most potent in-vitro activity against the clinical N. gonorrhoeae isolates, including those harbouring quinolone-resistance-associated alterations in GyrA and ParC.     

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.     

Alterations in the GyrA subunit of DNA gyrase and the ParC subunit of DNA topoisomerase IV associated with quinolone resistance in Enterococcus faecalis

The gyrA and parC genes of 31 clinical isolates of Enterococcus faecalis, including fluoroquinolone-resistant isolates, were partially sequenced and analyzed for target alterations. Topoisomerase IV may be a primary target in E. faecalis, but high-level fluoroquinolone resistance was associated with simultaneous alterations in both GyrA and ParC.     

Rapid emergence of high-level resistance to quinolones in Campylobacter jejuni associated with mutational changes in gyrA and parC

Quinolone resistance in clinical isolates of Campylobacter jejuni in Sweden increased more than 20-fold at the beginning of the 1990s. Resistance to 125 microgram of ciprofloxacin per ml in clinical isolates was associated with chromosomal mutations in C. jejuni leading to a Thr-86-Ile substitution in the gyrA product and a Arg-139-Gln substitution in the parC product.     

Primary targets of fluoroquinolones in Streptococcus pneumoniae

Mutants of wild-type Streptococcus pneumoniae IID553 with mutations in parC were obtained by selection with trovafloxacin, levofloxacin, norfloxacin, and ciprofloxacin. All of the parC mutants were cross-resistant to the selecting agents but were not resistant to gatifloxacin and sparfloxacin. On the other hand, gyrA mutants were isolated by selection with gatifloxacin and sparfloxacin. The gyrA mutants were cross-resistant to gatifloxacin and sparfloxacin but were not resistant to the other fluoroquinolones tested. These results suggest that in wild-type S. pneumoniae the primary target of trovafloxacin, levofloxacin, norfloxacin, and ciprofloxacin is topoisomerase IV, whereas the primary target of gatifloxacin and sparfloxacin is DNA gyrase.     

Resistance to fluoroquinolones in Escherichia coli isolated from poultry

Quinolone-resistant Escherichia coli strains were isolated from poultry clinical samples in Saudi Arabia. The poultry flocks had been treated with oxolinic acid or flumequine prophylaxis. The measure of the uptake of fluoroquinolones showed that none of the strains had a reduced accumulation of quinolones. The result of complementation with the wild-type E. coli gyrA gene, which restored fluoroquinolone susceptibility, and the isolation of DNA gyrase from six isolates indicated that the resistant strains had an altered DNA gyrase. The minimum effective dose of ciprofloxacin for inhibition of supercoiling catalyzed by the isolated gyrases varied from 0.085 microgram/ml for a susceptible isolate (MIC < 4 micrograms/ml) up to 96 micrograms/ml for the more resistant one (strain 215, MIC > 64 micrograms/ml). For the same two isolates, the minimum effective doses of sparfloxacin varied from 0.17 up to 380 micrograms/ml. The in vitro selection of spontaneous single-step fluoroquinolone-resistant mutants using ciprofloxacin suggested that the more resistant mutants are likely the result of several mutations. These results also show that, as in human medicine, cross-resistance between older quinolones and fluoroquinolones can exist in veterinary isolates and reiterate the need for the prudent use of these drugs.     

Development of a rapid assay for detecting gyrA mutations in Escherichia coli and determination of incidence of gyrA mutations in clinical strains isolated from patients with complicated urinary tract infections

The MICs of ofloxacin for 743 strains of Escherichia coli isolated from 1988 to 1994 were determined by testing. The strains were from patients with urinary tract infections complicated by functional or anatomical disorders of the urinary tract. Those determined to be ofloxacin resistant (MIC, > or =12.5 microg/ml) comprised 3 of 395 strains (1.3%) from the 1988 to 1990 group, 2 of 166 strains (1.2%) from the 1991 to 1992 group, and 7 of 182 strains (3.8%) from the 1993 to 1994 group. The incidence of resistant strains increased significantly during this period. The percentage of isolates with moderately decreased susceptibilities to ofloxacin (MIC, 0.39 to 3.13 microg/ml) also rose during the same period. To determine the incidence of gyrA mutations in urinary-tract-derived strains of E. coli, we developed a simple and rapid assay based on PCR amplification of the region of the gyrA gene containing the mutation sites followed by digestion of the PCR product with a restriction enzyme. Using this assay, we examined all 182 strains isolated in 1993 and 1994 for the presence of mutations at Ser-83 and Asp-87 in the gyrA gene. Of these strains, 33 (18.1%) had mutations in the gyrA gene. The incidences of mutations at Ser-83, at Asp-87, and at both codons were 10.4 (19 strains), 4.4 (8 strains), and 3.3% (6 strains), respectively. To determine the correlation of the mutations in the gyrA gene with susceptibilities to quinolones (nalidixic acid, ofloxacin, norfloxacin, and ciprofloxacin), we further examined 116 strains for which the MICs of ofloxacin were > or =0.2 microg/ml that were chosen from the isolates in the 1988 to 1992 group. The MICs of nalidixic acid for the strains without mutations at either Ser-83 or Asp-87 were < or =25 microg/ml, whereas those for the strains with single mutations or double mutations were from 50 to >800 microg/ml. For the fluoroquinolones, significant differences in the distributions of the MICs were observed among the strains without mutations, with single mutations, and with double mutations. The accumulation of mutations in the gyrA gene was associated with an increase in fluoroquinolone resistance. Ofloxacin MICs for the majority of the strains with single and double mutations were 0.39 to 3.13 and 6.25 to 100 microg/ml, respectively. This study demonstrates a chronological increase in the percentage of not only highly fluoroquinolone-resistant strains, corresponding to those with double mutations in the gyrA gene, but also strains with moderately decreased susceptibilities to fluoroquinolones, corresponding to those with single mutations. This increase in the incidence of strains with a single mutation in the gyrA gene portends a further increase in the incidence of strains with clinically significant resistance to fluoroquinolones.     

Antibacterial activity of gatifloxacin (AM-1155, CG5501, BMS-206584), a newly developed fluoroquinolone, against sequentially acquired quinolone-resistant mutants and the norA transformant of Staphylococcus aureus

Alternate mutations in the grlA and gyrA genes were observed through the first- to fourth-step mutants which were obtained from four Staphylococcus aureus strains by sequential selection with several fluoroquinolones. The increases in the MICs of gatifloxacin accompanying those mutational steps suggest that primary targets of gatifloxacin in the wild type and the first-, second-, and third-step mutants are wild-type topoisomerase IV (topo IV), wild-type DNA gyrase, singly mutated topo IV, and singly mutated DNA gyrase, respectively. Gatifloxacin had activity equal to that of tosufloxacin and activity more potent than those of norfloxacin, ofloxacin, ciprofloxacin, and sparfloxacin against the second-step mutants (grlA gyrA; gatifloxacin MIC range, 1.56 to 3.13 microg/ml) and had the most potent activity against the third-step mutants (grlA gyrA grlA; gatifloxacin MIC range, 1.56 to 6.25 microg/ml), suggesting that gatifloxacin possesses the most potent inhibitory activity against singly mutated topo IV and singly mutated DNA gyrase among the quinolones tested. Moreover, gatifloxacin selected resistant mutants from wild-type and the second-step mutants at a low frequency. Gatifloxacin possessed potent activity (MIC, 0.39 microg/ml) against the NorA-overproducing strain S. aureus NY12, the norA transformant, which was slightly lower than that against the parent strain SA113. The increases in the MICs of the quinolones tested against NY12 were negatively correlated with the hydrophobicity of the quinolones (correlation coefficient, -0.93; P < 0.01). Therefore, this slight decrease in the activity of gatifloxacin is attributable to its high hydrophobicity. Those properties of gatifloxacin likely explain its good activity against quinolone-resistant clinical isolates of S. aureus harboring the grlA, gyrA, and/or norA mutations.     

Reproducibility of in vivo carotid intima-media thickness measurements: a review

The gyrA gene of Campylobacter fetus subsp. fetus, which encodes the A subunit of DNA gyrase, was cloned, and its nucleotide sequence was determined. An open reading frame of 2,586 nucleotides which encodes a polypeptide of 862 amino acids with an Mr of 96,782 was identified. C. fetus subsp. fetus GyrA is most closely related to Campylobacter jejuni GyrA, with 73% homology at the nucleotide level and 78% identity between polypeptides. The next most closely related GyrA was that from Helicobacter pylori, with both DNA homology and amino acid identity of 63%. The gyrA and gyrB (DNA gyrase B subunit) genes were located on the genomic map of C. fetus subsp. fetus ATCC 27374 and shown to be separate. A clinical isolate of C. fetus subsp. fetus and a laboratory-derived mutant of ATCC 27374, both resistant to ciprofloxacin, had identical mutations within the quinolone resistance determining region. In both mutants a G-->T transversion, corresponding to a substitution of Asp-91 to Tyr in GyrA, was linked to ciprofloxacin resistance, giving MICs of 8 to 16 micrograms/ml.