In vitro activities of cephalosporins and quinolones against Escherichia coli strains isolated from diarrheic dairy calves.
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The in vitro activities of several cephalosporins and quinolones against 195 strains of Escherichia coli isolated from diary calves affected by neonatal diarrhea were determined. One hundred thirty-seven of these strains produced one or more potential virulence factors (F5, F41, F17, cytotoxic necrotizing factor, verotoxin, and the eae gene), but the remaining 58 strains did not produce any of these factors. From 11 to 18% of the E. coli strains were resistant to cephalothin, nalidixic acid, enoxacin, and enrofloxacin. However, cefuroxime, cefotaxime, and cefquinome were highly effective against the E. coli isolates tested. Some significant differences (P < 0.05) in resistance to quinolones between the strains producing potential virulence factors and nonfimbriated, nontoxigenic, eae-negative strains were found. Thus, eae-positive, necrotoxigenic, and verotoxigenic (except for nalidixic acid) E. coli strains were significantly more sensitive to nalidixic acid, enoxacin, and enrofloxacin than nonfimbriated, nontoxigenic, eae-negative strains. Moreover, eae-positive strains were significantly more sensitive to enoxacin and enrofloxacin than F5-positive strains. Thus, the result of this study suggest that the bovine E. coli strains that produce some potential virulence factors are more sensitive to quinolones than those that do not express these factors. (+info)
Cloning, expression, and enzymatic characterization of Pseudomonas aeruginosa topoisomerase IV.
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The topoisomerase IV subunit A gene, parC homolog, has been cloned and sequenced from Pseudomonas aeruginosa PAO1, with cDNA encoding the N-terminal region of Escherichia coli parC used as a probe. The homolog and its upstream gene were presumed to be parC and parE through sequence homology with the parC and parE genes of other organisms. The deduced amino acid sequence of ParC and ParE showed 33 and 32% identity with that of the P. aeruginosa DNA gyrase subunits, GyrA and GyrB, respectively, and 69 and 75% identity with that of E. coli ParC and ParE, respectively. The putative ParC and ParE proteins were overexpressed and separately purified by use of a fusion system with a maltose-binding protein, and their enzymatic properties were examined. The reconstituted enzyme had ATP-dependent decatenation activity, which is the main catalytic activity of bacterial topoisomerase IV, and relaxing activities but had no supercoiling activity. So, the cloned genes were identified as P. aeruginosa topoisomerase IV genes. The inhibitory effects of quinolones on the activities of topoisomerase IV and DNA gyrase were compared. The 50% inhibitory concentrations of quinolones for the decatenation activity of topoisomerase IV were from five to eight times higher than those for the supercoiling activities of P. aeruginosa DNA gyrase. These results confirmed that topoisomerase IV is less sensitive to fluoroquinolones than is DNA gyrase and may be a secondary target of new quinolones in wild-type P. aeruginosa. (+info)
Impact of gyrA and parC mutations on quinolone resistance, doubling time, and supercoiling degree of Escherichia coli.
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Isogenic mutants derived from quinolone-susceptible isolate WT by introducing gyrA (S83L, D87G) and parC (S80I, E84K) mutations associated with quinolone resistance were characterized with respect to quinolone resistance, growth rate, and degree of global supercoiling. The latter was determined by use of a pair of reporter plasmids carrying supercoiling-dependent promoters pgyrA and ptopA, respectively, transcriptionally fused to the reporter gene bla coding for TEM-1 beta-lactamase. The quotient (Qsc) of the beta-lactamase specific activity determined for a mutant carrying either plasmid was taken as a measure of the degree of global supercoiling. These Qsc data were comparable to results obtained from the separation of topoisomers of plasmid pBR322 on chloroquine-containing agarose gels and indicate a reduced degree of negative supercoiling in resistant mutants relative to the parent, WT. The S83L mutation in gyrA had the strongest influence on quinolone resistance while leaving other parameters nearly unaffected. The gyrA double mutation (S83L plus D87G) had an effect on quinolone resistance similar to that of a single mutation. Phenotypic expression of the parC mutation (S80I) was dependent on the presence of at least one gyrA mutation. Expression of high-level fluoroquinolone resistance (ciprofloxacin MIC, > 4 micrograms/ml) required a combination of the gyrA double mutation and one parC mutation (S80I or E84K). Such mutants showed considerable alterations of growth rate, global supercoiling, or both. Introduction of a parC mutation affected neither the doubling time nor the degree of supercoiling, while the presence of the gyrA D87G mutation was associated with a significant reduction in the degree of DNA supercoiling. (+info)
Penetration of moxifloxacin into peripheral compartments in humans.
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To characterize the penetration of moxifloxacin (BAY 12-8039) into peripheral target sites, the present study aimed at measuring unbound moxifloxacin concentrations in the interstitial space fluid by means of microdialysis, an innovative clinical sampling technique. In addition, moxifloxacin concentrations were measured in cantharides-induced skin blisters, saliva, and capillary plasma and compared to total- and free-drug concentrations in venous plasma. For this purpose, 12 healthy volunteers received moxifloxacin in an open randomized crossover fashion either as a single oral dose of 400 mg or as a single intravenous infusion of 400 mg over 60 min. An almost-complete equilibration of the free unbound plasma fraction of moxifloxacin with the interstitial space fluid was observed, with mean area under the concentration-time curve (AUC)(interstitial fluid)/AUC(total-plasma) ratios ranging from 0.38 to 0.55 and mean AUC(interstitial fluid)/AUC(free-plasma) ratios ranging from 0.81 to 0.86. The skin blister concentration/plasma concentration ratio reached values above 1.5 after 24 h, indicating a preferential penetration of moxifloxacin into inflamed lesions. The moxifloxacin concentrations in saliva and capillary blood were similar to the corresponding levels in plasma. Our data show that moxifloxacin concentrations attained in the interstitial space fluid in humans and in skin blister fluid following single doses of 400 mg exceed the values for the MIC at which 90% of isolates are inhibited for most clinically relevant bacterial strains, notably including penicillin-resistant Streptococcus pneumoniae. These findings support the use of moxifloxacin for the treatment of soft tissue and respiratory tract infections in humans. (+info)
Pharmacokinetics of antibiotics in burn patients.
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Drug pharmacokinetics are significantly altered in the burned patient but the interplay of a large number of variables is involved in deciding how an individual will deal with a drug. Consequently the burn patient population shows significant inter- and intrapatient variation. In 1976 altered aminoglycoside pharmacokinetics and the need for increased dosage in burn patients was reported but, despite this early study, a review of the currently available literature shows that for many drugs there is a paucity of information to support current dosage recommendations. In addition, many reports are based upon small numbers of patients, and even in larger studies there is no standardization of the study population with regard to the important variables known to affect drug handling. For the sub-population of burn patients who eliminate drugs extremely rapidly, a concern exists over the adequacy of antibiotic dosing. It is suggested that antibiotic serum concentrations be measured for all drugs in every patient to ascertain whether there is a significant problem with dosing. Additionally, future pharmacokinetic studies need to be standardized in burn patients. (+info)
Activity of moxifloxacin against mycobacteria.
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Moxifloxacin is an 8-methoxyquinolone compound with activity against a wide range of bacteria. We tested its activity in comparison with four other quinolones and isoniazid against clinical isolates of mycobacteria. It proved to be the most active of the quinolones tested against Mycobacterium tuberculosis (MIC90 0.25 mg/L), Mycobacterium avium-intracellulare (MIC90 1.0 mg/L), Mycobacterium kansasii (MIC90 0.06 mg/L) and Mycobacterium fortuitum (MIC90 1 mg/L). These data indicate that moxifloxacin merits further study as an antimycobacterial agent. (+info)
Characterization of MexT, the regulator of the MexE-MexF-OprN multidrug efflux system of Pseudomonas aeruginosa.
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We investigated the regulation of the MexEF-OprN multidrug efflux system of Pseudomonas aeruginosa, which is overexpressed in nfxC-type mutants and confers resistance to quinolones, chloramphenicol and trimethoprim. Sequencing of the DNA region upstream of the mexEF-oprN operon revealed the presence of an open reading frame (ORF) of 304 amino acids encoding a LysR-type transcriptional activator, termed MexT. By using T7-polymerase, a 34-kDa protein was expressed in Escherichia coli from a plasmid carrying the mexT gene. Expression of a mexE::lacZ fusion was 10-fold higher in nfxC-type mutants than in the wild-type strain; however, transcription of mexT as well as the mexT DNA region was unchanged. Located adjacent to mexT but transcribed in opposite direction, the beginning of an ORF termed qrh (quinone oxidoreductase homologue) was identified. Expression of a qrh::lacZ fusion was also found to be activated by MexT. Further, we present evidence for coregulation at the transcriptional and the posttranscriptional level between the MexEF-OprN efflux system and the OprD porin responsible for cross-resistance of nfxC-type mutants to carbapenem antibiotics. (+info)
Purification and inhibition by quinolones of DNA gyrases from Mycobacterium avium, Mycobacterium smegmatis and Mycobacterium fortuitum bv. peregrinum.
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The DNA gyrases from Mycobacterium avium, Mycobacterium smegmatis and Mycobacterium fortuitum bv. peregrinum, which are species naturally resistant, moderately susceptible and susceptible to fluoroquinolones, respectively, were purified by affinity chromatography on novobiocin-Sepharose columns. The DNA gyrase inhibiting activities (IC50 values) of classical quinolones and fluoroquinolones were determined from the purified enzymes and were compared to the corresponding antibacterial activities (MICs). Regarding M. fortuitum bv. peregrinum, which is nearly as susceptible as Escherichia coli, the corresponding MIC and IC50 values of quinolones were significantly lower than those found for M. avium and M. smegmatis (e.g. for ofloxacin, MICs of 0.25 versus 32 and 1 microg ml(-1), and IC50 values of 1 versus 8 and 6 microg ml(-1), respectively). Such a result could be related to the presence of Ser-83 in the quinolone-resistance-determining region of the gyrase A subunit of M. fortuitum bv. peregrinum, as found in wild-type E. coli, instead of Ala-83 in M. avium and M. smegmatis, as found in fluoroquinolone-resistant E. coli mutants. The IC50 values of quinolones against the M. avium and M. smegmatis DNA gyrases were similar, while the corresponding MICs were 32-fold higher for M. avium when compared to M. smegmatis, suggesting that an additional mechanism, such as a low cell wall permeability or a drug efflux, could contribute to the low antibacterial potency of quinolones against M. avium. (+info)