Development of in vitro resistance to fluoroquinolones in Pseudomonas aeruginosa.

Fluoroquinolone resistance in Pseudomonas aeruginosa typically arises through site-specific mutations and overexpression of efflux pumps. In this study, we investigated the dynamics of different resistance mechanisms in P. aeruginosa populations that have evolved under fluoroquinolone pressure, as well as the interactions between these mechanisms in evolutionary trajectories. Bacteria of strain ATCC27853 were selected under different concentrations of ciprofloxacin and levofloxacin for six parallel lineages, followed by amplification of four target genes in the quinolone-resistance determining region (QRDR) and Sanger sequencing to identify the mutations. The expression of four efflux pump proteins was evaluated by real-time polymerase chain reaction using the relative quantitation method, with the ATCC27853 strain used as a control. We found that ciprofloxacin killed P. aeruginosa sooner than did levofloxacin. Further, we identified five different mutations in three subunits of QRDRs, with gyrA as the main mutated gene associated with conferring fluoroquinolone resistance. Additionally, we found a larger number of mutations appearing at 2 mg/L and 4 mg/L of ciprofloxacin and levofloxacin, respectively. Moreover, we identified the main efflux pump being expressed as MexCD-OprJ, with initial overexpression observed at 0.25 mg/L and 0.5 mg/L of ciprofloxacin and levofloxacin, respectively. These results demonstrated gyrA83 mutation and MexCD-OprJ overexpression as the primary mechanism conferring ciprofloxacin and levofloxacin resistance in P. aeruginosa. In addition, we also show that ciprofloxacin exhibited a stronger ability to kill the bacteria while potentially rendering it more susceptible to resistance.

[In vitro antibiotic susceptibility to fluoroquinolones]. / Susceptibilidad antibiótica in vitro a fluoroquinolonas.

OBJECTIVE: To determine the antibiotic susceptibility of bacteria recovered from cultures of ocular infections in the Fundación Oftalmológica de Santander - Clínica Carlos Ardila Lulle (FOSCAL). MATERIALS AND METHODS: Retrospective descriptive study of a series of registries of cultures of samples from ocular surfaces and intraocular fluids from the OCULAB-FOSCAL laboratory in Floridablanca (Colombia) made between January and December of 2007. Antibiotic sensitivity screening by the method of Kirby-Bauer with impregnated Sensi-Discs™ of determined antibiotic concentrations was performed. RESULTS: A total of 352 samples were studied: 160 from conjunctiva, 150 from cornea and 42 from intraocular fluids. Of the total of the samples more than one microorganism was recovered 45.65% of the samples. Gram positive and Gram negative bacteria were identified in 78.7 and 18.4%, respectively. Resistance to gatifloxacin, moxifloxacin, ciprofloxacin and levofloxacin was observed in 6.3, 8.9, 33.2 and 35.6%, respectively, of Gram positive bacteria. Resistance to gatifloxacin, moxifloxacin, ciprofloxacin and levofloxacin was also observed in 7.4, 16.7, 16.7%and 25.9%, respectively, of Gram negative bacteria. The overall bacterial resistance (Gram positive and Gram negative) to moxifloxacin was 10.15%, and to gatifloxacin it was 6.46%, being which showed a statistically significant difference (P<.05). CONCLUSIONS: In our study the development of bacterial resistance to fourth generation fluoroquinolones was demonstrated in ocular samples. However, lower levels of resistance to fourth generation fluoroquinolones compared with that of third and second generation were found, particularly to Gram positive. Gatifloxacin showed lower resistance levels than moxifloxacin. Nevertheless, interpretation of this superiority must be made with caution in the clinical field, since other factors, like tissue penetration and in vivo activity, must be taken into account.

[Fluoroquinolones in ophthalmology: mechanisms of action and resistance]. / Les fluoroquinolones en ophtalmologie: modes d'actions et mécanismes de résistance.

The arrival of fluoroquinolones in the 1980s aroused enormous enthusiasm in the medical community, justified by the spectrum of antibacterial activity, good tolerance, and wide distribution in tissues, even after oral administration. However, the extensive use of these new antibiotiques finally ended in emerging resistance, which limits the efficiency of all the molecules in the class, including those that have appeared in the last few years. Ocular diseases also benefited from the emergence of fluoroquinolones, notably for topical treatments. It is now mandatory to adapt the prescriptions to the best indications, in order to protect the vast therapeutic potential of these molecules.

Contact lens-associated microbial keratitis / Ceratites microbianas relacionadas a lente de contato

PURPOSE: Contact lens-associated microbial keratitis is a severe condition with sight-threatening potential and increasing incidence. Information regarding the etiological agents is essential in guiding management and may vary geographically. The aim of this study was to analyze the microbiological results of corneal scrapings collected from patients presenting with contact lens-associated microbial keratitis. METHODS: Retrospective analysis of the records of all patients who were clinically diagnosed with contact lens-associated microbial keratitis and had corneal scrapings sent to the Laboratory of Ocular Microbiology, UNIFESP/EPM during a 5-year period from January 2002 to December 2007. RESULTS: The etiological agent was identified in 239 patients. Bacterial isolates accounted for 166 (69.46 percent) cases, Acanthamoeba for 95 (39.75 percent) cases and fungi for 4 (1.67 percent) cases. Among the bacterial infections, coagulase-negative Staphylococcus was demonstrated in 74 cases, while Pseudomonas spp was found in 32 patients. All coagulase negative Staphylococcus and Pseudomonas were susceptible to ciprofloxacin and ofloxacin. Resistance to gentamicin was documented in a single case of Pseudomonas. Fourth-generation flouoroquinolone resistance was not observed among Pseudomonas cases. CONCLUSION: Coagulase-negative Staphylococcus was the most frequent isolate, and such data must be considered when determining empiric treatment. Second-generation fluoroquinolones ciprofloxacin and ofloxacin and fourth-generation fluoroquinolones moxifloxacin and gatifloxacin showed a good antibacterial profile and therefore could be good options for initial management.

OBJETIVO: A ceratite microbiana associada ao uso de lente de contato é uma condição clínica grave, com risco de perda visual e cuja incidência vem aumentando progressivamente. Os principais agentes etiológicos podem variar geograficamente e informações referentes aos agentes causais são essenciais para tratamento apropriado. Este estudo se propõe a analisar os resultados microbiológicos de raspados de córnea coletados de pacientes usuários de lente de contato com quadro de ceratite microbiana. MÉTODOS: Foi realizada análise retrospectiva de todos os estudos microbiológicos de pacientes usuários de lente de contato com diagnóstico clínico de ceratite microbiana que tiveram material corneal enviado para o Laboratório de Microbiologia Ocular UNIFESP durante o período de 5 anos de janeiro de 2002 a dezembro de 2007. RESULTADOS: Dos 239 pacientes em que foi possível identificação, o agente era bactéria em 166 (69,46 por cento), Acanthamoeba em 95 (39,75 por cento) e fungo em 4 (1,67 por cento). Staphylococcus coagulase negativo foi encontrado em 74 casos, dos quais todos eram sensíveis tanto a ciprofloxacino quanto a ofloxacino. Dois casos de resistência a quinolonas de quarta geração (gatifloxacino e moxifloxacino) foram identificados entre os casos de Staphylococcus coagulase negativo. Pseudomonas spp foi isolada em 32 pacientes, dos quais todos eram sensíveis a quinolonas de segunda geração (ciprofloxacino e ofloxacino) e de quarta geração. Foi encontrado um único caso de Pseudomonas resistente a gentamicina. CONCLUSÃO: Staphylococcus coagulase negativo foi isolado em um número de casos superior a Pseudomonas, o agente tradicionalmente considerado o principal de ceratites microbianas associadas ao uso de lente de contato. Aminoglicosídeos, fluorquinolonas de segunda e de quarta geração apresentaram um perfil antibiótico satisfatório para o tratamento empírico de ceratites microbianas em usuários de lente de contato.

Contact lens-associated microbial keratitis.

PURPOSE: Contact lens-associated microbial keratitis is a severe condition with sight-threatening potential and increasing incidence. Information regarding the etiological agents is essential in guiding management and may vary geographically. The aim of this study was to analyze the microbiological results of corneal scrapings collected from patients presenting with contact lens-associated microbial keratitis. METHODS: Retrospective analysis of the records of all patients who were clinically diagnosed with contact lens-associated microbial keratitis and had corneal scrapings sent to the Laboratory of Ocular Microbiology, UNIFESP/EPM during a 5-year period from January 2002 to December 2007. RESULTS: The etiological agent was identified in 239 patients. Bacterial isolates accounted for 166 (69.46%) cases, Acanthamoeba for 95 (39.75%) cases and fungi for 4 (1.67%) cases. Among the bacterial infections, coagulase-negative Staphylococcus was demonstrated in 74 cases, while Pseudomonas spp was found in 32 patients. All coagulase negative Staphylococcus and Pseudomonas were susceptible to ciprofloxacin and ofloxacin. Resistance to gentamicin was documented in a single case of Pseudomonas. Fourth-generation flouoroquinolone resistance was not observed among Pseudomonas cases. CONCLUSION: Coagulase-negative Staphylococcus was the most frequent isolate, and such data must be considered when determining empiric treatment. Second-generation fluoroquinolones ciprofloxacin and ofloxacin and fourth-generation fluoroquinolones moxifloxacin and gatifloxacin showed a good antibacterial profile and therefore could be good options for initial management.

The categorization of prior antibiotic use: impact on the identification of risk factors for drug resistance in case control studies.

BACKGROUND: Analytic approaches to the identification of risk factors for the development of drug resistance vary and may affect study results. Using fluroquinolone-resistant Pseudomonas aeruginosa (FQRPA) and imipenem-resistant P. aeruginosa as models (IRPA), we aimed to examine the effect of different approaches to classification of prior antibiotic use (i.e., class versus spectrum) on the identification of risk factors for antibiotic resistance. METHODS: Case-control studies to identify risk factors for FQRPA and IRPA were performed. In each, two analytic models were utilized. In the first, prior antibiotic use was classified by class, and in the other, prior antibiotic use was classified by spectrum of activity. Risk factors identified by the two models were compared qualitatively for each resistant organism. RESULTS: 879 isolates of P. aeruginosa were included in the case-control studies. Risk factors for FQRPA and IRPA identified in multivariable analyses differed based on which method of classification of prior antibiotic use was utilized. CONCLUSIONS: The identification risk factors for the development of drug-resistant organisms could depend on the method of classification of prior antibiotic use. In studies of risk factors for resistant infections, the approach to classification of prior antibiotic use should be clearly stated and justified.

Effect of fluoroquinolone treatment on growth of and toxin production by epidemic and nonepidemic clostridium difficile strains in the cecal contents of mice.

Several recent outbreaks of Clostridium difficile-associated disease (CDAD) have been attributed to the emergence of an epidemic strain with increased resistance to fluoroquinolone antibiotics. Some clinical studies have suggested that fluoroquinolones with enhanced antianaerobic activity (i.e., gatifloxacin and moxifloxacin) may have a greater propensity to induce CDAD than ciprofloxacin and levofloxacin do. We examined the effects of subcutaneous fluoroquinolone treatment on in vitro growth of and toxin production by epidemic and nonepidemic C. difficile isolates in cecal contents of mice and evaluated the potential for these agents to inhibit fluoroquinolone-susceptible isolates during treatment. When C. difficile isolates were inoculated into cecal contents collected 2 days after the final antibiotic dose, gatifloxacin and moxifloxacin promoted significantly more growth and toxin production than ciprofloxacin and levofloxacin did. During treatment, gatifloxacin and moxifloxacin inhibited growth of fluoroquinolone-susceptible but not fluoroquinolone-resistant isolates. Ciprofloxacin and levofloxacin promoted growth of C. difficile when administered at higher doses (i.e., 20 times the human dose in mg/kg of body weight), and levofloxacin inhibited growth of fluoroquinolone-susceptible, but not fluoroquinolone-resistant, C. difficile isolates when administered in combination with ceftriaxone. Thus, fluoroquinolones with enhanced antianaerobic activity (i.e., gatifloxacin and moxifloxacin) promoted C. difficile growth to a greater extent than did ciprofloxacin and levofloxacin in this model. However, our findings suggest that fluoroquinolones may exert selective pressure favoring the emergence of epidemic fluoroquinolone-resistant C. difficile strains by inhibiting fluoroquinolone-susceptible but not fluoroquinolone-resistant isolates during treatment and that agents such as levofloxacin or ciprofloxacin can exert such selective pressure when administered in combination with antibiotics that disrupt the anaerobic microflora.

A topological substructural approach for the prediction of P-glycoprotein substrates.

A topological substructural molecular design approach (TOPS-MODE) has been used to predict whether a given compound is a P-glycoprotein (P-gp) substrate or not. A linear discriminant model was developed to classify a data set of 163 compounds as substrates or nonsubstrates (91 substrates and 72 nonsubstrates). The final model fit the data with sensitivity of 82.42% and specificity of 79.17%, for a final accuracy of 80.98%. The model was validated through the use of an external validation set (40 compounds, 22 substrates and 18 nonsubstrates) with a 77.50% of prediction accuracy; fivefold full cross-validation (removing 40 compounds in each cycle, 80.50% of good prediction) and the prediction of an external test set of marketed drugs (35 compounds, 71.43% of good prediction). This methodology evidenced that the standard bond distance, the polarizability and the Gasteiger-Marsilli atomic charge affect the interaction with the P-gp; suggesting the capacity of the TOPS-MODE descriptors to estimate the P-gp substrates for new drug candidates. The potentiality of the TOPS-MODE approach was assessed with a family of compounds not covered by the original training set (6-fluoroquinolones), and the final prediction had a 77.7% of accuracy. Finally, the positive and negative substructural contributions to the classification of 6-fluoroquinolones, as P-gp substrates, were identified; evidencing the possibilities of the present approach in the lead generation and optimization processes.

[Current evaluation of the fluoroquinolones]. / Chinolone--wann einsetzen, wie dosieren?

Originally, the fluoroquinolones were classified into the groups I-IV on the basis of their clinical field of application, pharmacokinetics, antibacterial spectrum, and bioavailability. In the present publication an evaluation taking account in particular of aspects of clinical application and dosage has been undertaken based on the 1998 classification by a group of PEG experts.

Guide to selection of fluoroquinolones in patients with lower respiratory tract infections.

Newer fluoroquinolones such as levofloxacin, moxifloxacin, gatifloxacin and gemifloxacin have several attributes that make them excellent choices for the therapy of lower respiratory tract infections. In particular, they have excellent intrinsic activity against Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and the atypical respiratory pathogens. Fluoroquinolones may be used as monotherapy to treat high-risk patients with acute exacerbation of chronic bronchitis, and for patients with community-acquired pneumonia requiring hospitalisation, but not admission to intensive care. Overall, the newer fluoroquinolones often achieve clinical cure rates in > or =90% of these patients. However, rates may be lower in hospital-acquired pneumonia, and this infection should be treated on the basis of anticipated organisms and evaluation of risk factors for specific pathogens such as Pseudomonas aeruginosa. In this setting, an antipseudomonal fluoroquinolone may be used in combination with an antipseudomonalbeta-lactam. Concerns are now being raised about the widespread use, and possibly misuse, of fluoroquinolones and the emergence of resistance among S. pneumoniae, Enterobacteriaceae and P. aeruginosa. A number of pharmacokinetic parameters such as the peak concentration of the antibacterial after a dose (C(max)), and the 24-hour area under the concentration-time curve (AUC24) and their relationship to pharmacodynamic parameters such as the minimum inhibitory and the mutant prevention concentrations (MIC and MPC, respectively) have been proposed to predict the effect of fluoroquinolones on bacterial killing and the emergence of resistance. Higher C(max)/MIC or AUC24/MIC and C(max)/MPC or AUC24/MPC ratios, either as a result of dose administration or the susceptibility of the organism, may lead to a better clinical outcome and decrease the emergence of resistance, respectively. Pharmacokinetic profiles that are optimised to target low-level resistant minor subpopulations of bacteria that often exist in infections may help preserve fluoroquinolones as a class. To this end, optimising the AUC24/MPC or C(max)/MPC ratios is important, particularly against S. pneumoniae, in the setting of lower respiratory tract infections. Agents such as moxifloxacin and gemifloxacin with high ratios against this organism are preferred, and agents such as ciprofloxacin with low ratios should be avoided. For agents such as levofloxacin and gatifloxacin, with intermediate ratios against S. pneumoniae, it may be worthwhile considering alternative dose administration strategies, such as using higher dosages, to eradicate low-level resistant variants. This must, of course, be balanced against the potential of toxicity. Innovative approaches to the use of fluoroquinolones are worth testing in further in vitro experiments as well as in clinical trials.

Action of fluoroquinolones and Linezolid on logarithmic- and stationary-phase culture of Mycobacterium tuberculosis.

BACKGROUND: The new challenges involved in the chemotherapy of tuberculosis make it necessary to find novel drugs, especially ones that are useful in the latent phase of the disease. METHODS: We evaluated the activity of linezolid and fluoroquinolones against logarithmic- and stationary-phase Mycobacterium tuberculosis. RESULTS: We observed that linezolid exhibits antibacterial action, although slowly, in both situations. Quinolones with an 8-methoxy group exhibit greater activity than levofloxacin in logarithmic growth phases, whereas levofloxacin exhibits greater activity in stationary-phase growth. CONCLUSION: The study of the activity of drugs against the M. tuberculosis microorganism in the latent phase is one of the most important tools available in the fight against the tuberculosis epidemic, and both linezolid and the new fluoroquinolones appear to be promising drugs.

Fluoroquinolones as chemotherapeutics against mycobacterial infections.

The antimicrobial agents used in the treatment of mycobacterial infections have remained largely unchanged for several decades. Primary treatment of tuberculosis relies on four drugs, isoniazid, a rifamycin, pyrazinamide, and ethambutol (or streptomycin), and generally results in >95% cure in uncomplicated tuberculosis infection. Drug resistance greatly complicates treatment of this disease. Treatment of tuberculosis caused by multiply drug-resistant strains with "second-line" drugs remains complex, and is generally tailored to the individual patient and strain. Several of the fluoroquinolones have shown promise as second line drugs for treatment of active disease and, in combination with clarithromycin or azithromycin, ethambutol, and other agents, for treatment of Mycobacterium avium complex infection. While large clinical trials are not possible with second line drugs, clinical treatment data are available and suggest that the quinolones have various degrees of promise in treatment of these infections. Bacterial type II DNA topoisomerases, DNA gyrase and topoisomerase IV, are the targets of quinolones, and provide the genetic basis for quinolone activity in mycobacteria. Mutations in these enzymes results in resistance, and characterization of resistant mutants allows correlation of genotype with susceptibility phenotype. Structure-activity relationship studies have provided further insight into optimal use of quinolones in mycobacterial infections. Care should be taken in treating pneumonia with fluoroquinolones if there is a degree of suspicion of tuberculosis, since quinolone monotherapy may rapidly select for quinolone resistance, thereby removing that class of antibiotic from the small range of treatment options.

Fourth-generation fluoroquinolones: new topical agents in the war on ocular bacterial infections.

PURPOSE OF REVIEW: The fourth-generation fluoroquinolones, moxifloxacin and gatifloxacin, were introduced in 2003 promising improved spectrum of activity and delayed development of resistance. Although these topical agents have recently been introduced in commercial form, there is already a growing body of evidence showing excellent potency in the war on ocular infections. The purpose of this review is to discuss the literature to date regarding these two agents. RECENT FINDINGS: Since their introduction in 1990 in the United States, fluoroquinolones have rapidly become the standard of care in the topical antibiotic arena. Unfortunately, recent evidence has shown the widespread use of fluoroquinolones, not only in eye care, but also in agriculture, and general medical and surgical use, has lead to decreasing susceptibilities of important ocular bacterial pathogens. Moxifloxacin and gatifloxacin have improved potency and are able to overcome resistant isolates. These agents also provide improved penetration into ocular tissues. SUMMARY: Moxifloxacin and gatifloxacin offer improved spectrum of activity, increased penetration into ocular tissues, and delayed propensity to the development of bacterial antibiotic resistance.

Fluoroquinolones: mechanism of action, classification, and development of resistance.

The fluoroquinolones represent an evolving class of broad-spectrum antimicrobial agents used in the prevention and treatment of a variety of ocular infections; however, resistance to currently available agents in the class has been emerging among ocular pathogens. This article reviews the mechanism of action of existing and new fluoroquinolones and discusses the structure-activity relationship of the fluoroquinolones as it relates to the classification of these compounds. This article also highlights the mechanism of resistance among common ocular pathogens and discusses the potential need for newer fluoroquinolones in ophthalmology.

Permeability classification of representative fluoroquinolones by a cell culture method.

This study was undertaken to categorize representative fluoroquinolone drug substance permeability based on the methods outlined in the Food and Drug Administration's biopharmaceutic classification system (BCS) Guidance for Industry. The permeability of ciprofloxacin, levofloxacin, lomefloxacin, and ofloxacin was measured in an in vitro Caco-2 assay with previously demonstrated method suitability. The permeability class and efflux potential were ascertained by comparing test drug results with standard compounds (metoprolol, atenolol, labetalol, and rhodamine-123). All 4 quinolones drugs demonstrated concentration-dependent permeability, indicating active drug transport. In comparing absorptive versus secretive in vitro transport, the tested fluoroquinolones were found to be subject to efflux in varying degrees (ciprofloxacin > lomefloxacin > rhodamine 123 > levofloxacin > ofloxacin). Based on comparison to labetalol, the high permeability internal standard, ciprofloxacin was classified as a low permeability drug, whereas lomefloxacin, levofloxacin, and ofloxacin were classified as high permeability drugs. The in vitro permeability results matched human in vivo data based on absolute bioavailabilities. This laboratory exercise demonstrated the applicability of an in vitro permeability method for classifying drugs as outlined in the BCS Guidance.

[Acute pyelonephritis: bacteriological data and general course of germ resistance]. / Pyélonéphrite aiguë: bactériologie et évolution des résistances.

Recent bacteriological data concerning pyelonephritis mainly focus on sensitivity to antibiotics and virulence factors. Epidemiologically, and regardless of age and sex of the patients, E. coli remains the most often isolated pathogen. Only 55% of E. coli strains (43% if isolated during a nosocomial infection) are sensitive to amoxicillin. Resistance to parenterally administered third generation cephalosporins remain exceptional. Fluoroquinolones remain very active, particularly in community-acquired infections (>95% sensitive strains). P. aeruginosa, S. aureus and enterococci are mainly isolated during nosocomial infections or in patients with a history of previous hospital admissions. Adhesion factors to urothelial cells are of uppermost importance in the pathogenesis of urinary infection. Adhesins G borne by fimbriae P are mainly concerned. Also, hemolysin is very often present in strains responsible for pyelonephritis. As to the search for these factors in the diagnosis of pyelonephritis, particularly in children, further studies are needed. The choice of antibiotherapy not only rests upon antibiogram data, but also upon pharmacological characteristics of the antibiotic.

High-performance liquid chromatographic separation of fluoroquinolone enantiomers: a review.

Fluoroquinolones are antibacterial agents widely used clinically. In recent years, there has been an important development of new derivatives, and more than 7000 analogues have been described today. Different fluoroquinolones (FQ) have one or two chiral centers in their chemical structure and are available as racemates, diastereoisomers, or pure enantiomers. The clinical and pharmaceutical uses of these compounds need effective analytical procedures for quality control and pharmacodynamic and pharmacokinetic studies. This review article focuses on the high-performance liquid chromatographic separation of fluoroquinolone stereoisomers by the use of derivatization methods and ligand exchange (LE) or chiral liquid chromatography.