氟喹诺酮类药物(FQs)耐药性产生的分子机制

随着氟喹诺酮类药物的广泛使用,细菌对该类药物产生耐药性日趋严重,这引起了国内外的高度重视。本文就FQs耐药性产生的分子机制,即药靶的改变,药物在菌体内蓄积浓度下降及由质粒介导的耐药等机制做了综述。

Susceptibilities of Mycoplasma Hominis and Ureaplasma Urealyticum to Fluoroquinolones

Objectives: To determine the susceptibilities of M.hominis and U. urealyticum to fluoroquinolones forthe instruction of reasonable clinical application ofantibiotics.Method: The susceptibilities of M. hominis and U.urealyticum to six fluoroquinolones were determinedby the broth dilution method.Results: Sparfloxacin and gatifloxacin were veryactive with MIC50S of 0.03125 and 0.25 μg/ml againstM. hominis, 0.25 and 0.5 μg/ml against U. urealyticum,respectively. Levofloxacin and ofloxacin had MIC50S of1 μg/ml and 2 μg/ml, respectively against both species.Norfloxacin was less effective against both species at16 and 32 μg/ml. Ciprofloxacin was unusual in thatthe MIC50S varied fourfold between species, with 2 μg/ml against M. hominis and 8 μg/ml against U.urealyticum.Conclusions: The results can provide useful infor-mation for selecting fluoroquinolones for treatmentof urogenital mycoplasma infections.

错配PCR方法快速检测耐FQs沙门氏菌基因突变的初步研究

根据耐药沙门氏菌基因突变位点碱基变化情况,设计合适的引物进行错配PCR,鉴别突变位点,使其结果与测序结果相吻合,以建立错配PCR方法快速检测耐氟喹诺酮类药物沙门氏菌基因突变,判断菌株是否耐药。错配PCR检测方法条件优化成熟之后,对经过测序的菌株进行耐药突变位点的快速检测,检测结果与测序结果符合率高达96%。

海洋环境中氟喹诺酮类抗生素(FQs)分析的样品前处理与检测技术

氟喹诺酮类(FQs)药物是一种广泛使用的人工合成类抗生素,存在于水体、沉积物等各种环境介质中,并在水生生物体内得到富集,对人类健康和全球生态系统的可持续发展有重要的影响。环境中FQs残留的分析检测是了解其环境生物地球化学行为和潜在生态环境风险的基础,本文系统总结了近几年海洋水体、沉积物和生物体样品中FQs的残留特征、样品前处理与检测技术,在此基础上,前瞻分析了海洋环境中FQs残留分析检测技术的发展趋势。分析表明,FQs的分离富集和测定必须充分考虑FQs的物理化学性质和样品成分的复杂性。海水样品准备应注意过滤膜的选择和pH的调节;沉积物和生物体的样品准备应考虑水分、萃取溶剂、基质效应和pH的影响,并使用超声萃取。固相萃取、QuEChERS萃取、磁性固相萃取是分离富集FQs较常用的方法,吸附剂、淋洗溶液和洗脱溶液的选择和优化是提高样品回收率的关键。FQs的检测大多通过液质联用或液相色谱结合荧光检测器进行,其中色谱柱的选择、离子对试剂的添加和进样pH值的调整都是优化的关键因素。研究指出海洋领域FQs在线自动SPE技术的开发以及新型萃取吸附剂的研制应在未来研究中被重点关注。

Determination of fluoroquinolones, sulfonamides, and tetracyclines multiresidues simultaneously in porcine tissue by MSPD and HPLC-DAD

An efficient method is provided to detect simultaneously some important veterinary drugs from different classes in highly complex animal tissue matrix. This method using matrix solid-phase dispersion (MSPD) and high performance liquid chromatography (HPLC) with diode array detection (DAD) is developed to effectively determine two fiuoroquinolones (enoxacin and lomefioxacin), two sulfonamides (sulfanilamide and sulfamethoxazole) and one tetracycline (tetracycline) simultaneously in porcine tissues. In the process, MSPD methodology was used to treat samples, washed by n-hexane to remove lipid, eluted the analytes with acetonitrile–dichloromethane (1:1, v/v). Solvent acetonitrile and solvent acetic acid (0.1%) were combined in a gradient. HPLC–DAD analysis of the tissue samples was performed within 15 min at a fiow rate of 1.0 mL/min. The results showed that a recovery at 0.1, 0.5 and 1.0 mg/g fortification levels ranged from 80.6% to 99.2% with satisfactory relative standard deviations (RSDs) (below 6.1%, nfi3) and the limits of quantitation (LOQ) ranged from 7 mg/kg to 34 mg/kg in porcine tissues. Utilization of the method in successfully simultaneous analysis of porcine tissue incurred with veterinary drug multiresidues is described.

广东省镇海湾红树林根域中氟喹诺酮类(FQs)抗生素残留特征

对广东省江门市镇海湾红树林自然保护区两种优势红树植物——桐花树Aegiceras corniculatum、秋茄Kandelia candel的根域(根际土、非根际土)中氟喹诺酮类(Fluoroquinolones,FQs)抗生素展开分析。采用高效液相质谱联用法测定14个根域土样品、6个排污口沉积物和3个光滩沉积物样品中4种FQs—氧氟沙星(Ofloxacin,OFL)、诺氟沙星(Norfloxacin,NOR)、环丙沙星(Ciprofloxacin,CIP)、恩诺沙星(Enrofloxacin,ENR)的残留特征,并与根域土理化因子pH、有机质(Soil organicmatter,SOM)、阳离子交换量(Cationex changecapacity,CEC)、过氧化氢酶(Catalase,CAT)、蔗糖酶(Sucrase,SUC)、土壤有效态Cu、Zn、Fe进行典范对应分析(canonical correspondence analysis,CCA)。结果表明:桐花树、秋茄根际土中4种FQs质量分数均值分别为55.22、29.25μg·kg^(-1),非根际土中4种FQs均值分别为22.45、18.66μg·kg^(-1),两种红树根域中4种FQs含量桐花树>秋茄(P<0.05),4种FQs含量均为根际土>非根际土;对FQs残留影响程度的环境因子权重排序为:p H>Fe>SOM≥CEC>Zn>SUC≥CAT;研究结果对阐释红树林对有机污染物(包括抗生素)的吸收净化作用具有重要理论意义。

Fluoroquinolones for the treatment of latent Mycobacterium tuberculosis infection in liver transplantation

Solid organ transplantation(SOT)is the best treatment option for end-stage organ disease.Newer immunosuppressive agents have reduced the incidence of graft rejection but have increased the risk of infection,particularly due to the reactivation of latent infections due to opportunistic agents such as Mycobacterium tuberculosis.Active tuberculosis(TB)after SOT is a significant cause of morbidity and mortality.Most cases of posttransplant TB are secondary to reactivation of latent tuberculosis infection(LTBI)due to the effects of long-term immunosuppressive therapy.Risk minimization strategies have been developed to diagnose LTBI and initiate treatment prior to transplantation.Isoniazid with vitamin B6 supplementation is the treatment of choice.However,liver transplantation(LT)candidates and recipients have an increased risk of isoniazid-induced liver toxicity,leading to lower treatment completion rates than in other SOT populations.Fluoroquinolones(FQs)exhibit good in vitro antimycobacterial activity and a lower risk of drug-induced liver injury than isoniazid.In the present review,we highlight the disease burden posed by posttransplant TB and summarize the emerging clinical evidence supporting the use of FQs for the treatment of LTBI in LT recipients and candidates.

GyrA and ParC Gene Mutation of Clinically Isolated Fluoroquinolones-resistant Strain of Salmonella

Nine strains resistant to five fluoroquinolones （Ciprofloxacin, Ofloxacin, Enrofloxacin, Danofloxacin, Sarafloxacin） were isolated from clinical samples and extracted the chromosomal DNA of these strains. Designed primers to amplify the Quinolone-resistance-determining region （QRDR） of gyrA and par（？,, then the PCR products were sequenced and analyzed. In comparision with NCTC5776, a single mutation was found at base 371 in gyrA of strain 38 which changed from C to T, and a single mutation was found at base 350 in gyrA of strain 60 which changed from A to C. No mutation was found in gyrA of the rest The mutation of strain 38 led to an amino acid substitution of Arg99Cys and the mutation of 60 led to an amino acid substitution of Met 92 Leu. No mutation was found in parC QRDR of all the isolates. These results indicats that the DNA gyrase will be the primary target to salmonella of fluoroquinolone.

Comparative clinical study of conjunctival toxicities of newer generation fluoroquinolones without the influence of preservatives

AIMTo compare the conjunctival epithelial toxicities of three newer-generation fluoroquinolones without preservatives.METHODSIn a prospective, randomized, double blind comparative study, 47 eyes of 47 patients with a primary pterygium were enrolled, and divided randomly into three groups (levofloxacin 0.5%, gatifloxacin 0.3%, and moxifloxacin 0.5%). After pterygium surgery with the same conjunctival autograft technique, each patient maintained a regimen with a randomly assigned fluoroquinolone eye drop. Patients were examined every other day after surgery until the epithelium had completely healed. Photos were taken and used to measure the area of residual epithelial defects. Conjunctival healing time and speed (initial defect area/healing time (mm2/d) compared in each group using Kruskal-Wallis tests.RESULTSThere were no significant differences in mean age, gender, and conjunctival defect size of the donor site between these groups. However, the mean of conjunctival healing time and speed were statistically different in each group. The mean of conjunctival epithelial healing time was 8.93±2.69d (levofloxacin group), 10.31±2.96d (gatifloxacin group), and 13.50±4.10d (moxifloxacin group), P=0.006. The mean conjuctival epithelial healing speed was 6.18±1.39 mm2/d (levofloxacin group), 5.52±1.68 mm2/d (gatifloxacin group), and 4.40±1.30 mm2/d (moxifloxacin group), P=0. 003.CONCLUSIONWithout the influence of preservatives, levofloxacin and gatifloxacin might be less toxic to the regeneration of conjunctival epithelial cells and cause a faster conjunctival wound healing relative to moxifloxacin.

QSAR Studies on 7-Substituted Fluoroquinolones

The PM3 and B3LYP methods were employed to calculate the properties of 18 7-substituted fluoroquinolones. The correlation between biological activity （against gram-positive organisms or gram-negative organisms） and structural properties was obtained by using multiple linear regression （MLR） methods： The best model generated correlates the antibacterial activity with EHOMO and QF8 for gram-positive organisms, and EHOMO and dipole moment for gram-negative organisms, respectively. It suggests that the interaction mechanisms ,of fluoroquinolons with gram-positive and gram-negative organisms are different.

Effect and Mechanism Analysis of Solvent on the Electron Transition of Fluoroquinolones Based on Quantum Chemical Calculation

Ciprofloxacin（CIP）, moxifoxacin（MOX） and enrofloxacin（ENR） were selected as typical fluoroquinolones（FQs） to analyze the excitation-enhancing effect and mechanism of solvents on FQs＇ electron transition based on quantum chemical calculations. The UV spectra of three FQs in gas and five different solvents（water, cyclohexane, dimethylsulfoxide, methanol, acetone） were calculated using Gaussian 09 software. The transition mechanisms of FQs＇ main electron transitions were analyzed by natural bond orbital（NBO） theory, and the solvent effect on each electron transition was assessed qualitatively and quantitatively by sensitivity analysis and an established index system. The excitation enhancing mechanism of solvent on electron transitions of FQs was analyzed from the view of photo-induced reactions between solvent and FQs molecules. The results show that there are two main transitions located in the spectrum ranges of 300~380 and 240~300 nm for each FQ in any medium, which are assigned as n →π＊ and π→π＊ electron transitions, respectively. By comparison, the n →π＊ transition is more sensitive to solvent because of the energy transfer between solvent molecules and FQs, but the solvent effect on the π→π＊ transition is stronger than on the n →π＊ transition. The sequence of affected extent of solvent effect on electron transition was CIP 〉 MOX 〉 ENR, and the sequence of solvent effect was water 〉 DMSO 〉 methanol 〉 acetone 〉 cyclohexane（stronger solvent effect with increasing the dielectric constant of solvent）. From the view of photo-induced reactions, the reaction between FQs＊T1 and solvent＊T1 has the decisive regulatory effect on the n →π＊ transition of FQs in solvent, and the reaction between FQsS0 and solvent＊TI has an enhancing effect on the π→π＊ transition.

Study on Rapid Detection of Tetracyclines,Fluoroquinolones and Sulfonamides in Milk

Aiming at the market demand for rapid detection of tetracyclines,fluoroquinolones and sulfonamides in milk,a golloidal gold immunochromatography test strip for simultaneous detection of tetracyclines,fluoroquinolones and sulfonamides in milk was prepared based on the principle of competitive inhibition immunochromatography. The performance indicators of the test strip were verified. The results showed that the test strip can simultaneously detect 4 tetracyclines,13 fluoroquinolones and 13 sulfonamides,and the detection limits all can meet the national residue limits; the tests strip exhibited false positive rate≤5% and false negative rate = 0; and no cross-reaction with other drugs was commonly found in milk,indicating good specificity. The method is simple,rapid,and has low cost and easy popularization. It provides a means for realizing on-site rapid detection and is of important practical significance to guarantee of safety of milk and dairy products in China.

Sensitive Spectrofluorimetric Method for Determination of Fluoroquinolones through Charge-Transfer Complex Formation

A sensitive spectrofluorimetric method was developed for determination of ciprofloxacin (CPFX), levofloxacin (LEV), gatifloxacin (GAT) and moxifloxacin (MOX) in pure, commercial formulations, human urine and plasma. The method is based on charge-transfer (CT) complex with chloranilic acid. Fluorescence intensity of the complexes was measured at emission wavelength ranging from 445-492 nm with excitation wavelengths from 285-330 nm. At optimum experimental conditions, a linear calibration plot was obtained in the concentration range of 20-1000 ng·mL-1, 60-320 ng·mL-1, 20-800 ng·mL-1 and 20 -00 ng·mL-1 for CPFX, LEV, MOX and GAT, respectively with good correlation coefficient in the range of 0.9929-1.0 in methanolic medium. The limit of detection and limit of quantification were found to be 5 ng·mL-1 and 18 ng·mL-1 for CPFX, 12 ng·mL-1 and 40 ng·mL-1 for LEV, 8 ng·mL-1 and 19 ng·mL-1 for MOX, 6 ng·mL-1 and 19 ng·mL-1 for GAT, respectively. The method was found free of interferences from excipients used as additive in pharmaceutical preparations, some common cations and compounds present in urine and plasma as well as co-administered analgesic, vitamins and other drugs. The method was successfully applied for quantification of selected fluoroquinolones in commercial formulations and also in spiked human urine and plasma samples with percent recoveries of 100.0 ± 1.56 and 100.2 ± 1.29 respectively.

Detection of Helicobacter pylori resistance to clarithromycin and fluoroquinolones in Brazil:A national survey

AIM To evaluate bacterial resistance to clarithromycin and fluoroquinolones in Brazil using molecular methods.METHODS The primary antibiotic resistance rates of Helicobacter pylori(H. pylori) were determined from November 2012 to March 2015 in the Southern,South-Eastern,Northern,North-Eastern,and Central-Western regions of Brazil. Four hundred ninety H. pylori patients [66% female,mean age 43 years(range: 18-79)] who had never been previously treated for this infection were enrolled. All patients underwent gastroscopy with antrum and corpus biopsies and molecular testing using Geno Type Helico DR(Hain Life Science,Germany). This test was performed to detect the presence of H. pylori and to identify point mutations in the genes responsible for clarithromycin and fluoroquinolone resistance. The molecular procedure was divided into three steps: DNA extraction from the biopsies,multiplex amplification,and reverse hybridization. RESULTS Clarithromycin resistance was found in 83(16.9%) patients,and fluoroquinolone resistance was found in 66(13.5%) patients. There was no statistical difference in resistance to either clarithromycin or fluoroquinolones(P = 0.55 and P = 0.06,respectively) among the different regions of Brazil. Dual resistance to clarithromycin and fluoroquinolones was found in 4.3%(21/490) of patients. The A2147 G mutation was present in 90.4%(75/83),A2146 G in 16.9%(14/83) and A2146 C in 3.6%(3/83) of clarithromycin-resistant patients. In 10.8%(9/83) of clarithromycin-resistant samples,more than 01 mutation in the 23 S r RNA gene was noticed. In fluoroquinolone-resistant samples,37.9%(25/66) showed mutations not specified by the Geno Type Helico DR test. D91 N mutation was observed in 34.8%(23/66),D91 G in 18.1%(12/66),N87 K in 16.6%(11/66) and D91 Y in 13.6%(9/66) of cases. Among fluoroquinolone-resistant samples,37.9%(25/66) showed mutations not specified by the Geno Type Helico DR test. CONCLUSION The H. pylori clarithromycin resistance rate in Brazil is at the borderline(15%-20%) for applying the standard triple therapy. The fluoroquinolone resistance rate(13.5%) is equally concerning.

Cloning and Sequence Analysis of gyrB Gene of Fluoroquinolones-resistant Salmonella Isolated from Chickens

Nine strains resistant to five fluoroquinolones (Ciprofloxacin, Ofloxacin, Enrofloxacin, Danofloxacin, Sarafloxacin) were isolated from clinical samples and extracted the chromosomal DNA of these strains. Designed primers to amplify the Quinolone-resistance-determining region(QRDR) of gyrB gene, then the PCR products were cloned and the sequence was analyzed. In comparison with the standarded strain NCTC5776, no mutation was found in the QRDR of gyrB gene of all resistant strains. The result indicated that the QRDR of gyrB has little relationship with fluoroquinolone resistance to salmonella.

Colloidal Gold Test Strip for the Detection of Fluoroquinolones in Foods Using an Analyzer

A test strip was developed to rapidly detect fluoroquinolones in foods by colloidal gold immunochromatography method in combination with a food safety analyzer. The results showed that the test strip has the detection sensitivity of 20 μg/L for enrofloxacin, sarafloxacin, difloxacin, ofloxacin, norfloxacin, ciprofloxacin, pefloxacin, flumequine and danofloxacin, and the detection sensitivity of 40 μg/L for enoxacin and oxolinic acid. The test strip consumes only 10 min for detection, and the false positive rate and the false negative rate are both zero. The test strip can accurately, reliably and easily detect residual fluoroquinolones in foods, and is suitable for on-site detection of a large number of samples.

Identification of Quinolones/Fluoroquinolones Resistance Genes from Staphylococci Strains Isolated at the University Hospital of Brazzaville, Republic of the Congo

Staphylococci strains, like the majority of bacterial strains, have developed the resistance to several antibiotics, including Quinolones and Fluoroquin-olones In the Republic of the Congo, cases of resistance leading to treat-ment failures have been observed during the treatment of staphylococcal infections with antibiotics in hospitals. The objective of this study was to identify the Quinolone/Fluoroquinolone resistance genes from staphylo-cocci strains isolated in hospitals. A total of 51 strains of Staphylococci were isolated, including 16 (31.37%) community strains, and 35 (68.62%) clinical strains. 46 strains of Staphylococcus aureus (S. aureus) and 5 SCNs were identified. A total of 34 DNA fragments from different strains resistant to Quinolones/Fluoroquinolones, including 21 (61.67%) DNA fragments from clinical S. aureus and 13 (38.23%) from community SCN strains were analyzed by the molecular method (genotypic detection) by PCR. The genotypic results made it possible to identify the gyrA, grLA and norA genes and to show that these genes are involved in the resistance of the strains to the various antibiotics used. The grLA gene was the most identified gene with a frequency of 75%. The gyrA and grLA genes have been identified in Staphylococcus aureus and Coagulase Negative Staphy-lococci. The norA gene, on the other hand, has only been identified in Staphylococcus aureus. Two mechanisms are essentially involved in the resistance of Staphylococci to quinolones/Fluoroquinolones, the mecha-nism of resistance by efflux, which takes place thanks to a transmembrane protein coded by the norA gene and by point mutations (substitution and deletion of acids or nucleotides) observed within the protein and nucleic sequences of the chromosomal gyrA and grLA genes.

Analysis on Influential Factors for Anti-Infection Efficacy of Fluoroquinolones

Objective: To investigate factors contributed to anti-infection efficacy of fluoroquinolones (FQNS), and ultimately to provide guidelines for the application of such drugs. Methods: Clinical data of 519 infected patients who were treated with fluoroquinolones were analyzed retrospectively. According to the therapeutic efficacy of the drugs, cases were divided into 3 groups: clinical inefficient, improved and cured. 11 potential factors were investigated. The data were analyzed through logistic regression analysis to determine the main factors which influence therapeutic effects. Results: Ordinal logistic regression revealed that age (OR = 0.979, 95% CI: 0.969, 0.989), a variety of medicine (moxifloxacin-OR = 3.465, 95% CI: 1.396, 8.601;levofloxacin-OR = 4.605, 95% CI: 1.971, 10.760;ciprofloxacin-OR = 3.220, 95% CI: 1.089, 9.552;compared to lomefloxacin) (levofloxacin-OR = 2.591, 95% CI: 1.130, 5.944;compared to fleroxacin) and site of infection (respiratory system-OR = 3.016, 95% CI: 1.737, 5.236;urological system-OR = 4.077, 95% CI: 1.981, 8.391;digestive system-OR = 3.740, 95% CI: 1.849, 7.565) are main factors which influence the efficacy. Conclusion: Fluoroquinolones are more effective in the treatment of bacterial infection within drug’s indications in young population. Variety, dosage and intervals of the drugs should be adjusted according to disease condition.

Inhibitory Effects of Several Fluoroquinolones on Feline CYP1A and 3A in Hepatic Microsomes

In this study, the effects of several fluoroquinolones (FQs), such as Ciprofloxacin (CPFX);Orbifloxacin (OBFX);Norfloxacin (NFX);Ofloxacin (OFX);and Enerofloxacin (EFX) on activities of both Cytochrome P450 1A (CYP1A) and Cytochrome P450 3A (CYP3A) of feline microsomes by in vitro tests were studied. Ethoxyresorufin O-deethylation (EROD) and Midazolam 1' hydroxylation and 4-hydroxylation (MDZ1'H and MDZ4H) were analyzed by High Performance Liquid Chromatography (HPLC). All the FQs inhibited the reactions by a competitive or noncompetitive and irreversible manner. The inhibitory constants (Ki) were as followings: CYP1A;ranged from 0.12 to 1.23 mM for NFX, OBFX, EFX, CPFX, OFX and CYP3A, for MDZ1'H;ranged from 5.8 to 35 and MDZ4H;9 to 29 mM, respectively. As these values are higher by 24 to 200-times of given single clinical dose of serum levels after application of FQs. It indicates that if co-administrated with these FQs by reversible inhibitory manner, the inhibition of CYP1A and CYP3A effect on CYP1A and 3A actions is not very significant to cause drug interaction with above mentioned enzyme substrates. Out of the FQs tested, CPFX and NFX for CYP1A, and CPFX for CYP3A showed irreversible inhibitory effects (time-dependent), so it has been concluded that these drugs may cause drug-drug interaction by accumulation, when they are repeatedly administrated. Since EFX is biotransformed to CPFX by the liver, it could have the identical risk too.