Quinolones in vitro

The first quinolone compound, nalidixic acid, showed activity against a limited number of Gram-negative micro-organisms. One step resistance developed hiin vitro and during treatment. Resistance was not mediated by transfer of R-plasmids, which is a characteristic of all quinolones. Newer quinolones like oxolinic acid, piromidic acid, cinoxacin and pipemidic acid exhibit an extended spectrum of activity against Gram-negative bacteria at lower MIC values. In recent years fluorinated quinolones were introduced like ciprofloxacin, norfloxacin, pefloxacin, ofloxacin, enoxacin and amifloxacin. These compounds exhibitin vitro a broad spectrum of activity against Gram-negative and Gram-positive bacteria at MIC values seventy to four hundred times less than those for nalidixic acid. Thein vitro activity of these compounds has been investigated in a large study of uncomplicated urinary tract infections in general practice (PINISU). No resistance was found. The fluorinated quinolones are very promising antimicrobial agents for a limited number of indications.     

2018—2021年北京市沙门菌血清型及喹诺酮类耐药表型和基因型分析

目的 分析2018—2021年北京市肠道门诊哨点医院分离到的沙门菌血清型及喹诺酮类药物耐药表型和耐药基因的流行情况。方法 采用玻片凝集法鉴定血清型;微量肉汤稀释法测定药物敏感性;全基因组测序数据通过与MLST和ResFinder数据库比对,查询ST型别和耐药基因。结果 228株沙门菌共检出42种血清型,51种ST型。其中肯塔基沙门菌、肠炎沙门菌和黄金海岸沙门菌等9种血清型比较常见。77株沙门菌（33.8%）对萘啶酸耐药;40株（17.5%）对环丙沙星和左氧氟沙星耐药;38株（16.7%）对吉米沙星耐药。不同血清型对喹诺酮类药物的耐药性不同。228株沙门菌共筛选出5种喹诺酮耐药基因:qnrS（23.7%）、aac(6’)-Ib-cr（8.3%）、qnrB（6.6%）、OqxAB（1.3%）和qepA（0.4%）;喹诺酮耐药决定区存在gyrA和parC的7种氨基酸突变:以parC:T57S（70.2%）最常见,其次是gyrA:D87N（14.9%）、parC:S80I（14.5%）、gyrA:S83F（14.5%）、gyrA:D87Y（7.9%）、gyrA:D87G（5.3%）和gyrA:S83Y（1.8%）。在组合突变中,以parC:S80I-gyrA:D87N-gyrA:S83F三突变,并伴随parC:T57S最多。耐药基因和突变位点在9种常见血清型中的检出率存在差异。结论 北京市沙门菌血清型及ST型种类繁多,携带喹诺酮耐药基因以qnrS、aac(6’)-Ib-cr和 qnrB为主,存在gyrA和parC多个位点突变并协同作用。应持续监测血清型和耐药性,以指导临床有针对性合理用药。     

Selective antagonism of the GABAA receptor by ciprofloxacin and biphenylacetic acid

1. Previous studies have shown that ciprofloxacin and biphenylacetic acid (BPAA) synergistically inhibit γ-aminobutyric acid (GABA)_A receptors. In the present study, we have investigated the actions of these two drugs on other neuronal ligand-gated ion channels.
2. Agonist-evoked depolarizations were recorded from rat vagus and optic nerves in vitro by use of an extracellular recording technique.
3. GABA (50 μM)-evoked responses, in the vagus nerve in vitro, were inhibited by bicuculline (0.3–10 μM) and picrotoxin (0.3–10 μM), with IC₅₀ values and 95% confidence intervals (CI) of 1.2 μM (1.1–1.4) and 3.6 μM (3.0–4.3), respectively, and were potentiated by sodium pentobarbitone (30 μM) and diazepam (1 μM) to (mean±s.e.mean) 168±18% and 117±4% of control, respectively. 5-Hydroxytryptamine (5-HT; 0.5 μM)-evoked responses were inhibited by MDL 72222 (1 μM) to 10±4% of control; DMPP (10 μM)-evoked responses were inhibited by hexamethonium (100 μM) to 12±5% of control, and αbMeATP (30 μM)-evoked responses were inhibited by PPADS (10 μM) to 21±5% of control. Together, these data are consistent with activation of GABA_A, 5-HT₃, nicotinic ACh and P2_X receptors, respectively.
4. Ciprofloxacin (10–3000 μM) inhibited GABA_A-mediated responses in the vagus nerve with an IC₅₀ (and 95% CI) of 202 μM (148–275). BPAA (1–1000 μM) had little or no effect on the GABA_A-mediated response but concentration-dependently potentiated the effects of ciprofloxacin by up to 33,000 times.
5. Responses mediated by 5-HT₃, nicotinic ACh and P2_X receptors in the vagus nerve and strychnine-sensitive glycine receptors in the optic nerve were little or unaffected by ciprofloxacin (100 μM), BPAA (100 μM) or the combination of these drugs (both at 100 μM).
6. GABA (1 mM)-evoked responses in the optic nerve were inhibited by bicuculline with an IC₅₀ of 3.6 μM (2.8–4.5), a value not significantly different from that determined in the vagus nerve. Ciprofloxacin also inhibited the GABA-evoked response with an IC₅₀ of 334 μM (256–437) and BPAA (100 μM) potentiated these antagonist effects. However, the magnitude of the synergy was 48 times less than that seen in the vagus nerve.
7. These data indicate that ciprofloxacin and BPAA are selective antagonists of GABA_A receptors, an action that may contribute to their excitatory effects in vivo. Additionally, our data suggest that the molecular properties of GABA_A receptors in different regions of the CNS influence the extent to which these drugs synergistically inhibit the GABA_A receptor.

     

Evaluation of in vitro activity of a new fluoroquinolone trovafloxacin (cp-99,219) compared with other anti-anaerobic antimicrobials against members of the Bacteroides fragilis group

The in vitro activity of a new fluoroquinolone, trovafloxacin (CP-99,219) was compared with that of ten other agents against 100 clinical isolates in the Bacteroides fragilis group. Trovafloxacin was the most active quinolone (MIC₉₀, 1 μg/ml) followed by sparfloxacin (MIC₉₀, 8 μg/ml), levofloxacin (MIC₉₀, 16 μg/ml) and ofloxacin (MIC₉₀, 32 μg/ml). Ciprofloxacin was the least active quinolone (MIC₉₀, 64 μg/ml). Metronidazole, chloramphenicol, imipenem and piperacillin/tazobactam, showed excellent activity with an MIC₉₀ of 1, 8, 0.25 and 16 μg/ml, respectively. Cefoxitin showed good activity and piperacillin was the least active compound. B. vulgatus and B. ovatus were the most resistant species to trovafloxacin among those of the B.fragilis group with an MIC₉₀ of 4 μg/ml while B. fragilis and B. thetaiotaomicron were the most susceptible (MIC₉₀, 1 μg/ml).     

Characteristics of antibiotic resistance and sequence type of Acinetobacter baumannii clinical isolates in Japan and the antibacterial activity of DS-8587

DS-8587 is a novel broad-spectrum fluoroquinolone with extended antimicrobial activity against both Gram-positive and Gram-negative pathogens. In this study, we evaluated the antibacterial activity and mechanism of DS-8587 in 31 quinolone-resistant Acinetobacter baumannii clinical isolates. Efflux pump and qnr genes, mutations in quinolone resistance-determining regions of target enzymes, and sequence types determined by multilocus sequence typing were analyzed. Forty-two quinolone-susceptible clinical isolates were analyzed for comparison. For susceptibility testing, DS-8587 exhibited more effective antibacterial activity when compared with ciprofloxacin and levofloxacin. When combined with the efflux pump inhibitor 1-(1-napthylmethyl)-piperazine, the MIC of DS-8587 was less affected when compared with the MIC exhibited by combined ciprofloxacin and 1-(1-napthylmethyl)-piperazine. The efflux pump genes adeA/adeB/adeC and regulatory elements adeR/adeS were detected in 23 of 31 quinolone-resistant isolates. The qnrA/qnrB/qnrS genes were not detected in any A. baumannii isolates analyzed. Mutations in quinolone resistance-determining regions were observed in all 31 quinolone-resistant isolates. Multilocus sequence typing analyses revealed that 22 of 31 quinolone-resistant isolates belonged to ST-2, corresponding to international clonal lineage II. In conclusion, DS-8587 exhibits potent antibacterial activity against quinolone-resistant A. baumannii isolates that harbor mutations in quinolone resistance-determining regions. In the presence of the efflux pump inhibitor 1-(1-napthylmethyl)-piperazine, no significant changes were observed in the MIC for DS-8587. DS-8587 should be considered as a treatment option for A. baumannii including ST-2 strains that are predominant among the quinolone-resistant A. baumannii isolates found in Japan.     

荧光PCR熔解曲线法检测耐多药肺结核患者对左氧氟沙星和莫西沙星耐药性的效能研究

目的: 探究荧光PCR熔解曲线法快速检测耐多药肺结核患者对左氧氟沙星(Lfx)和莫西沙星(Mfx)耐药性的临床价值。 方法: 以2018年11月至2019年12月在黑龙江省传染病防治院采用BACTEC MGIT 960全自动分枝杆菌培养及比例法药物敏感性试验(简称“药敏试验”)确诊为耐多药肺结核的195例患者为研究对象,同时经荧光PCR熔解曲线法进行筛查,通过与药敏试验结果进行对比,分析荧光PCR熔解曲线法检测结核分枝杆菌对Lfx和Mfx耐药性的准确性。结果: 195株耐多药结核分枝杆菌经比例法药敏试验检测,对Lfx和Mfx耐药率分别为46.15%(90/195)和48.72%(95/195);以比例法药敏试验结果为标准,荧光PCR熔解曲线法检测结核分枝杆菌对Lfx和Mfx耐药性的敏感度分别为95.56%(86/90)和95.79%(91/95),特异度分别为73.33%(77/105)和77.00%(77/100),一致率分别为83.59%(163/195)和86.15%(168/195),Kappa值分别为0.83和0.85。 结论: 荧光PCR熔解曲线法检测耐多药肺结核患者对Lfx和Mfx耐药的结果与比例法药敏试验结果高度一致,适用于筛查耐多药肺结核患者对Lfx和Mfx的耐药性。     

针对gyrA基因突变的反向斑点杂交技术快速检测结核分枝杆菌喹诺酮耐药性的研究

目的 建立快速检测结核分枝杆菌喹诺酮耐药性的反向斑点杂交(reverse dot blot hybridization,RDBH)技术,并观察其效果。方法 针对结核分枝杆菌gyrA基因序列及常见的突变位点,分别设计1条野生型和7条突变型探针,建立RDBH技术,对临床分离结核分枝杆菌菌株进行喹诺酮耐药性检测,以比例法药敏试验和DNA测序做对照。结果 应用比例法药敏试验、DNA测序、RDBH三种方法分别检测59株喹诺酮耐药株和51株喹诺酮敏感株,与比例法相比,RDBH试验灵敏度和特异度分别为69.49%(41/59)、100%(51/51),符合度为83.63%;而RDBH与DNA测序结果比较,敏感度和特异度分别为97.56%(40/41),98.55%(68/69),符合度达98.18%。结论 RDBH技术检测结核分枝杆菌喹诺酮耐药具有良好的灵敏度、特异度和符合度。     

大肠埃希菌和肺炎克雷伯菌中质粒介导的喹喏酮类耐药研究

目的:研究国内11家医院分离大肠埃希菌(ECO)和肺炎克雷伯菌(KPN)中质粒介导的喹喏酮类耐药。方法:收集北京大学第三医院无重复临床分离产超广谱β-内酰胺酶(ESBL s)的ECO和KPN共84株,另10家医院相应菌株共155株,北京协和医院对头孢西丁不敏感的ECO和KPN共25株。采用琼脂稀释法测定抗生素最低抑菌浓度M IC;接合实验判断该基因位置和移动性;聚和酶链反应(PCR)及其产物测序确定基因型。结果:北医三院84株菌中1株KPN(编号24)阳性,ECO的发生率是0;余10家医院155株产ESBL s菌株中没有阳性结果;协和医院25个临床株中3株KPN(编号P 3、P 6、P 10)阳性。这4株菌只有P 6接合试验阳性,相应接合子qnr-PCR阳性。环丙沙星C IP对该菌的M IC是0.5μg/m l,对该接合子的M IC是1μg/m l,而对受体菌EC 600的M IC是0.125μg/m l。对24号标本和P 6标本及其接合子进行测序证实:二者的qnr↑序列和AY 878718(qnrA)100%同源,并且qnr基因的上游都有ORF 513存在。结论:北医三院和北京协和医院分类株中存在质粒介导的喹喏酮类耐药基因qnr,其他医院分离株本次研究中未发现该基因。     

新生大鼠血清和关节软骨喹诺酮类药物浓度与关节软骨细胞外基质变化的相关性研究

目的 探讨喹诺酮类药物对新生大鼠关节软骨的损伤作用及其损伤机制.方法 将60只日龄2天的新生大鼠随机分为对照组、小剂量组和大剂量组各20只,每组又根据处死动物的时间随机分为7天、14天两个亚组,每个亚组10只.对照组不注射任何药物,小剂量组、大剂量组自实验开始第1天腹腔分别注射左氧氟沙星（商品名：左克）10 mg/（kg＆#183;d）、20 mg/（kg＆#183;d）,每天1次,连用7天.各组动物分别在实验第7、14天取血清及关节软骨组织标本,采用高效液相色谱法测定血清、关节软骨左氧氟沙星药物浓度;采用酶联免疫吸附法（ELISA）测定血清Ⅱ型胶原（COLⅡ）、Ⅰ型前胶原羧基端前肽（PICP）及金属蛋白酶-3（MMP-3）水平.结果 7天时对照组血清和关节软骨组织中均未检测出左氧氟沙星,小剂量组血清和关节软骨组织左氧氟沙星含量均低于大剂量组[血清：（0.99＆#177;0.33） mg/L比（2.22＆#177;0.96） mg/L,关节软骨：（0.77＆#177;0.30）tμg/g比（1.82＆#177;0.30） μg/g,P均＜0.01];14天时各组血清和关节软骨组织均未检测出左氧氟沙星.血清、关节软骨左氧氟沙星药物浓度与血清COLII含量呈负相关（P＜0.05）,与血清PICP、MMP-3含量无相关性（P＞0.05）.结论 血清和关节软骨组织喹诺酮类药物浓度与血清COLII含量呈负相关,血清和软骨组织高浓度喹诺酮类药物对新生大鼠关节软骨有明显损伤作用.     

大肠埃希菌对喹诺酮类抗菌药耐药机制的研究近况

喹诺酮类药物是一类化学合成药物,由于其具有较强抗菌活性,临床上已广泛用于各种细菌性感染。随着喹诺酮类药物在临床上越来越广泛的应用,喹诺酮类药物的耐药也逐渐上升。已知细菌对喹诺酮类药物的主要耐药机制为:①靶位基因Ⅱ型拓扑异构酶即DNA回旋酶基因(gyrA和gyrB)和拓扑异构酶Ⅳ基因(parC和parE)突变;②细菌膜通透性的降低和膜上药物主动外排泵的激活;③质粒上qnr基因介导细菌对喹诺酮类耐药。