肺炎支原体对大环内酯类抗生素耐药性及耐药机制研究

目的 了解肺炎支原体(mycoplasma pneumoniae,MP)对大环内酯类抗生素的耐药情况及耐药机制.方法 对370份咽拭子标本进行MP分离培养,应用套式PCR扩增MP种特异16S核蛋白体RNA(16SrRNA)基因对临床分离株进行分子鉴定;通过药物敏感实验测定MP分离株对大环内酯类药物的MIC并筛选出耐药株;设计套式PCR扩增红霉素作用靶位23S核蛋白体RNA(23SrRNA)基因,扩增产物进行全自动DNA测序,测得序列与NCBI已登录的MP标准株M129(登录号X68422)23SrRNA基因作比对.结果 370份临床标本中分离MP 50株,分离阳性率为13.5%.50株中敏感株4株,耐药株46株(占92%).耐药菌株的红霉素、阿奇霉素、交沙霉素MIC值均升高.4株敏感株和肺炎支原体国际标准株FH的23SrRNA基因序列与基因库的MP基因序列相同,46株耐药株的23SrRNA基因发生点突变,41株突变位点在23SrRNA V区中心环的2063位,其中40株发生了A→G的点突变,1株发生了A→C的点突变;另5株突变位点在2064位,A→G.结论 MP对大环内酯类抗生素耐药率高,耐药性的分子基础是23SrRNA基因的点突变,其中2063位点突变占主导地位.23SrRNA基因发生点突变的肺炎支原体对红霉素、阿奇霉素及交沙霉素的MIC值均升高.     

肺炎支原体23SrRNA基因突变位点与耐药表型的分析

目的 了解本地区社区呼吸道感染肺炎支原体(Mycoplasma pneumoniae,Mp)感染状况,探讨肺炎支原体对大环内酯类抗菌药物的耐药分子机制,并分析肺炎支原体耐药菌株23SrRNA基因突变位点与耐药表型之间的关系.方法 对400例社区获得性呼吸道感染患儿咽拭子标本进行分离培养,应用巢式聚合酶链反应(PCR)对临床分离株进行分子鉴定;通过体外药物敏感试验测定Mp临床分离株对大环内酯类抗菌药物的最小抑菌浓度(MIC),并筛选出耐药株;检测耐药株23SrRNA基因序列,并与标准菌株M129基因序列对比分析,分析突变位点与耐药表型的关系.结果 400例咽拭子标本中分离Mp 50株.其中敏感株32株,耐药株18株.18株耐药株分别出现A2063G、A2064G、A2067G 位点突变.A2063G突变株表现出对14元环大环内酯类抗菌药物耐药,A2064G突变株表现为对14、16元环大环内酯类抗菌药物的耐药,A2067G突变株表现出对交沙霉素耐药.结论 Mp对大环内酯类抗菌药物耐药现象严重,23SrRNA基因位点突变是耐药性产生的主要机制.通过对23SrRNA基因突变位点与耐药表型的分析研究,初步了解临床肺炎支原体耐药现状,并且为抗菌药物的合理选择和应用提供理论指导.     

一种快速同步检测肺炎支原体及其大环内酯类耐药突变的新方法

目的 建立基于CycleavePCR技术的肺炎支原体及其大环内酯类耐药突变株快速检测方法.方法 收集102株分离自2005-2008年上海市交通大学附属儿童医院住院患儿的肺炎支原体及136份2009年11-12月上海市交通大学附属儿童医院呼吸道感染的住院患儿经鼻采集鼻咽部痰标本,根据肺炎支原体无突变敏感株与突变耐药株23S rRNA基因2063/2064位的碱基差异及上下游保守序列分别设计新型特异性探针(Cycling探针)及引物,建立肺炎支原体及其大环内酯类耐药突变株检测方法,以含有被检测靶序列的重组质粒为阳性对照,以常见呼吸道病原菌DNA为阴性对照,评价其敏感度及特异度.采用自建方法检测所有标本,并与常规PCR及测序结果比较.结果 该方法可准确检出并鉴别大环内酯类敏感及耐药突变株阳性对照,102株临床分离株中83株对大环内酯类耐药,测序表明82株发生23S rRNA基因A2063G突变,1株A2064G突变,与CycleavePCR检测结果相符,136份痰标本检测结果也与常规PCR扩增及测序结果相符.所有阴性对照样品均未出现假阳性.与常规PCR及测序方法相比,CycleavePCR法的敏感度和特异度均达100%.该方法的检测下限为10拷贝/PCR反应,扩增检测可在1.5 h内完成.结论 建立了一种基于CycleavePCR技术的肺炎支原体及其大环内酯类耐药突变株快速检测方法,可同步提供病原菌及其耐药性信息,为肺炎支原体感染诊断及临床合理选用抗菌药物提供参考.     

实时荧光定量PCR结合探针检测MP的临床价值及MP耐药情况分析

目的评价实时荧光定量聚合酶链反应(PCR)结合探针检测对肺炎支原体(MP)的诊断价值,并分析MP的耐药突变情况,指导MP感染患儿的临床用药。方法选取2017年9月—2019年8月奉城医院收治并经诊断确诊的70例MP感染患儿及30例非MP感染患儿,对所有患儿咽拭子标本进行MP-DNA扩增,采用荧光探针法对MP与大环内酯类抗菌药物耐药突变率进行检测。收集所有患儿临床资料,并分析其与MP-DNA阳性检出及MP耐药突变的相关性。结果实时荧光定量PCR结合探针检测MP的敏感性和特异性分别为80.0%(56/70)和96.7%(29/30);双份血清MP-IgM抗体滴度呈4倍或4倍以上(升高或降低)变化组MP-DNA阳性检出率明显较单份血清滴度≥1∶160组高;患儿性别、年龄、入院时病程及入院前大环内酯类抗菌药物治疗史对于MP-DNA阳性的检出率均无显著影响;MP-DNA耐药突变率高达85.7%,入院前规律完成大环内酯类抗菌药物1个疗程组DNA耐药突变率为90.5%,明显高于未完成1个疗程组(80.0%)和未服用组(50.0%)。结论实时荧光定量PCR结合探针可作为检测MP的一种有效方法,为临床对MP感染患儿抗菌药物的使用提供依据,具有较高的临床应用价值。     

肺炎支原体对红霉素体外耐药性与耐药基因相关性研究

目的探讨肺炎支原体(Mp)对红霉素耐药与耐药基因之间的关系。方法通过对46株红霉素耐药Mp临床分离株的23SrRNAⅤ区进行聚合酶链反应(PCR)扩增,并将扩增产物进行基因测序,确定其基因型。结果 46株红霉素耐药Mp临床分离株中有44株(95.65%)发生A2063G突变,2株(4.35%)A2064G突变,未见A2063C和C2617G/A位点突变。结论 Mp临床分离株对红霉素耐药与23SrRNAⅤ区基因2063、2064位点A→G突变有密切关系,其中最主要的突变是A2063G突变。快速、准确识别Mp的23SrRNAⅤ区基因突变可在诊断的同时给出耐药信息,有效指导临床合理用药。     

上海地区幽门螺杆菌对克拉霉素耐药的相关基因检测

目的通过荧光PCR熔解曲线方法直接检测临床样本中幽门螺杆菌(Hp)对克拉霉素耐药基因,了解上海地区Hp耐克拉霉素基因的分布特征,并为临床快速检测Hp对克拉霉素耐药性提供一种有效的方案。方法收集2017年9-12月复旦大学附属中山医院消化科就诊并接受胃镜检查的快速尿素酶试验Hp阳性患者的胃黏膜组织标本80份,使用荧光PCR熔解曲线法直接检测黏膜样本中Hp种特异性23S rRNA的区域并对扩增的核酸进行荧光标记,通过检测荧光变化绘制其熔解曲线,以确定Hp的23S rRNA突变情况;采用DNA测序技术直接检测标本中Hp克拉霉素23S rRNA耐药基因突变位点。并对两种方法的结果进行Kappa检验统计学比较。结果 80份胃黏膜组织标本使用荧光PCR熔解曲线法共检测69份Hp阳性,11份Hp阴性,阳性率为86.2%。在69份Hp阳性标本中24份标本存在23SrRNA第2142或2143位点的变异,突变率34.8%。使用DNA测序法共检测出64份Hp阳性,阳性率为80.0%。其中23例患者的标本检测到23S rDNA耐药基因的突变,突变率35.9%。两种方法检测Hp耐药基因突变的阳性符合率、阴性符合率和总符合率分别为87.0%、95.1%和92.2%,Kappa系数为0.829,两者一致性较好。结论上海地区克拉霉素耐药Hp菌株基因型以23S rRNA基因的A2143G突变占主导。荧光PCR熔解曲线法可直接检测胃黏膜活检组织中Hp及其23S rRNA突变基因,该方法结果准确可靠。     

RTFQ-PCR检测儿童肺炎支原体16S rRNA的临床意义

目的研究实时荧光定量聚合酶链反应(RTFQ-PCR)检测儿童肺炎支原体(MP)16SrRNA的临床意义。方法回顾性分析2014年12月至2016年12月在该院接受治疗的肺炎患儿156例,将以上患儿分为MP感染肺炎(MPP)组,非MP感染肺炎(非MPP)组;另选24例健康儿童作为对照组。使用颗粒凝集试验检测血清MP抗体,使用RTFQ-PCR检测患儿MP 16SrRNA基因,并与血清学的检测结果进行对比,分析患儿病情和基因拷贝数量的联系。结果 156例患儿中两份血清MP抗体有4倍以上升高者112例,两份和单份血清MP抗体检测结果差异有统计学意义(P0.05);单份血清检测的阴性预测值为37.11%,阳性预测值为62.02%,MP诊断的正确率为50.89%;112例确诊的MP感染患儿中RTFQ-PCR检测为阳性109例,敏感度为97.30%;对照组和非MPP组检测为阴性68例,其特异度为100.00%;RTFQ-PCR检测阴性预测值为97.10%,阳性预测值为100.00%;难治性MPP检测出基因拷贝数量为(6.32±1.80)copy/mL,高于普通性MPP[(1.51±0.02)copy/mL],差异有统计学意义(P0.05)。结论 RTFQ-PCR检测儿童MP 16SrRNA基因是诊断前期MP感染肺炎的可靠方法,效果好于血清MP抗体的检测,值得临床关注。     

应用巢氏PCR对成人社区获得性肺炎患者咽拭子中肺炎支原体DNA的检测及耐药分析

目的 了解肺炎支原体（MP）在成人社区获得性肺炎（CAP）患者中的感染情况与成人MP耐大环内脂类抗生素的分子机制.方法 收集北京友谊医院成人CAP住院患者咽拭子标本,巢氏PCR扩增红霉素作用靶位23S rRNA基因,电泳检测阳性标本行双脱氧末端终止法全自动DNA测序,测序结果与NCBI已登录的MP标准株23S rRNA基因作比对,分析差异基因.结果 共收集成人CAP住院病例97例,其中咽拭子阳性25例,阳性率25.8%.15例为耐药株,耐药率为60.0%,均出现2 063位A-G的点突变.结论 北京地区成人MP存在耐药现象,主要对大环内酯类抗菌药物耐药,耐药分子机制与国内外文献报道基本相似.     

1379例儿童肺炎支原体快速培养鉴定结果分析

目的探讨肺炎支原体(MP)快速鉴定培养在儿童MP感染早期的临床诊断价值。方法采用MP快速鉴定培养基对本院2008年1月至2009年12月患急性呼吸道感染的1379例患儿咽拭子标本进行MP培养检测。结果 1 379例急性呼吸道感染患儿的咽拭子标本MP培养阳性494例,总阳性率35.82%;其中男性阳性率36.48%,女性阳性率35.11%,男、女比较差异无统计学意义(P0.05)。在各年龄组中,4～8岁组阳性率最高(45.72%),2岁组最低(17.23%),两组间比较差异有统计学意义(P0.05)。1年中,11月至次年1月阳性率最高(42.62%),5～7月阳性率最低(23.74%),两组间比较差异有统计学意义(P0.05)。结论 MP快速鉴定培养基检测肺炎支原体感染总检出率较高,结果准确、简便、快速,为临床尽快诊断肺炎支原体感染具有重要的参考价值,可作为一般实验室诊断MP感染的首选试验。     

儿童呼吸道分离肺炎支原体药物敏感性分析

目的了解儿童呼吸道肺炎支原体临床分离株对常用抗菌药物的敏感性。方法采用微量稀释法测定分离自呼吸道感染患儿的112株肺炎支原体对大环内酯类、四环素类和氟喹诺酮类药物的敏感性。对肺炎支原体核糖体23S rRNA全序列进行扩增及测序。结果 98株(87.5%)肺炎支原体临床分离株对红霉素和阿奇霉素耐药,耐药株均存在A2063G或A2064G的23S rRNA核苷酸点突变。四环素类和氟喹诺酮类对肺炎支原体仍具有良好的抗菌活性。结论呼吸道感染患儿肺炎支原体临床分离株对大环内酯类耐药率高,核糖体23S rRNA核苷酸点突变是导致其对红霉素和阿奇霉素耐药的原因。     

Mutations in domain V of Mycoplasma pneumoniae 23S rRNA and clinical characteristics of pediatric M. pneumoniae pneumonia in Nanjing,China

ObjectiveTo investigate the prevalence of mutations in domain V of Mycoplasma pneumoniae (MP) 23S ribosomal RNA (rRNA) and the clinical characteristics of pediatric MP pneumonia (MPP) in Nanjing, China.MethodsDomain V of 23S rRNA was sequenced in MP strains collected from children diagnosed with MPP in Nanjing. Clinical and laboratory data were obtained.ResultsAmong the 276 MP strains, 255 (92.39%) harbored mutations, primarily A2063G in domain V of MP 23S rRNA. When children were stratified according to the presence or absence of mutations, no significant differences were found in sex, age, the MP DNA load at enrollment, lymphocyte counts, pulmonary complications, immunomodulator levels, fever duration, the duration of fever after macrolide therapy, and hospital stay. The prevalence of refractory MPP in the two groups was similar. Children with refractory MPP exhibited higher MP DNA loads than those with non-refractory MPP.ConclusionsDespite the high prevalence of the A2063G mutation in domain V of MP 23S rRNA, mutations were not associated with the clinical characteristics of MPP. The MP DNA load significantly differed between refractory and non-refractory MPP.     

Reversion to susceptibility in a linezolid-resistant clinical isolate of Staphylococcus aureus

OBJECTIVES: Linezolid resistance in rare isolates of Staphylococcus aureus has been associated with G2576T mutations in domain V of the 23S rRNA gene. We report the analysis of a clinical S. aureus isolate that developed linezolid resistance (MIC of linezolid of 12 mg/L) after a 25 day course of the drug. Sequencing identified G2576T mutations in four of the five copies of the 23S rRNA gene. METHODS: To examine the stability of this resistance, we serially passaged this original isolate 60 times over a 75 day period on antimicrobial-free medium. RESULTS: After 30 passages, the MIC of linezolid had decreased to 8 mg/L and only two of the five copies of the 23S rRNA gene contained the G2576T mutation. After 60 passages, the MIC of linezolid fell to 2 mg/L and only one of the five 23S rRNA gene copies contained the mutation. The original and two passaged staphylococci were indistinguishable by pulsed-field gel electrophoresis. CONCLUSIONS: In the absence of antibiotic pressure, linezolid resistance was unstable in a clinical isolate that did not have all copies of the 23S rRNA gene mutated, although a single copy of mutant rDNA was maintained. Gene conversion was probably the mechanism for this reversion, using the wild-type 23S rRNA gene sequences to replace the G2576T mutation by homologous recombination.     

Explaining the Bias in the 23S rRNA Gene Mutations Associated with Clarithromycin Resistance in Clinical Isolates of Helicobacter pylori

A single point mutation in the 23S rRNA gene of Helicobacter pylori is known to confer resistance to clarithromycin. Most prevalent among clarithromycin-resistant clinical H. pylori isolates are the mutations from A-2142 to G and A-2143 to G in the 23S rRNA gene. The bias in the 23S rRNA gene mutations conferring clarithromycin resistance may result from the higher MIC, stability of resistance, and growth rate found for the strains with the above-mentioned mutations.     

Involvement of the CmeABC efflux pump in the macrolide resistance of Campylobacter coli

OBJECTIVES: This study was conducted to examine the role of the CmeABC efflux pump in decreasing the susceptibility of Campylobacter coli to macrolides and ketolides in the context of absence or presence of mutations in the 23S rRNA genes. METHODS: The cmeB gene was inactivated in strains of C. coli showing two different patterns of erythromycin resistance (low or high level of resistance) associated with the absence or presence of a A2075G mutation in the 23S rRNA genes. MICs of erythromycin, azithromycin, tylosin, telithromycin and ciprofloxacin were compared for wild-type (with or without efflux pump inhibitor) and mutant strains. RESULTS: The cmeB gene inactivation (or addition of efflux pump inhibitor) led to the restoration of susceptibility of the low-level-resistant strains (no A2075G mutation in the 23S rRNA genes). In the highly resistant strains (A2075G mutation in the 23S rRNA genes), the MICs of erythromycin decreased 128- to 512-fold upon inactivation of the cmeB gene. MICs of azithromycin, tylosin and telithromycin were also affected by both addition of efflux pump inhibitor and cmeB gene inactivation, revealing these molecules as substrates of the CmeABC efflux pump. Compared with azithromycin, MICs of telithromycin drastically decreased upon cmeB gene inactivation even in the presence of a A2075G mutation in 23S rRNA genes. CONCLUSIONS: The CmeABC efflux pump acts synergically with 23S rRNA mutations to drastically increase the MICs of erythromycin and tylosin in C. coli. In contrast, azithromycin was less affected by efflux and telithromycin, although being a good substrate for the CmeABC efflux pump, was less affected by an A2075G mutation in 23S rRNA genes.     

成人非典型社区获得性肺炎患者病原体感染情况分析

目的分析肺炎支原体(MP)等经典微生物引起的非典型肺炎患者的实验室检查结果,为临床诊治提供参考。方法采用实时荧光定量聚合酶链反应(qPCR)检测188例成人社区获得性肺炎患者急性期咽拭子和/或痰液样本中的MP、肺炎衣原体和嗜肺军团菌,分析每位患者的临床资料、实验室指标、影像学检查结果。结果MP核酸检测阳性41例(21.8%),肺炎衣原体核酸检测阳性1例(0.5%),未检出嗜肺军团菌;细菌培养阳性23例(12.23%)。MP阳性患者年龄较阴性患者小,较少伴有基础疾病,肺部以单侧受累为主,其他临床症状和实验室相关指标差异均无统计学意义。秋季、冬季MP阳性率明显高于春季、夏季(P<0.05)。结论MP是成人社区获得性肺炎的主要病原体,qPCR对MP急性感染的早期诊断具有明显的优势。     

沈阳地区非综合征性耳聋患者的基因芯片检测结果分析

目的：探讨基因芯片技术在耳聋基因筛查的临床应用价值,了解沈阳地区非综合征性耳聋患者致病基因的分子特征。方法采集沈阳地区门诊或病房散发的非综合征性耳聋患者100例的外周血并提取DNA ,应用晶芯遗传性耳聋基因芯片试剂盒检测GJB2、GJB3、SLC26A4和线粒体12SrRNA 4个常见耳聋基因中的9个突变位点。结果在100例患者中,36例耳聋患者检出遗传性耳聋基因突变,检出阳性率为36．0％（36/100）;其中GJB2、SLC26A4、线粒体12S rRNA 和 GJB3基因突变的阳性检出率分别为20．0％（20/100）、14．0％（14/100）、2．0％（2/100）和0．0％（0/100）。检出致聋基因突变型19例,占被检出阳性总数的52．8％（19/36）,杂合基因突变型17例,占被检出阳性总数的47．2％（17/36）。结论中国人常见的4个致聋基因突变在沈阳地区人群中除GJB3外都有一定的检出率。     

Site-Specific Mutations in the 23S rRNA Gene of Helicobacter pylori Confer Two Types of Resistance to Macrolide-Lincosamide-Streptogramin B Antibiotics

Clarithromycin resistance in Helicobacter pylori is mainly due to A-to-G mutations within the peptidyltransferase region of the 23S rRNA. In the present study, cross-resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics (MLS phenotypes) has been investigated for several clinical isolates of H. pylori. Two major types of MLS resistance were identified and correlated with specific point mutations in the 23S rRNA gene. The A2142G mutation was linked with high-level cross-resistance to all MLS antibiotics (type I), and the A2143G mutation gave rise to an intermediate level of resistance to clarithromycin and clindamycin but no resistance to streptogramin B (type II). In addition, streptogramin A and streptogramin B were demonstrated to have a synergistic effect on both MLS-sensitive and MLS-resistant H. pylori strains. To further understand the mechanism of MLS resistance in H. pylori, we performed in vitro site-directed mutagenesis (substitution of G, C, or T for A at either position 2142 or 2143 of the 23S rRNA gene). The site-directed point mutations were introduced into a clarithromycin-susceptible strain, H. pylori UA802, by natural transformation followed by characterization of their effects on MLS resistance in an isogenic background. Strains with A-to-G and A-to-C mutations at the same position within the 23S rRNA gene had similar levels of clarithromycin resistance, and this level of resistance was higher than that for strains with the A-to-T mutation. Mutations at position 2142 conferred a higher level of clarithromycin resistance than mutations at position 2143. All mutations at position 2142 conferred cross-resistance to all MLS antibiotics, which corresponds to the type I MLS phenotype, whereas mutations at position 2143 were associated with a type II MLS phenotype with no resistance to streptogramin B. To explain that A-to-G transitions were predominantly observed in clarithromycin-resistant clinical isolates, we propose a possible mechanism by which A-to-G mutations are preferentially produced in H. pylori.     

PCR-高效液相色谱法检测线粒体DNA A1555G突变

目的 探讨PCR-变性高效液相色谱(PCR-DHPLC)分析技术在检测线粒体(mt)12S rRNA 基因第1 555位A-G均质点突变中的应用.方法 4例临床诊断为氨基糖苷类抗生素耳聋的患者及40例听力正常体检者,分别运用PCR-DHPLC技术、酶切技术及DNA测序技术进行mtDNA1555位点检测.结果 PCR-D...