Whole Genome Analysis Reveals New Insights into Macrolide Resistance in Mycoplasma pneumoniae

Objective Mutations in 23 S rRNA gene are known to be associated with macrolide resistance in Mycoplasma pneumoniae(M. pneumoniae). However, these mutations alone do not fully explain the high resistance rates in Asia. The aim of this study was to investigate other possible mutations involved in macrolide resistance in M. pneumoniae. Methods The whole genomes of 10 clinical isolates of M. pneumoniae with macrolide resistance were sequenced by Illumina Hi Seq2000 platform. The role of the macrolide-specific efflux transporter was assessed by efflux-pump inhibition assays with reserpine and carbonyl cyanide m-chlorophenyl-hydrazone(CCCP). Results A total of 56 single nucleotide polymorphisms(SNPs) were identified in 10 clinical isolates in comparison to the reference strains M129 and FH. Strikingly, 4 of 30 SNPs causing non-synonymous mutations were clustered in macrolide-specific efflux system gene mac B encoding macrolide-specific efflux pump protein of the ATP-binding cassette transporter family. In assays of the minimal inhibitory concentrations(MIC) of macrolide antibiotics in the presence of the efflux pump inhibitors caused a significant decrease of MICs, even under detectable levels in some strains. Conclusion Our study suggests that macrolide efflux pump may contribute to macrolide resistance in M. pneumoniae in addition to the common point mutations in 23 S r RNA gene.     

Mycoplasma pneumoniae Macrolide Resistance and MLVA Typing in Children in Beijing,China, in 2016: Is It Relevant*

ObjectiveThe aim of this study is to investigate the macrolide resistance rate and molecular type with multiple-locus variable-number tandem-repeat analysis (MLVA) of Mycoplasma pneumoniae of Beijing in 2016 in pediatric patients.MethodsReal-time quantitative polymerase chain reaction (PCR) was used to identify M. pneumoniae, and MLVA was performed. The domain V of the 23S rRNA was sequenced to detect macrolide-resistant point mutations. We also investigated the activities of antibiotics against M. pneumoniae isolates in vitro.ResultsThe PCR detection rate of M. pneumoniae in children in Beijing was 40%, and the macrolide resistance rate was 66%. The A2063G mutation in the 23S rRNA V region is the dominant mutation (137/146, 93.84%), whereas the A2064G mutation is rare (9/146, 6.16%). Seventy-three samples were typed successfully by MLVA typing, including 86.3% (63/73) were MLVA type 4-5-7-2, and 13.7% (10/73) were MLVA type 3-5-6-2. No other types were found. No strains were resistant to levofloxacin or tetracycline.ConclusionIn 2016, a specific decrease in the macrolide resistance rate occurred in Beijing. The detection rate and macrolide resistance rate of outpatients are lower than those of inpatients. The A2063G mutants M. pneumoniae have high levels of resistance to erythromycin and azithromycin. The primary MLVA type is 4-5-7-2, followed by 3-5-6-2. No other MLVA types were detected. No strains resistant to tetracycline or levofloxacin were found in vitro.     

肺炎支原体耐药性及分子流行病学研究进展

肺炎支原体是临床上非典型性肺炎常见的致病菌,主要采用大环内酯类抗菌药物治疗。近年来,肺炎支原体对大环内酯类耐药性日趋严重,且全世界各地耐药率存在差异,目前认为其耐药机制主要与23S rRNA V区基因位点突变（2 063位和2 064位）相关。根据肺炎支原体菌株P1基因序列的不同,应用聚合酶链式反应-限制片段长度多态性分析（PCR-RFLP）可分为Ⅰ型和Ⅱ型,其流行基因型随地区和时间的不同而不同。另外,为了解肺炎支原体耐药菌株之间是否存在克隆传播,可采用多位点可变数量串联重复序列分析（MLVA）方法分析肺炎支原体克隆型。加强肺炎支原体的耐药性及流行基因型的监测,有助于了解肺炎支原体的流行病学特点,为新型抗菌药物及疫苗的研发提供依据。     

耐碳青霉烯类鲍曼不动杆菌外排泵AdeABC的研究

目的从外排泵表型和基因型两个方面探讨外排泵系统在鲍曼不动杆菌对碳青霉烯类抗生素耐药机制中所起的作用。方法外排泵抑制剂羰基氰氯苯腙(CCCP)对138株鲍曼不动杆菌的外排泵进行表型检测;荧光定量PCR测定外排泵adeABC在51株鲍曼不动杆菌中的表达情况。结果泵抑制剂羰基氰氯苯腙(CCCP)和美罗培南协同实验的结果显示:138株临床分离菌中,CCCP抑制实验共筛选出泵阳性菌株28株,其中对美罗培南耐药的39株菌中有15株即38.4%的菌株为泵阳性株,对美罗培南敏感的99株菌中有13株即13.1%的菌株为泵阳性株。经卡方检验统计学分析,差异有显著意义(χ2=12.477b,P=0.01);荧光定量PCR检测菌株的adeB和16s rRNA后,根据检测结果计算出adeB表达量差异。经t检验统计学分析结果显示碳青霉烯类抗生素敏感组的adeB的表达量是0.899±1.172,耐药组的表达量是21.101±21.443,敏感组的表达量显著低于耐药组的表达量,差异有统计学意义(t=4.403,P=0.000)。结论主动外排泵机制在临床分离的鲍曼不动杆菌对碳青霉烯类抗生素的耐药机制中起作用。adeB基因或者adeABC外排泵机制可能在临床菌株对碳青霉烯类抗生素的耐药机制中起作用。     

肺炎支原体23S rRNA突变与患儿临床特征及耐药性的相关性

  目的  观察肺炎支原体(Mycoplasma pneumoniae,MP)肺炎患儿23S rRNA基因突变特征,探讨该基因突变与患儿临床特征及耐药性的相关性。  方法  对2016年1月—2019年1月于温州市中西医结合医院住院治疗的214例小儿肺炎支原体肺炎病例MP菌株进行23S rRNA基因突变检测,并分别纳入无突变组(36例)和突变组(178例),通过病历收集患儿的一般信息、临床资料和药敏试验结果,分析23S rRNA基因突变与患儿临床特征及耐药性的相关性。  结果  共检测到23S rRNA基因突变178例(83.18%)。与无突变组相比,突变组患儿的重症肺炎比例(83.18%)更高,平均发热时间[(7.22±2.13)d]、住院时间[(8.30±3.25)d]及体温恢复正常的时间[(6.45±2.33)d]更长,差异均有统计学意义(均P < 0.05)。药敏试验结果显示突变组阿奇霉素、罗红霉素的耐药率(38.20%、25.84%)显著高于无突变组(13.89%、8.33%,均P < 0.05)。多因素logistic回归分析显示肺炎严重程度、体温恢复正常时间和23S rRNA基因突变是MP耐药性的独立相关因素(OR=1.693、1.285、3.338,均P < 0.05)。  结论  23S rRNA的A2063G基因突变是MP的主要突变类型,对大环内酯类抗生素具有明显耐药性,可导致重症肺炎患儿比例增高,并可显著延长患儿的临床治疗时间。      

AdeABC外排泵系统与鲍曼不动杆菌对碳青霉烯类药物耐药的关系

目的：探讨外排泵AdeABC系统与鲍曼不动杆菌对碳青霉烯类药物耐药的关系。方法：采用美罗培南 多步法体外诱导敏感鲍曼不动杆菌获得对碳青霉烯类药物耐药的菌株；采用E-test法定量检测诱导前后菌株的敏感 性；羰基氰化物间氯苯腙(carbonylcyanide-m-chlorophenylhydrazone，CCCP)抑制试验筛查外排泵；PCR及测序分析诱 导前后AdeABC系统的调控基因adeS，adeR及主要碳青霉烯酶基因的变化；荧光定量PCR检测诱导前后adeA，adeB， adeR和adeS基因mRNA的表达量。结果：亲代敏感菌株S25595和S7257的美罗培南最低抑菌浓度(minimal inhibitory concentration，MIC)分别为0.38和0.25 μg/mL，诱导后MIC均>32 μg/mL；与亲代敏感株相比，诱导耐药株adeA，adeB， adeR和adeS基因的mRNA表达量上升2.45~9.44倍，但调控基因adeS和adeR没有基因突变或插入序列。结论：外排泵 AdeABC系统高表达与鲍曼不动杆菌对美罗培南耐药密切相关，其表达水平升高不是由调控基因adeS和adeR序列中基 因突变或插入序列引起，可能存在其他机制。     

利奈唑胺体外诱导粪肠球菌中介耐药及机制研究

目的 研究利奈唑胺对粪肠球菌中介耐药的体外诱导作用并探讨其耐药机制.方法 采用多步诱导法对4株临床分离粪肠球菌以及质控菌株进行体外诱导耐药试验,采用琼脂平皿二倍稀释法测定诱导前后的MIC值,采用PCR测序法检测粪肠球菌4个23S rRNA V区基因的变异.结果 4株临床分离粪肠球菌和质控菌株均诱导出中介株和稳定耐药株,在中介株和耐药株中均检测到了G2576U、G2505A和C2424U变异,2株中介株和耐药株还同时存在G2576U和C2424U变异.结论 利奈唑胺可体外诱导粪肠球菌产生中介株和稳定性耐药株,粪肠球菌中介株即可出现23S rRNA V区基因变异.     

Preliminary Study on Drug Susceptibility Profile and Resistance Mechanisms to Macrolides of Clinical Isolates of Non-tuberculous Mycobacteria from China

Objective

Macrolide susceptibility and drug resistance mechanisms of clinical non-tuberculous mycobacteria (NTM) isolates were preliminarily investigated for more accurate diagnosis and treatment of the infection in China.

91株耐利福平结核分枝杆菌的rpoB基因突变分析

目的分析利福平耐药结核菌株rpoB基因突变特点,探讨DNA序列分析在药敏测定中的意义。方法对101株结核分枝杆菌临床分离株rpoB基因的PCR产物进行测序分析,观察其rpoB基因突变的规律。结果 101株临床分离株中,耐利福平91株,经DNA序列分析有85.71%(78/91)菌株存在rpoB基因突变的突变,主要集中在531位(44.87%)和526位(28.21%),未检测到发生缺失或插入碱基突变的菌株,10株敏感株均无突变。结论 rpoB基因核心突变区发生突变是结核分枝杆菌对利福平耐药的主要原因,其中531位丝氨酸和526位组氨酸是最常见的突变位点。     

Clinical and laboratory evaluation of penicillin resistant Streptococcus pneumoniae in relation to the mutations of pbp 1a, pbp2b and pbp2x

A total of 210 isolates of Streptococcus pneumoniae (S. pneumoniae) were selected randomly to examine drug susceptibility which were obtained from out- and in-patients who visited Kurume University Hospital and other affiliated hospitals through May 1998 to September 1999. The prevalence of penicillin resistant S. pneumoniae in this study was 39.5% and was compatible with those of previous reports in Japan. From the clinical aspects, the resistant strains of S. pneumoniae have been shown not to be so highly virulent comparing with sensitive strains that only few severe or mortal cases had been reported. Carbapenems, glycopeptides, and fluoroquinolones were shown to be highly active against penicillin-resistant S. pneumoniae strains as evidenced by the low minimum inhibitory concentrations (MICs) though LVFX showed 4 micrograms/ml or higher MICs against some strains. Regarding to the mechanisms of penicillin resistance, penicillin binding proteins coding gene (pbp1a, pbp2x and pbp2b) mutations that cause modifications in these proteins were also examined for all isolates. The multivariate analysis showed statistically significant correlation between higher MIC of penicillin and cephem and pbp1a mutation while no significant contribution of pbp2x and pbp2b to the resistance was demonstrated. Additionally, no significant correlation between pbp mutation and MIC of carbapenem was observed. Furthermore, there were two highly penicillin resistant S. pneumoniae strains with no pbp mutations. Thus the pbp mutations alone were not responsible for the elevation of MIC all beta-lactams. Nevertheless the pbp mutations were detected in advance of actual MIC elevations by inducement experiment in vitro. It indicated that penicillin resistance might be detected earlier than conventional methods. In previous reports some other responsible genes for penicillin resistance were demonstrated. Therefore, it might be possible to presume exact values of MICs of beta-lactams against resistant strains of S. pneumoniae by detecting still unknown genes other than pbps.     

大环内酯类药物次抑菌浓度诱导沙眼衣原体耐药的实验研究

目的：探讨沙眼衣原体对大环内酯类药物的分子耐药机制。方法：13例沙眼衣原体临床敏感株经红霉素、阿奇霉素、交沙霉素诱导耐药后与未耐药之前的敏感株和标准株E—UW-5／Cx比较,检测基因23SrRNA和核糖体蛋白L4的位点突变。结果：耐药株显现出对红霉素、阿奇霉素和交沙霉素低耐药性,MIC较敏感株分别增加了16、16、8倍。药物敏感性结果显示相对于红霉素、阿奇霉素,沙眼衣原体治疗交沙霉素的易感性最好。敏感株和耐药株的L4的PCR扩增产物的序列均出现G274A、C276T、C339T、C466G位点突变,考虑与大环内酯类药物耐药无关。耐药株的23SrRNA基因PCR扩增产物中出现了A2057G、A2059G和T2611C的突变。结论：沙眼衣原体对大环内酯类药物的耐药分子基础是23SrRNA基因的点突变。     

尿源产超广谱β-内酰胺酶肠杆菌科细菌中16S rRNA甲基化酶基因及菌株克隆测定

目的 了解16S rRNA甲基化酶基因在尿源产超广谱β-内酰胺酶（ESBLs)肠杆菌科细菌中的分布及菌株序列型别,为临床用药提供依据。 方法 采用琼脂稀释法及PCR法对引起尿路感染的74株产ESBLs肠杆菌科细菌进行体外药敏、16S rRNA 甲基化酶基因检测与分型,MLST法进行溯源性序列分型研究。 结果 74株产ESBLs肠杆菌科细菌中93.4%耐庆大霉素、18.4%耐奈替米星、13.2%耐妥布霉素、仅5.3%耐阿米卡星与异帕米星。74株菌中22株（29.7%）检出16S rRNA甲基化酶基因,其中7株检出rmtB基因, 18株检出armA基因,3株同时检出rmtB和armA基因。22株16S rRNA甲基化酶基因阳性菌株对庆大霉素、奈替米星耐药率为100%,对妥布霉素者为59.1%,对阿米卡星及异帕米星者仅为18.2%。多位点序列分型显示19株甲基化酶基因阳性大肠埃希菌序列型为:12株为ST117,2株为ST2003,ST3843、ST915、ST844、ST2581、ST2922各1株;3株甲基化酶阳性肺炎克雷伯菌分为ST1184、ST490、ST337。 结论 大部分尿源性大肠埃希菌可能源于同一克隆。16S rRNA甲基化酶基因在尿源产ESBLs肠杆菌科细菌中分布广泛,与氨基糖苷类药物耐药有明显相关性。     

广泛耐药鲍曼不动杆菌对氨基糖苷类耐药机制

目的 调查广泛耐药鲍曼不动杆菌（Extensively drug Resistant Acinetobacter Baumannii,XDR-ABA）临床分离菌株中氨基糖苷类修饰酶基因、16SrRNA甲基化酶基因和外排泵基因的存在情况.方法 收集2011年1-12月临床标本中分离的XDR-ABA菌20株,采用微量肉汤稀释法进行抗菌药物敏感性试验,聚合酶链反应（PCR）方法分析9种氨基糖苷类修饰酶基因、6种16SrRNA甲基化酶基因和外排泵基因adeB.结果 20株XDR-ABA菌均检出aac（6＇）-Ⅰb、ant（2＂）-Ⅰ、ant（3＂）-Ⅰ、aphA1和adeB外排泵基因,其余5种氨基糖苷类修饰酶基因和6种16SrRNA甲基化酶基因和均未检出.结论该组20株XDR-ABA菌耐多种氨基糖苷类药物与细菌产aac（6＇）-Ⅰb、ant（2＂）-Ⅰ、ant（3＂）-Ⅰ、aphA1 4种氨基糖苷类修饰酶和存在adeABC外排泵系统相关.     

肺炎链球菌临床菌株Tn1545和Tn917介导的大环内酯类耐药性研究

目的探讨上海地区大环内酯类耐药肺炎链球菌ermB基因表达及传播特性。方法收集上海地区对红霉素耐药的肺炎链球菌共86株，用E试验和K—B纸片扩散法检测其对12种抗生素的敏感性；用双纸片法（D试验）确定大环内酯类耐药表型；用PCR扩增检测耐药基因ermB、mefE和Tn1545、Tn917中ermB基因调控序列，经测序、BLAST比对分析调控序列多态性并按菌株携带Tn1545和Tn917的不同分为Tn1545组和Tn917组，分析二者介导的耐药表型的不同；用BOX—PCR分析不同菌株遗传背景。结果（1）86株红霉素耐药肺炎链球菌中ermB、mefE、Tn1545、Tn917检出率分别为94％、46％、87％、7％，基因型为ermB^- mefE^＋的5个菌株未检出Tn1545和Tn917。（2）Tn1545组和Tn917组Sp大多对红霉素高度耐药（最低抑菌浓度MIC50 256μg／ml），Tn917组对β内酰胺类抗生素的MIC低于Tn1545组，对四环素、复方磺胺及左氧氟沙星耐药率前者亦低于后者。（3）Tn917组中三株菌发生ermB基因启动子区194bp片段的删除和前导肽N末端TAAA认插入，可能导致ermB基因由诱导型转变成组成型表达；Tn1545组菌株主要表现为cMLSB表型。（4）BOX—PCR图谱呈散发性，未发现单一的耐药克隆株流行。结论上海地区大环内酯类耐药肺炎链球菌主要是由ermB基因介导的高水平、组成型耐药，ermB基因大多由Tn1545携带并水平转移传播。     

幽门螺杆菌的耐药性分析

目的:分析幽门螺杆菌(Hp)菌株对阿莫西林、克拉霉素、甲硝唑、呋喃唑酮的耐药情况,并探讨Hp对克拉霉素耐药与23S rRNA基因点突变的关系.方法:分离培养Hp,药敏纸片法进行药敏实验,按酚-氯仿法提取Hp的DNA,PCR方法扩增23S rRNA基因中的片段,用限制性片段长度多态性(PCR-RFLP)检测克拉霉素耐药菌株的点突变.结果:Hp菌株对阿莫西林、克拉霉素、甲硝唑、呋喃唑酮的耐药率分别为9.3%、18.6%、53.5%、0%;8个克拉霉素耐药的Hp菌株有7个存在23S rRNA基因的A2143G点突变,进行PCR-RFLP的29个敏感菌株均无23S rRNA的点突变.结论:Hp菌株对甲硝唑耐药率高,对克拉霉素、阿莫西林和呋喃唑酮敏感;23S rRNA基因A2143G点突变与Hp对克拉霉素的耐药相关.     

志贺菌主动外排泵acrAB-tolC及调控基因marOR突变与耐药的关系

目的：研究志贺菌的耐药状况,检测临床分离志贺菌主动外排泵acrAB-tolC基因及调控基因marOR的突变情况,并探讨其与耐药性的关系。方法：对159株临床分离的志贺菌进行四环素、氯霉素、氨苄青霉素、庆大霉素、诺氟沙星、复方新诺明6种药物的药敏试验和有机溶剂耐受试验,对其acrAB-tolC基因和marOR基因进行聚合酶链反应（PCR）扩增,并进行限制性内切酶酶切与单链构象多态性（SSCP）分析。结果：159株临床分离的志贺菌属细菌筛选出4株敏感株、18株耐单药株和137株耐多药株,耐多药率为86.2%(137/159);159株中有122株对有机溶剂耐受,耐受率为76.7%(122/159);159株均扩增出acrAB-tolC基因和marOR基因,未发现基因缺失株;单链构象多态性分析,155株耐药菌株中,marOR基因的突变率为17.4%,acrA基因的突变率为5.8%,acrB基因的突变率为3.9%,tolC基因的突变率为2.6%;4株敏感株未发现marOR、acrA、acrB和tolC基因突变。结论：临床分离的志贺菌耐多药率和有机溶剂耐受率均较高,且存在较高的marOR基因突变率。     

The action of Pseudomonas aeruginosa biofilms in intrinsic drug resistance

Background There is a growing interest in studying the relationship between intrinsic resistance and biofilms resistance to drugs. However, the relationship still remains unclear in the macroscopic bacterial growth. Our study is to illuminate the change of bacterial drug resistance of gyrA mutant and active efflux pump during the development of Pseudomonas aeruginosa （ P. aeruginosa ） biofilms. Methods The strains of typeⅡ topoisomerase gene mutant （gyrA mutant） and multidrug resistance （MDR） efflux pump were clinical isolates and detected by polymerase chain reaction （PCR）. The process of bacterial biofilms development was observed by scanning electron microscope. Triparental mating experiments were performed to transfer report gene of green fluorescent protein （GFP） into P. aeruginosa biofilms strains and followed by analysis of bacterial survival rate between intrinsic resistance and biofilms resistance. Results The fluorescent strains with pGFPuv could develop mature biofilms on Teflon surface. Before a period of 72 hours, the survival rate of biofilms bacteria and intrinsic resistance strains in ciprofloxacin solution was significantly different （ P 〈 0.05）. The survival number of intrinsic resistance strains （gyrA mutation and active efflux pump） was illustriously higher than biofilm strain in the initial stage of biofilms development. After 72 hours incubation, there was no clearly difference between mutants and biofilms strains in the survival rate （P 〉 0.05）. The carbonyl cyanide m-chlorophenylhydrazone and azithromycin could significantly reduce the drug resistance of biofilm strains and efflux pump strains. Conclusions In the development of P. aeruginosa biofilms, the strains of gyrA mutation and MDR efflux could be conferred with new level of drug resistance. When co-cultured mutated strains with biofilm strains, biofilms may play a major role in bacterial resistance. But after 72 hours incubation （ a mature biofilms had been developed） , there was no clearly difference between the number of mutant strains and biofilm strains.     

泵抑制剂对嗜麦芽寡养单胞菌氟喹诺酮主动外排表型特征的影响

目的:探讨主动外排泵在嗜麦芽寡养单胞菌对氟喹诺酮类药物耐药中的作用。方法:用泵抑制剂氰氯苯腙(CCCP)和利血平对5种氟喹诺酮类药物抗菌活性进行干预。采用琼脂二倍稀释法检测泵抑制剂存在时,嗜麦芽寡养单胞菌对莫西沙星、加替沙星、左氧氟沙星、洛美沙星、环丙沙星敏感性的变化。结果:主动外排泵不仅存在于耐药菌株,而且也存在于敏感菌株,但对耐药菌株影响更大。与泵阴性组比较,泵阳性组敏感率降低在左氧氟沙星、洛美沙星、环丙沙星组有统计学差异;耐药率增加在洛美沙星、环丙沙星组有统计学差异。结论:主动外排泵抑制剂CCCP和利血平可增强氟喹诺酮类药物对嗜麦芽寡养单胞菌的抗菌活性,对其耐药表型有影响。主动外排机制是嗜麦芽寡养单胞菌对氟喹诺酮类药物耐药的原因之一。     

Macrolide resistance among invasive Streptococcus pneumoniae isolates

CONTEXT: Macrolide antibiotics, including erythromycin, clarithromycin, and azithromycin, are the mainstays of empirical pneumonia therapy. Macrolide resistance among Streptococcus pneumoniae, the most common cause of community-acquired pneumonia, is increasing in the United States. Whether resistance is a significant problem or whether macrolides remain useful for treatment of most resistant strains is unknown. OBJECTIVE: To examine the epidemiology of macrolide-resistant pneumococci in the United States. DESIGN AND SETTING: Analysis of 15 481 invasive isolates from 1995 to 1999 collected by the Centers for Disease Control and Prevention's Active Bacterial Core surveillance system in 8 states. MAIN OUTCOME MEASURES: Trends in macrolide use (1993-1999) and resistance and factors associated with resistance, including examination of 2 subtypes, the M phenotype, associated with moderate minimum inhibitory concentrations (MICs), and the MLS(B) phenotype, associated with high MICs and clindamycin resistance. RESULTS: From 1993 to 1999, macrolide use increased 13%; macrolide use increased 320% among children younger than 5 years. Macrolide resistance increased from 10.6% in 1995 to 20.4% in 1999. M phenotype isolates increased from 7.4% to 16.5% (P<.001), while the proportion with the MLS(B) phenotype was stable (3%-4%). The median erythromycin MIC (MIC(50)) of M phenotype isolates increased from 4 microg/mL to 8 microg/mL. In 1999, M phenotype strains were more often from children than persons 5 years or older (25.2% vs 12.6%; P<.001) and from whites than blacks (19.3% vs 11.2%; P<.001). CONCLUSIONS: In the setting of increasing macrolide use, pneumococcal resistance has become common. Most resistant strains have MICs in the range in which treatment failures have been reported. Further study and surveillance are critical to understanding the clinical implications of our findings.     

黄柏提取物抗耐大环内酯类药物肺炎支原体的实验研究

目的 探讨黄柏提取物体外对大环内酯类药物耐药肺炎支原体(MP)的抗菌活性,并分析黄柏提取物对其耐药相关基因MPN421 mRNA表达的影响,通过检测黄柏提取物作用前后MPN421 mRNA表达改变情况,初步阐述黄柏提取物抗MP的相关分子机制。 方法 用实时荧光定量PCR鉴定45例MP临床分离株对大环内酯类药物耐药突变(23S rRNA区域),鉴定肺炎支原体的突变株(23S rRNA 2063点突变)和野生株;体外药物敏感试验检测45株肺炎支原体对黄柏提取物的最小抑菌浓度(MIC);实时荧光PCR检测黄柏提取物作用前后对MP MPN421 mRNA表达改变。 结果 45例MP临床分离株中发现30株23S rRNA 发生突变(突变株),15株MP 未发生突变(野生型);15株MP野生株的MIC值为0.036~0.576 mg/ml,30株肺炎支原体突变株的MIC值为0.036~1.152 mg/ml。15株野生株及30株突变株在无黄柏提取物作用下MPN421 mRNA相对表达量分别为0.079(0.071,0.089),0.083(0.062,0.084),P>0.05;黄柏提取物作用6 h和9 h后,无论是野生株还是突变株的MPN421 mRNA相对表达量均显著下降(均P<0.01)。 结论 MP野生株及突变株对黄柏提取物均敏感,其机制可能是降低MP MPN421 mRNA表达,对大环内酯类药物耐药MP感染有潜在治疗价值。