铜绿假单胞菌对亚胺培南耐药机制的研究

铜绿假单胞菌是引起医院内感染的主要病原菌。亚胺培南(IMP)作为革兰阴性菌的最有效的抗生素，随着青霉烯类抗生素的广泛应用，铜绿假单胞菌的耐药菌株逐渐增多，给临床治疗带来困难。这与铜绿假单胞菌的主动外排机制以及特异性膜蛋白OprD2的缺失有关，能够水解碳青霉烯类抗生素的新β-内酰胺酶一金属酶的产生，对碳青霉烯类抗生素的使用造成了更大威胁。本研究分析耐亚胺培南铜绿假单胞菌产生金属酶合并外膜蛋白OprD2的缺失。     

铜绿假单胞菌oprD2基因突变及表达量改变与碳青霉烯类耐药的关系

目的 研究铜绿假单胞菌外膜通道蛋白OprD2的表达减弱或缺失,以及OprD2蛋白自身突变是否会影响铜绿假单胞菌对碳青霉烯类药物的耐药性.方法 收集分离自临床对亚胺培南(IPM)的最低抑菌浓度(MIC)值≥8μg/m1的铜绿假单胞菌共101株,采用肉汤稀释法检测菌株对比阿培南(BPM)、美罗培南(MEM)、帕尼培南(PEM)的MIC值;荧光定量RT-PCR检测铜绿假单胞菌膜通道蛋白oprD2基因的表达量情况;针对oprD2相对表达量正常并对亚胺培南耐药的铜绿假单胞菌,采用普通PCR的方法扩增oprD2全长基因并测序.结果 根据铜绿假单胞菌的OprD2蛋白相对表达量结果,将101株铜绿假单胞菌分成两组,组1为OprD2相对表达量降低组;组2为OprD2相对表达量正常组;组1与组2相比,对IPM、BPM、MEM、PEM的MIC值≥16μg/ml的菌株比例差异均有统计学意义(P＜0.01).组1中,28株同时对4种药物的MIC均≥16 μg/ml,其中有25株的OprD2的相对表达量明显减低(＜0.4);外膜孔蛋白OprD2转录水平与4种碳青霉烯类药物MICs之间呈负相关趋势.组2中,有16株9prD2基因发生突变,按照突变的类型主要分成4组;与PAO1相比,这些菌株对IPM、BPM、MEM、PEM的MIC值有不同程度的增加.结论 OprD2外膜蛋白的表达量减少或缺失是铜绿假单胞菌对亚胺培南耐药的主要机制,可能也与其他3种碳青霉烯类药物耐药有密切关系;铜绿假单胞菌的oprD2基因发生突变,可能会降低铜绿假单胞菌对这儿种碳青霉烯类药物的敏感性.     

汕头地区耐亚胺培南铜绿假单胞菌耐药分析

目的：了解汕头地区对亚胺培南耐药的铜绿假单胞菌（PA）的耐药情况及耐药机制。方法：收集临床分离耐亚胺培南的PA共141株，双纸片协同实验检测金属酶表型，PCR法检测外膜孔蛋白OprD2和金属β内酰胺酶（IMP、VIM、SPM）基因。结果：耐亚胺培南的铜绿假单胞菌均为多重耐药茵，对头孢哌酮／舒巴坦的耐药率较低，未发现产金属酶菌株，仅22株菌株扩增出OprD2基因。结论：头孢哌酮／舒巴坦可作为本地区临床治疗耐亚胺培南铜绿假单胞茵所致感染的首选经验用药，OprDa表达减少或不表达可能是临床分离铜绿假单胞茵对亚胺培南耐药的主要机制。     

老年人亚胺培南耐药铜绿假单胞菌耐药性研究

目的 明确我院老年病人临床分离铜绿假单胞菌的耐药性、同源性及耐碳青霉烯菌株的基因型。方法 收集我院2006年5月-2009年5月自临床老年病人分离的262株铜绿假单胞菌,纸片扩散法测定其对16种抗菌药物的耐药性;琼脂稀释法和E test法测定耐碳青霉烯菌株对14种抗菌药物的MIC值,PCR扩增及克隆测序分析金属酶基因型。脉冲场凝胶电泳(PFGE)分析携带金属酶基因型菌株的同源性。结果 262株铜绿假单胞菌中筛选到104株耐碳青霉烯。104株耐碳青霉烯铜绿假单胞菌对氨苄西林/舒巴坦、头孢哌酮/舒巴坦两个含舒巴坦制剂药物耐药率分别为78.9％和35.9％,对多黏菌素E耐药率最低为6.0％,对米诺环素耐药率58.3％,其余抗菌药物耐药率均大于70.0％;104株亚胺培南耐药铜绿假单胞菌中12株携带金属酶基因,10株检测到有携带VIM-2基因的1类整合子。PFGE分型中12株菌株属于5个克隆株。结论 在我院流行的亚胺培南耐药铜绿假单胞菌中,金属酶基因不是最主要的基因型,金属β-内酰胺酶均为VIM-2型金属酶,耐药基因盒分布于不同的1类整合子中,整合子播散是最主要的流行方式。     

社区医疗机构多重耐药铜绿假单胞菌产金属β内酰胺酶检测

目的 了解多重耐药铜绿假单胞菌(M RPA)的社区感染情况及产金属β内酰胺酶(MBL)情况.方法 2011年1月至12月在广州市多个社区医院收集分离出MRPA 54株.采用MicroScan Walk Away 40S1全自动细菌鉴定与药敏检测系统检测其对亚胺培南等11种抗菌药物的耐药性,比较亚胺培南耐药铜绿假单胞菌与亚胺培南敏感铜绿假单胞菌对常用9种抗菌药物的耐药率.改良三维实验分析亚胺培南耐药铜绿假单胞菌产MBL情况.结果 54株MRPA中有29株对亚胺培南耐药,占53.7％( 29/54).三维试验显示有9株亚胺培南耐药铜绿假单胞菌产MBL,占31.0％( 9/29).亚胺培南耐药铜绿假单胞菌对常用7种抗菌药物的耐药率显著高于亚胺培南敏感铜绿假单胞菌(头孢他啶:65.5％比36.0％;头孢噻肟:89.7％比44.0％;头孢曲松:100.0％比52.0％:氨曲南:72.4％比36.0％,氧氟沙星:70.0％比32.0％;哌拉西林-他唑巴坦:58.6％比16.0％;阿米卡星:62.1％比32.0％,均P＜0.05).结论 社区医疗机构临床分离MRPA 产MBL的比率较高.     

河源地区铜绿假单胞菌碳青霉烯耐药机制分析

目的研究本院铜绿假单胞菌耐碳青霉烯类抗生素的耐药现况及主要耐药机制。方法用E-test方法检测铜绿假单胞菌对哌拉西林、头孢他啶、亚胺培南、美洛培南、庆大霉素、妥布霉素、环丙沙星7种抗生素的最小抑菌浓度,用EDTA双纸片扩散法及三维实验分别对金属酶及AmpC、KPC酶表型进行确证。结果从1 068例致病菌中共分离出108例铜绿假单胞菌,18例是对亚胺培南和/或美罗培南不敏感的菌株,耐药率为16.7%,其中有9例金属酶确证实验阳性,3例为AmpC酶持续高产型菌株,KPC酶确证实验尚没有检测出阳性菌株。结论耐碳青霉烯类铜绿假单胞菌多表现为多重耐药,这是多因素共同作用的结果 。     

对碳青霉烯类耐药的铜绿假单胞菌的金属β-内酰胺酶研究

目的：研究耐碳青霉烯类的铜绿假单胞菌产金属β-内酰胺酶的情况及铜绿假单胞菌的耐药机制。方法：微量二倍稀释法测定亚安培南及美洛培南对铜绿假单胞菌的MIC值；DETA纸片法检测产金属β-内酰胺酶的铜绿假单胞菌。结果：从62株临床分离的铜绿假单胞菌中共检测到28株对亚胺培南耐药，耐药率为45％，18株对美洛培南耐药，耐药率为29．03％。从18株同时对亚胺培南及美洛培南耐药株中共检测到16株产金属β-内酰胺酶，占同期分离铜绿假单胞菌的25．8％。结论：产生金属β-内酰胺酶是铜绿假单胞菌对碳青霉烯类药物耐药的机制之一。     

10-23脱氧核酶抑制铜绿假单胞菌耐药基因VIM2作用的初步研究

目的 探讨10-23脱氧核酶(DRz)抑制铜绿假单胞菌耐药基因VIM2的表达及其在逆转铜绿假单胞菌耐药中的作用.方法 根据计算机模拟的 VIM2 mRNA 的二级结构设计合成 5 条VIM2特异的10-23DRz(DRz1～DRz5),在无细胞反应体系中鉴定10-23DRz对VIM2 mRNA的切割活性后,将其导入铜绿假单胞菌,利用E-test法检测亚胺培南对处理前后铜绿假单胞菌的MIC值.结果 DRz3、DRz4和DRz5在无细胞反应体系中可对VIM2 mRNA进行有效切割,且其活性具有高度特异性.与对照相比,1O-23DRz可以降低亚胺培南对铜绿假单胞菌的MIC值.结论 实验中所设计的10-23DRz能在细胞外高效特异性的切割VIM2 mRNA;在细胞内能协同业胺培南抑制铜绿假单胞菌耐药.     

鲍曼不动杆菌和铜绿假单胞菌临床分离株对碳青霉烯抗生素和头孢他啶的耐药性和金属β内酰胺酶的检测

目的 :了解我院鲍曼不动杆菌和铜绿假单胞菌对碳青霉烯和头孢他啶的耐药性以及与金属 β内酰胺酶的关系。方法 :对 2 0 0 2年 4月～ 2 0 0 3年 1月我院临床分离的 1 0 1株鲍曼不动杆菌以及 2 0 0 3年 2月～ 4月我院临床分离的以MicroScanWalkAway～ 40全自动鉴定药敏仪确定的对亚胺培南和 (或 )对头孢他啶耐药的 69株鲍曼不动杆菌和 2 3株铜绿假单胞菌采用美国国家临床实验室标准委员会 (NCCLS)推荐的琼脂对倍稀释法测定了对亚胺培南、美罗培南和头孢他啶的最低抑菌浓度 (MICs) ,将对亚胺培南耐药和(或 )对头孢他啶高度耐药 (MIC >1 2…     

2014~2018年某三甲医院产生物膜铜绿假单胞菌耐药性及患者临床特征分析

目的 分析某三级甲等医院分离的产生物膜铜绿假单胞菌耐药性及患者的临床特征。方法 收集2014~2018年某三甲医院临床分离的产生物膜铜绿假单胞菌,进行菌种鉴定、抗菌药物敏感性试验并对药物的耐药率进行分析;收集患者的临床资料,并对感染患者临床特征进行分析。结果 2014~2018年临床分离的423株产生物膜铜绿假单胞菌均来自下呼吸道标本,来源以呼吸内科为主,年龄＞60岁、合并有支气管扩张等肺部基础疾病的患者易引起产生物膜铜绿假单胞菌的感染;2015~2018年产生物膜铜绿假单胞菌对亚胺培南、美罗培南的耐药率逐年下降。结论 产生物膜铜绿假单胞菌对碳青霉烯类抗生素及氨基糖苷类抗生素耐药率较低,低于非产生物膜的铜绿假单胞菌,临床微生物学实验室应选用可靠的药敏方法和合适的培养时间,为临床医生提供准确的药敏结果。     

志贺菌临床分离多重耐药株mar基因突变研究

目的 研究志贺菌mar基因突变与其多重耐药的关系.方法 对54株临床分离的志贺菌进行四环素、氯霉素、氨苄西林、环丙沙星、诺氟沙星等5种药物的药敏试验和有机溶剂耐受实、验(OST),对其,mar基因的marOR区进行聚合酶链反应-单链构象多态性分析(PCR-SSCP)及核酸序列分析.结果 筛选出35株多重耐药株,多重耐药率为64.8%.54株志贺菌株中有38株对有机溶剂耐受,其中有33株为多重耐药株,3株为单耐药株,仅有2株为敏感野生株.54株志贺菌临床分离株均扩增出MAR基因,未发现该基因缺失株,SSCP分析发现,35株多重耐药株中marOR基因突变率为14.29%.核酸测序分析发现志贺菌多重耐药株marO区基因1376-1379处4个碱基缺失,导致其后编码氨基酸出现移码突变,而标准株S51250和敏感野生株不存在该移码突变;marR区基因存在10处点突变,第1752碱基(G→A,Gly→Ser)和第1854碱基(T→c,Tyr→His)两处点突变导致编码氨基酸改变,但该突变也同时存在于标准株和敏感野生株中,余者均为无义突变,且有些核苷酸改变也发生于敏感野生株或同时存在于多株菌株中.结论 ,marO区基因突变可能是志贺菌多重耐药的调控机制之一.     

体内诱导耐碳青霉烯类鲍曼不动杆菌膜蛋白质组学研究

目的 研究膜蛋白在耐碳青霉烯类抗生素鲍曼不动杆菌中的作用.方法 从同一住院病人体内分别收集碳青霉烯类敏感和耐药的鲍曼不动杆菌各1株.经多序列位点测序分型(MIST)和细菌基因组外回文结构重复序列分型(REP-PCR)分析后,等电聚焦电泳检测其已知碳青霉烯类水解酶的表达,在此进行膜蛋白二维电泳和质谱鉴定分析,并最后应用PABN(Phe-Arg-β-naphthylamide)外排泵抑制剂检测其外排泵相关膜蛋白表达.结果 MIST和REP-PCR结果表明耐药株来源与敏感菌株属于同一型别;等电聚焦电泳在P17.6和P19.0处两株菌中检测到β-内酰胺酶,没有检测到任何已知的碳青霉烯类水解酶;耐约株和敏感株的差异膜蛋白组学鉴定出相对分子质量(M_r)为34×10~3外排泵膜蛋白和0prD与CarO膜孔蛋白,且后续的外排泵抑制剂试验表明,在PAβN存在的情况下,耐药株的亚胺培南最低抑菌浓度(MIC)由大于32 μg/ml下降到8 μg,/ml.结论 本研究发现外排泵膜蛋白的过度表达伴随OprD和CarO膜孔蛋白的下调是临床分离耐碳青霉烯类抗生素鲍曼不动杆菌主要耐药机制.     

Antibiotic- and metal-resistant strains of Vibrio parahaemolyticus isolated from shrimps

Enteropathogenic strains of Vibrio parahaemolyticus were isolated from shrimps, Penaeus monodon collected from the region of the Deltaic Sundarbans (West Bengal, India). About 63% of the isolated strains were resistant to ampicillin, cephalexin, and kanamycin. However, all these strains were sensitive to nitrofurantoin, nalidixic acid, tetracycline, and norfloxacin. The isolated strains were resistant to Ni2+] (75%), Cu2+ (87%), and Co2+ (37%), but all the strains were resistant against Cd2+, Zn2+, and Pb2+ at 10 mM concentration.     

大肠埃希菌尿液分离株中检出gyrA基因新变异株

目的 调查大肠埃希菌尿液分离株中喹诺酮类耐药相关基因的存在与变化状况.方法 收集宁波市第一医院2008年10月到2009年3月患者尿液标本中分离的大肠埃希菌共28株,采用聚合酶链反应(PCR)及序列分析的方法分析1种染色体介导的喹诺酮类耐药相关基因(gyrA基因)和5种质粒介导的喹诺酮类耐药相关基因[qnrA、qnrB、qnrS、aac(6＇)-Ⅰb、qepA].结果 28株大肠埃希菌检测到1株aac(6＇)-Ⅰb-Cr基因阳性株(经测序比对证实),qnrA、qnrB、qnrS、qepA基因均未检出.gyrA基因83位密码子28株菌都有突变(100.0%),其突变方式为TCG-83→HTG,导致氨基酸从丝氨酸(S)-83→亮氨酸(L);87位密码子22株菌(78.6%)有突变,可分为两种突变方式:21株(75.0%)突变方式为GAC-87→AAC,导致氨基酸从天冬氨酸(D)-87→天冬酰胺(N);5号株gyrA基因(3.6%)为新亚型,其突变方式为GAC-87→TAC,导致氨基酸从天冬氨酸(D)-87→脯氨酸(Y),另6株菌87位密码子无突变.结论 本组大肠埃希菌gyrA基因突变率为100.0%,是喹诺酮类耐药的主要原因.其他耐药相关基因阳性率很低.     

Detecting drug resistant genetic mutation among pneumoconiosis patients complicated with tuberculosis in Mycobacterium tuberculosis L-forms application of PCR-SSCP technique in Huainan mining district

Objective: To study the relationship between drug resistant genetic mutation and drug resistance in Mycobacterium tuberculosis L-form, discuss the internal relationship between drug resistances and drug-resistant related genes and explore the value of PCRSSCP to clinical application. Methods: A total of 52 clinically isolated strains of tuberculosis L-form were collected among 97pneumoconiosis patients complicated with tuberculosis. The gene mutations of katG, rpoB and rpsL were detected by PCR-SSCP,and the results were compared with those analyzed by traditional antimicrobial susceptibility test(AST). Results: The gene mutation rates of katG, rpoB and rpsL by PCR-SSCP were respectively 57.70% (30/52), 65.38% (32/52) and 40.38% (21/52). The rate of reversion was 78.85%(41/52) and the result of drug-resistant genes was invariable. The results of AST showed that there were 40 (76.92%) multi-drug resistant strains in 52 clinically isolated strains. The number for three-drug resistant strain was 21(40.38%) and that of two-drug resistant was 19(36.54%), but only 12 (23.08%) strains were one drug resistant. The rate of total drug-resistance was 100%, but there were 15 strains of allied mutation of three genes, 16 of two mutations and 6 of only one by PCR-SSCP. The coincidences were respectively 71.43%, 84.12% and 50.00%. Then there was no significant difference between the allied mutations of multi-drug resistant gene and the mutations of only one drug resistant gene (P ＞ 0.05). Conclusion: PCRSSCP technique has a higher sensibility and specificity to detect the genes of katG, rpoB and rpsL in tuberculosis L-form among pneumoconiosis complicated with tuberculosis,and the detecting rate of two drug resistant strains and three drug resistant strains was higher. The combined application of PCR-SSCP and AST has advantages at earlier diagnosis and guidance of clinical medications.     

体外构建喹诺酮耐药肺炎克雷伯菌模型及gyrA、parC基因突变与耐药性关系

目的 体外构建喹诺酮耐药肺炎克雷伯菌模型并研究其gyrA、parC基因突变与唪诺酮耐药的关系.方法 运用亚抑菌浓度的左旋氧氟沙星与肺炎克雷伯菌在培养基中共培养,并倍比增加诱导药物的浓度,直至肺炎克雷伯菌能够在含高浓度的左旋氧氟沙星培养基上良好生长.收集耐药菌株,用PCR扩增和DNA测序法榆测gyrA、parC基因喹诺酮耐约决定区域(QRDR)的突变情况.结果 体外成功构建喹诺酮高浓度耐药肺炎克雷伯菌模型,所有耐药菌株QRDR均发生了变异,绝大部分为gyrA和parC双重突变.gyrA基因以Ser83、Asp87突变为主,Ser83→Ile,Asp87→Arg、Gly,也有菌株86位氨基酸发生了突变,由Tyr86→Ser.几乎所有菌株parC基因80位氨基酸的突变均为Ser80→Ile.结论 长期低浓度用药可以诱导喹诺酮耐药性的产生.QRDR突变与喹诺酮耐药性有关,gyrA及parC的双重突变可以导致高水平耐药,同时町能存在其他耐药机制.     

Novel mutations in the promoter and coding region of the human 5-HT1A receptor gene and association analysis in schizophrenia

Dysfunction of serotonin systems has been implicated in schizophrenia. In the present study, the human 5-HT_1A receptor gene containing the 5′ untranslated region was screened in order to detect genetic variations, through which alteration of protein function or level of expression might contribute to schizophrenia. Genomic DNAs were isolated from whole-blood samples of 61 unrelated schizophrenic patients and 100 healthy controls. Genetic variations were screened systematically by single-strand conformational polymorphism (SSCP) analysis, followed by direct sequencing of polymerase chain reaction (PCR) product as well as restriction fragment-length polymorphism (RFLP). The novel mutations (−51T → C, −152C → G, −321G → C, −480delA, and −581C → A) were found in the 5′ untranslated region. Furthermore, we found a novel missense mutation (Gly272Asp) in the coding region in addition to the mutations (Pro16Leu, 294G → A, and 549C → T) reported previously. No significant differences in genotype frequencies as well as allele frequencies were found between patients and controls. Our data provided no evidence of association between schizophrenia and the variants in the 5′ untranslated region as well as the coding region of the human 5-HT_1A receptor gene. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81:434–439, 1998. © 1998 Wiley-Liss, Inc.     

Systematic screening for mutations in the promoter and the coding region of the 5-HT1A gene

In the present study we sought to identify genetic variation in the 5-HT_1A receptor gene which through alteration of protein function or level of expression might contribute to the genetic predisposition to neuropsychiatric diseases. Genomic DNA samples from 159 unrelated subjects (including 45 schizophrenic, 46 bipolar affective, and 43 patients with Tourette's syndrome, as well as 25 healthy controls) were investigated by single-strand conformation analysis. Overlapping PCR (polymerase chain reaction) fragments covered the whole coding sequence as well as the 5′ untranslated region of the 5-HT_1A gene. The region upstream to the coding sequence we investigated contains a functional promoter. We found two rare nucleotide sequence variants. Both mutations are located in the coding region of the gene: a coding mutation (A→G) in nucleotide position 82 which leads to an amino acid exchange (Ile→Val) in position 28 of the receptor protein and a silent mutation (C→T) in nucleotide position 549. The occurrence of the Ile-28-Val substitution was studied in an extended sample of patients (n = 352) and controls (n = 210) but was found in similar frequencies in all groups. Thus, this mutation is unlikely to play a significant role in the genetic predisposition to the diseases investigated. In conclusion, our study does not provide evidence that the 5-HT_1A gene plays either a major or a minor role in the genetic predisposition to schizophrenia, bipolar affective disorder, or Tourette's syndrome. © 1995 Wiley-Liss, Inc.