致泻性大肠埃希菌的分布及耐药特点研究

目的 监测致泻性大肠埃希菌(diarrheagenic Escherichia coli,DEC)的分布特点及耐药趋势,为流行病学研究、疫苗制备及合理使用抗生素提供依据.方法 收集2002-2010年我院肠道门诊腹泻患者的大便标本,沙门-志贺菌培养基培养,筛出可疑菌落后经生化及血清学试验进一步鉴定血清型,用纸片扩散法测定抗菌药物的灵敏性.结果 共分离肠道病原菌2104株,其中DEC 118株(5.61%),包括致病性大肠埃希菌(enteropathogenic E.coli,EPEC)78株(66.10%),侵袭性大肠埃希菌(enteroinvasive E.coli,EIEC)29株(24.     

多重耐药菌的抗菌治疗

多重耐药菌是感染控制的严峻挑战.本文对临床重要的多重耐药菌包括鲍曼不动杆菌、铜绿假单胞菌、产超广谱β-内酰胺酶肠杆菌和产碳青霉烯酶肠杆菌的抗菌治疗进行综述.     

血流感染产超广谱β-内酰胺酶大肠埃希菌耐药性及临床特征分析

目的 分析血流感染产超广谱β-内酰胺酶(extended spectrumβ-lactamases, ESBLs)大肠埃希菌的耐药性及临床特点, 为临床合理应用抗菌药物提供依据. 方法 对我院疑为血流感染患者的血液用BacT/Alert全自动血培养仪细菌培养,用VitekⅡ细菌鉴定仪进行细菌鉴定,采用Kirby-Bauer纸片进行药物敏感性试验和ESBLs的筛选及确认. 对ESBLs阳性病例临床特点进行分析. 结果 2009—2012年我院从血流感染病例中共分离获得大肠埃希菌235株,产ESBLs为90株,阳性率38.3%. 产酶菌株对氨苄西林、头孢噻肟和头孢曲松耐药率为100%;对亚胺培南/西司他丁敏感率为100%,对美罗培南、头孢美唑和阿米卡星敏感率超过90%;对含有β-内酰胺酶抑制剂的复合抗生素头孢哌酮/舒巴坦、替卡西林/克拉维酸和哌拉西林/他唑巴坦的耐药率分别为6.7%、29.2%和34.4%.感染者中,50岁以上占57.8%.感染发生时间多出现在入院后1周内,占58.9%.原发感染来自自发性细菌性腹膜炎者占46.7%.出现严重并发症患者35例,其中20例医治无效或死亡,占57.1%. 结论 血流感染产ESBLs大肠埃希菌患者病情危重,预后不良,临床用药时应密切结合患者的病情和感染特征及感染菌的耐药表型等综合情况,制定合理的个体化治疗措施.     

婴幼儿大肠埃希菌性肺炎177例临床分析

目的分析婴幼儿大肠埃希菌性肺炎患儿的临床特征及药敏情况,指导临床肺炎诊治。方法回顾性分析177例住院婴幼儿大肠埃希菌性肺炎患儿的临床表现、X线表现、细菌耐药情况及治疗预后。结果患儿的临床表现主要的症状为咳嗽、咳痰、发热,容易出现呼吸困难。 X线主要表现为片状影或间质渗出。大肠埃希菌多数菌株对泰能、美平、头孢他定、丁胺卡那霉素、左氧氟沙星、哌拉西林/他唑巴坦、头孢哌酮/舒巴坦敏感性高,对大多数β内酰胺类抗生素普遍耐药。产超广谱β内酰胺酶（ ESBLs ）菌33例,阳性率为18.6％。痰液培养细菌阳性转阴比较慢,治疗主要以经验疗法和药敏试验结果结合,病程迁延,预后尚好。结论婴幼儿大肠埃希菌肺炎是儿科常见病,临床诊治相对容易。但大肠埃希菌抗菌药物的耐药率高,需引起重视。     

大肠埃希菌和肺炎克雷伯菌耐药性和耐药质粒谱分析

目的研究大肠埃希菌(Escherichiacoli)和肺炎克雷伯菌(Klebsiella peumoniae)临床分离株耐药性及其质粒谱变化。方法对福建医科大学2所附属医院分离的E.coli和K.peumoniae共53株行药敏试验,检出产超广谱β-内酰胺酶(ESBLs)的菌株用碱变性法提取质粒,采用0.8%琼脂糖凝胶电泳分析质粒谱。结果产ESBLs菌株对多数青霉素和头孢菌素类抗生素耐药,对碳青霉烯类和酶抑制剂敏感(P0.05)。不产ESBLsE.coli菌对喹诺酮类的敏感性高于产酶菌株(P0.01)。受检菌株对丁胺卡那霉素的敏感性较高。产ESBLs菌株60.0%检出质粒,其中50%携带0.9 kb质粒。对环丙沙星耐药的E.coli,50%携带4.2 kb质粒;4株对丁胺卡那霉素耐药的E.coli中,2株携带9.4kb质粒。结论E.coli和K.peumoniae产ESBLs主要由质粒介导。0.9 kb质粒可能与产ESBLs有关;4.2 kb的质粒可能与环丙沙星耐药有关;9.4 kb质粒可能与丁胺卡那霉素耐药有关。     

大肠埃希菌对含酶抑制剂药物耐药性分析

检测216株大肠埃希菌，超广谱β-内酰胺酶（ESBLs）阳性68株，阳性率为31．5％。大肠埃希菌对含酶抑制剂复合剂的耐药性不同，非产生ESBLs菌株的耐药率均较低。     

外排泵抑制剂对幽门螺杆菌多重耐药性的影响

目的:探索多种外排泵抑制剂对H.pylori多重耐药性的影响.方法:临床分离培养H.pylori,对分离敏感株进行氯霉素耐药性诱导,筛选出多重耐药(MDR)株;琼脂二倍稀释法对敏感菌株和MDR株进行9种抗生素的药敏试验,分别加入外排泵抑制剂碳酰氰基-对-氯苯腙(CCCP)、利血平、泮托拉唑后再次进行药敏试验,比较分析外排泵抑制剂对H.pylori耐药性的影响;同样方法检测泮托拉唑、雷贝拉唑、奥美拉唑、兰索拉唑和埃索美拉唑对MDR-H.pylori株抗生素最小抑菌浓度(MIcs)值的影响.结果:诱导出4株MDR-H.pylori;CCCP及泮托拉唑能够部分恢复MDR-H.pylon株对抗生素的敏感性,而利血平对MDR-H.pylori耐药性无明显影响;这三种外排泵抑制剂对敏感株的MICs值影响不大;5种质子泵抑制剂中,雷贝拉唑能使甲硝唑和阿莫西林的MIC值分别降低至1/4和1/3,泮托拉唑能使其均降低至1/2左右.结论:外排泵抑制剂能够在体外提高多重耐药H.pylori株的药物敏感性;在所有质子泵抑制荆中雷贝拉唑对降低MDR-H.pylori对抗生素的耐药性作用最强,其次为泮托拉唑.     

内蒙古中东部地区乳源性大肠埃希菌PCR检测及其耐药性分析

目的分离牛乳源性大肠埃希菌(E.coli)并进行PCR检测及耐药性分析。方法根据GenBank上公布的牛源致病性大肠埃希菌基因序列,利用Primer5.0和DNAMAN生物信息学软件,根据该致病菌基因组序列16S～23SrRNA间隔区保守序列设计并合成1对引物,对牛乳中分离的大肠埃希菌进行基因扩增和序列测定,同时利用琼脂平板扩散方法分析阳性菌株的耐药状况。结果大肠埃希菌基因PCR扩增产物为396bp,与预期片段大小相符;测序结果与GenBank上公布的牛源大肠埃希菌(X80731.1)基因序列相似性为97.22%,与标准菌株大肠埃希菌(CVCC247)基因序列相似性为100%。分离菌株普遍对卡那霉素、氧氟沙星、氟哌酸高度敏感,对四环素、氨苄青霉素高度耐药。结论成功分离出牛乳源大肠埃希菌菌株,该组分离菌对四环素和氨苄青霉素高度耐药,为进一步建立牛乳中致病性大肠埃希菌的分子检测方法及指导临床用药提供了依据。     

院内多重耐药大肠埃希菌肺炎的耐药现状及影像学特征分析

目的 分析院内多重耐药大肠埃希菌(MDR-ECO)肺炎的耐药现状及影像学特征,并探讨发生MDR-ECO感染的危险因素.方法 选择2018-07-01～2020-06-30潍坊市人民医院经呼吸道或者血标本分离检测确认的大肠埃希菌所致院内获得性肺炎(HAP)患者178例的病历资料,根据致病菌是否为多重耐药菌将其分为MDR-...     

源自同一患者的二株血和粪便对碳青霉烯类耐药大肠埃希菌

目的 了解对碳青霉烯类抗菌药物耐药大肠埃希菌的耐药机制及与内源性感染的关系.方法 将临床分离的来源于同一患者血培养和粪便培养的大肠埃希菌2株,采用浓度梯度法(E-test)测定亚胺培南、美罗培南的最低抑菌浓度(MIC)值,纸片扩散法测定其他16种抗菌药物的敏感性,等电聚焦电泳(IEF)检测所产生的β-内酰胺酶,PCR及序列分析确定其基因型,接合试验和Southern杂交进行耐药基因定位,脉冲场凝胶电泳(PFGE)分析2株菌株的同源性.结果 亚胺培南、美罗培南对2株大肠埃希菌的MIC值均≥32 mg/L,均产KPC-2(等电点值为6.7)和SHV-12(等电点值为8.2).blaKPC-2基因位于可转移的54 kb质粒上.PFGE显示2株大肠埃希菌为同一克隆株.结论 2株大肠埃希菌的耐药性及染色体DNA酶切图谱均一致,该患者大肠埃希菌败血症极可能是肠道的大肠埃希菌移位引起的内源性感染.     

Structure of the multidrug resistance efflux transporter EmrE from Escherichia coli

Multidrug resistance efflux transporters threaten to reverse the progress treating infectious disease by extruding a wide range of drug and other cytotoxic compounds. One such drug transporter, EmrE, from the small multidrug resistance family, utilizes proton gradients as an energy source to drive substrate translocation. In an effort to understand the molecular structural basis of this transport mechanism, we have determined the structure of EmrE from Escherichia coli to 3.8 A. EmrE is a tetramer comprised of two conformational heterodimers related by a pseudo two-fold symmetry axis perpendicular to the cell membrane. Based on the structure and biochemical evidence, we propose a mechanism by which EmrE accomplishes multidrug efflux by coupling conformational changes between two heterodimers with proton gradient. Because of its simplicity and compact size, the structure of EmrE can serve as an ideal model for understanding the general structural basis of proton:drug antiport for other drug efflux systems.     

Bypassing the periplasm: Reconstitution of the AcrAB multidrug efflux pump of Escherichia coli

AcrAB is a constitutively expressed, major multidrug efflux system of Escherichia coli. We have purified the cytoplasmic membrane component, AcrB, to near homogeneity, and reconstituted the protein into proteoliposomes. In the presence of DeltapH (outside acid), the protein catalyzed the extrusion of fluorescent phospholipids, which were then trapped by protein-free acceptor vesicles. Known substrates of AcrAB, such as bile acids, erythromycin, and cloxacillin, inhibited this activity. Addition of various drugs to AcrB-containing proteoliposomes, in the presence of DeltapH (inside acid) resulted in proton efflux, suggesting that AcrB is a proton antiporter. Interestingly, fluorescent lipid extrusion was accelerated strongly by the periplasmic protein AcrA in the presence of Mg2+, and at pH 5.0 AcrA alone produced a slow mixing of lipids of different vesicles, without causing the mixing of intravesicular material. These results suggest that AcrA brings two membranes together, and under certain conditions may even cause the fusion of at least the outer leaflets of the membranes, contributing to the ability of the AcrAB-TolC system to pump drugs out directly into the medium.     

多重耐药大肠杆菌中的主动外排机制

目的研究主动外排系统在多重耐药大肠杆菌中的作用。方法用抽滤法测定了３Ｈ四环素和３Ｈ青霉素在临床分离的大肠杆菌中的积聚。结果在临床分离的多重耐药大肠杆菌中四环素和青霉素稳态浓度均显著低于敏感株;供能时稳态浓度明显下降,给予能量抑制剂后细胞内药物浓度则又显著上升,而敏感株变化不明显。ＬＭ３１３．１（大肠杆菌野生株ＡＧ１００经诱导产生的多重耐药株）对四环素的积聚及对ＣＣＣＰ的反应与临床分离多重耐药株一致,而ＬＭ３１３．５（ＬＭ３１３．１经噬菌体将Ｔｎ５插入ｍａｒＡ并使之失活的突变株）与临床分离的敏感株一致。结论多重耐药大肠杆菌中存在四环素和青霉素的主动外排系统。主动外排系统是其形成多重耐药的机制之一。     

Influence of efflux pump inhibitors on the multidrug resistance of Helicobacter pylori

AIM:To evaluate the effect of efflux pump inhibitors (EPIs) on multidrug resistance of Helicobacter pylori (H. pylori).METHODS: H. pylori strains were isolated and cultured on Brucella agar plates with 10% sheep‘s blood. The multidrug resistant (MDR) H. pylori were obtained with the inducer chloramphenicol by repeated doubling of the concentration until no colony was seen, then the susceptibilities of the MDR strains and their parents to 9 antibiotics were assessed with agar dilution tests. The present stud...     

Global dissemination of a multidrug resistant Escherichia coli clone

Escherichia coli sequence type 131 (ST131) is a globally disseminated, multidrug resistant (MDR) clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with several factors, including resistance to fluoroquinolones, high virulence gene content, the possession of the type 1 fimbriae FimH30 allele, and the production of the CTX-M-15 extended spectrum β-lactamase (ESBL). Here, we used genome sequencing to examine the molecular epidemiology of a collection of E. coli ST131 strains isolated from six distinct geographical locations across the world spanning 2000–2011. The global phylogeny of E. coli ST131, determined from whole-genome sequence data, revealed a single lineage of E. coli ST131 distinct from other extraintestinal E. coli strains within the B2 phylogroup. Three closely related E. coli ST131 sublineages were identified, with little association to geographic origin. The majority of single-nucleotide variants associated with each of the sublineages were due to recombination in regions adjacent to mobile genetic elements (MGEs). The most prevalent sublineage of ST131 strains was characterized by fluoroquinolone resistance, and a distinct virulence factor and MGE profile. Four different variants of the CTX-M ESBL–resistance gene were identified in our ST131 strains, with acquisition of CTX-M-15 representing a defining feature of a discrete but geographically dispersed ST131 sublineage. This study confirms the global dispersal of a single E. coli ST131 clone and demonstrates the role of MGEs and recombination in the evolution of this important MDR pathogen.Many multidrug-resistant (MDR) bacterial strains are now recognized as belonging to clones that originate in a specific locale, country, or even globally. Escherichia coli sequence type 131 (ST131) is one such recently emerged and globally disseminated MDR pandemic clone responsible for community and hospital-acquired urinary tract and bloodstream infections. E. coli ST131 was identified in 2008 as a major clone linked to the spread of the CTX-M-15 extended-spectrum β-lactamase (ESBL) resistance (1–3). Since then, E. coli ST131 has also been strongly associated with fluoroquinolone resistance, and coresistance to aminoglycosides and trimethoprim-sulfamethoxazole (4–6). Alarmingly, strains of E. coli ST131 resistant to carbapenems have also been reported (7, 8), further limiting treatment options for this clone.E. coli ST131 belongs to the B2 phylogenetic subgroup I, with most isolates characterized as serotype O25b:H4 (1). Epidemiology studies using pulse-field gel electrophoresis (PFGE) have demonstrated that E. coli ST131 strains exhibit diversity, with some dominant PFGE pulsotypes including the UK epidemic strain A (9) and pulsotype 968 (10, 11) widely distributed across the globe. More recently, a typing scheme using the type 1 fimbriae fimH adhesin gene revealed that a large subclonal lineage of E. coli ST131 strains possess the FimH30 allele, which is also associated with specific mutations in the gyrA and parC genes that confer resistance to fluoroquinolones (12).Several whole genome (13–16) and PCR (1, 17–20) studies have revealed that E. coli ST131 strains possess a variable complement of genes encoding established virulence factors commonly associated with extraintestinal pathogenic E. coli (ExPEC). Indeed, few virulence genes appear to be uniformly present in E. coli ST131 and, thus, it is likely that differences in virulence gene content contribute to the variable virulence potential that has been reported. For example, although some ST131 strains cause rapid death in a mouse sepsis infection model (21), this phenotype is not consistent among all strains (22). The E. coli ST131 strain EC958, which is a representative of the FimH30-fluoroquinolone resistant subgroup, has been characterized at the molecular level (15). E. coli EC958 contains an insertion in the type 1 fimbriae regulator gene fimB (15) that is also common to other strains in the FimH30 subgroup (23) and colonizes the mouse bladder in a type 1 fimbriae-dependent manner (15). In mice, E. coli EC958 establishes acute and chronic urinary tract infection (UTI), forms intracellular bacterial communities in the bladder (24), and causes impairment of ureter contractility (25). E. coli EC958 is also resistant to the bactericidal action of human serum (26).The rapid global dissemination of E. coli ST131, combined with its MDR phenotype and the lack of new antimicrobial drugs in the developmental pipeline, highlights the urgent need to understand this pathogen and combat its spread. Here, we sequenced the genomes of 95 E. coli ST131 strains from six geographical regions (isolated from 2000 to 2011) to examine the spatial and temporal relationships of E. coli ST131. Our data supports the rapid and recent global dispersal of E. coli ST131 as a single clone.     

主动外排泵外膜蛋白编码基因hefA在幽门螺杆菌多重耐药中的重要作用

目的: 研究幽门螺杆菌(H pylori)分离株的多重耐药机制.方法:用氯霉素对临床分离株H pylori和标准株H pylori进行体外诱导,琼脂稀释法测定诱导后菌株对甲硝唑、四环素、琥乙红霉素、环丙沙星和青霉素G的耐药性,从而筛选出多重耐药株;实时定量PCR检测多重耐药株和敏感株中编码主动外排泵外膜蛋白的结构基因hefA的mRNA水平.通过构建hefA基因敲除株,测定敲除前后菌株对10种抗生素的敏感性.PCR扩增20株H pylori临床分离株主动外排泵结构基因hefA和hefC.结果:经氯霉素诱导后,筛选出的耐药菌株均表现出相似的多重耐药性,6株多重耐药株hefA基因mRNA水平显著高于敏感株(5.8466±2.9370 vs 2.6356±1.7245,P=0.033);成功构建了HefA基因敲除株(△HpLZ1026),△HpLZ1026对4种抗生素的敏感性明显增加;所有20株临床分离株中均检测出hefA和hefC基因,未发现hefABC基因缺失株.结论:主动外排系统hefA基因高表达导致体外人工诱导H pylori多重耐药的产生;hefABC基因在H pylori中普遍存在,且在H pylori多药耐药机制中起重要作用.     

Emr, an Escherichia coli locus for multidrug resistance.

An Escherichia coli chromosomal DNA fragment cloned on a multicopy plasmid conferred resistance to carbonylcyanide m-chlorophenylhydrazone, nalidixic acid, and a number of other toxic compounds. The sequence of the cloned emr locus located at minute 57.5 of the chromosome revealed two open reading frames, emrA and emrB. emrB encodes a highly hydrophobic 56.2-kDa peptide, with 14 potential alpha-helices to span the inner membrane. The peptide is homologous to QacA, a multidrug-resistant pump from Staphylococcus aureus, and belongs to a gene family that includes tetracycline-resistant pumps of Gram-positive bacteria and the galactose/H+ symporter of E. coli. emrA encodes a putative 42.7-kDa peptide containing a single hydrophobic domain and a large C-terminal hydrophilic domain. An active pho-fusion to the C domain suggested that EmrA is a membrane protein. Disruption of emrB significantly increased sensitivity of cells to uncouplers. The cellular content of uncoupler increased in the order: overexpressed emrB cells greater than wild type greater than emrB-.