1株产GES-1型碳青霉烯酶铜绿假单胞菌的全基因组测序分析

目的了解产GES-1型碳青霉烯酶铜绿假单胞菌的分子流行特征。方法采用Illumina HiSeq X10测序平台对1株产GES-1型碳青霉烯酶铜绿假单胞菌进行全基因组测序,通过Kleborate和ResFinder分析菌株的多位点序例分型(MLST)、毒力基因和抗菌药物耐药基因,并分析bla_(GES-1)基因的周围环境。结果经比对,本菌为ST235型,血清分型为O11型,共携带12个抗菌药物耐药基因,分别为aac(6′)-Ⅰb3、aadA10、aph(3′)-Ⅱb、aph(3′)-XV、bla_(GES-1)、bla_(OXA-50)、bla_(PAO)、crpP、sul1、tet(G)、fosA、catB7;bla_(GES-1)位于含有3 150 bp的NODE_219片段中,该片段与GenBank中的KY860573菌株的72068—75217序列99.94%一致。其基因环境为位于上游的整合子1(intl1),β-内酰胺类耐药基因bla_(GES-1),氨基糖苷类/氟喹诺酮类耐药基因aac(6′)-Ⅰb3,氨基糖苷类耐药基因aph(3′)-XV及位于下游的插入序列ISPa21e。该菌包含有357个毒力基因,为exoU~+/exoS~-基因型。结论本菌为全球流行的高风险克隆ST235,exoU~+/exoS~-基因型。全基因组测序分析提供了bla_(GES-1)基因环境、基因分型、耐药基因、毒力基因等多种信息。     

铜绿假单胞菌耐药性及产碳青霉烯酶研究

目的了解医院2010年分离的铜绿假单胞菌的耐药性及耐亚胺培南菌株产碳青酶烯酶情况。方法 K-B纸片扩散法进行药敏试验;碳青霉烯酶检测采用改良Hodge试验。结果 2010年分离铜绿假单胞菌145株,主要来自呼吸道标本、脓汁及分泌物。药敏结果显示铜绿假单胞菌氨苄西林耐药率最高,达88.97%。对阿莫西林/克拉维酸、头孢噻肟、环丙沙星和头孢他啶的耐药率均超过50%。对洛美沙星和头孢哌酮/舒巴坦较敏感;耐亚胺培南铜绿假单胞菌31株,耐药率21.38%,改良Hodge试验阳性10株,阳性率为32.26%。结论分离的铜绿假单胞菌耐药性和产碳青霉烯酶率均较高,铜绿假单胞菌耐药严重     

Combination antibiotic therapy for multidrug-resistant Gram-negative bacteria

Combination antibiotic therapy for Gram-negative sepsis is controversial. The present review provides a brief summary of the existing knowledge on combination therapy for severe infections with multidrug-resistant Pseudomonas spp., Acinetobacter spp., and Enterobacteriaceae. Empirical combination antibiotic therapy is recommended for severe sepsis and septic shock to reduce mortality related to inappropriate antibiotic treatment. Because definitive combination therapy has not been proven superior to monotherapy in meta-analyses, it is generally advised to de-escalate antibiotic therapy when the antibiotic susceptibility profile is known, although it cannot be excluded that some subgroups of patients might still benefit from continued combination therapy. Definitive combination therapy is recommended for carbapenemase-producing Enterobacteriaceae and should also be considered for severe infections with Pseudomonas and Acinetobacter spp. when beta-lactams cannot be used. Because resistance to broad-spectrum beta-lactams is increasing in Gram-negative bacteria and because no new antibiotics are expected to become available in the near future, the antibacterial potential of combination therapy should be further explored. In vitro data suggest that combinations can be effective even if the bacteria are resistant to the individual antibiotics, although existing evidence is insufficient to support the choice of combinations and explain the synergistic effects observed. In vitro models can be used to screen for effective combinations that can later be validated in animal or clinical studies. Further, in the absence of clinical evidence, in vitro data might be useful in supporting therapeutic decisions for severe infections with multidrug-resistant Gram-negative bacteria.     

某院铜绿假单胞菌体外药敏试验检测结果分析

目的：了解近年来临床分离的铜绿假单胞菌对常用抗菌药物的敏感性,为临床合理使用抗菌药物提供依据。方法选择解放军第一一八医院2010年1月至2012年12月临床3177份标本,运用法国ATB Expres-sion微生物鉴定系统对铜绿假单胞菌进行菌株鉴定及15种抗菌药物的药敏试验。所有数据均在 Excel软件进行统计学分析。结果临床共分离铜绿假单胞菌263株,分离自痰液211株,占80．22％;分泌物25株,占9．51％;脓液10株,占3．80％;中段尿8株,占3．04％;留置针5株,占1．90％;血液4株,占1．52％。铜绿假单胞菌对多黏菌素E、阿米卡星及妥布霉素敏感率分别为95．06％、94．30％及93．16％;美洛培南和亚胺培南敏感率均在50％以下。除复方磺胺甲噁唑和复方氨苄西林以外,其余均为70％～90％。阿米卡星和妥布霉素男女之间敏感率比较,差异具有统计学意义（U＝2．74,P＜0．01;U＝2．08,P＜0．05）;其余均无统计学意义（ P＞0．05）。结论运用细菌耐药预警监测系统了解和掌握感染耐药和药物敏感情况,对指导临床合理使用抗菌药物具有重要意义。     

The Role of Elastase in Corneal Epithelial Barrier Dysfunction Caused by Pseudomonas aeruginosa Exoproteins

PurposeTo investigate the role of elastase in corneal epithelial barrier dysfunction caused by the exoproteins secreted by Pseudomonas aeruginosa.MethodsExoproteins obtained from Pseudomonas aeruginosa culture supernatant were analyzed by shotgun proteomics approach. In vitro multilayered rabbit corneal epithelial barrier model prepared by air-liquid interface technique (CECs-ALI) were treated with 2 µg/ml exoproteins and/or 8 mM elastase inhibitor. Then the epithelial barrier function was evaluated by transepithelial electrical resistance (TEER) assay and tight junction proteins immunofluorescence. Cell viability and the apoptosis rate were examined by CCK8 assay and flow cytometry. TNF-α, IL-6, IL-8, and IL-1β levels were measured by ELISA. Mice cornea treated with exoproteins and/or elastase inhibitor were evaluated in vivo and in vitro.ResultsElastase (24.2%) is one of the major components of exoproteins. After 2 µg/ml exoproteins were applied to CECs-ALI for two hours, TEER decreased from 323.2 ±  2.7 to 104 ± 6.8 Ω/cm² (P < 0.001). The immunofluorescence results showed a distinct separation in tight junction and significant degradation of ZO-1 and occludin (P < 0.05). Elastase inhibitor (8 mM) alleviated the decrease in TEER value (234 ± 6.8 Ω cm²) induced by exoproteins. Inhibition of elastase decreased the apoptosis rate of CECs treated with exoproteins from 30.2 ± 3.8% to 7.26 ± 1.3% and the levels of inflammatory factors (P < 0.05). Mice corneal epithelium defect could be induced by exoproteins and protected by elastase inhibitor.ConclusionsElastase plays a critical role in corneal epithelial barrier dysfunction caused by Pseudomonas aeruginosa exoproteins via damaging tight junctions. The inhibition of elastase could protect the corneal epithelial barrier via reducing virulence and inflammation.     

绿脓假单胞菌多重耐药机制的研究

目的调查我院2003-2005年临床分离多重耐药绿脓假单胞菌的氨基糖苷类修饰酶基因、β内酰胺酶编码基因和外膜通道蛋白oprD2基因存在状况；并研究整合子参与绿脓假单胞菌多重耐药的机制。方法纸片扩散法测定绿脓假单胞菌对14种抗菌药物的药物敏感性，并筛选出14株多重耐药菌株；聚合酶链反应检测氨基糖苷类修饰酶基因、β内酰胺酶编码基因和外膜通道蛋白oprD2基因；聚合酶链反应检测整合子5’保守区的整合酶基因和3’保守区的qacE△1-sulI基因。对整合酶基因的阳性扩增产物的限制性片段长度多态性分析进行整合子分类，整合子可变区扩增并测序。结果14株绿脓假单胞菌对14种抗菌药（哌拉西林等）的耐药率为14．3％-100．0％；氨基糖苷类修饰酶基因ant（2”）-Ⅰ、aac（3）-Ⅱ、aac（6’）-Ⅱ、aac（6’）-Ⅰ、ant（3”）-Ⅰ和aac（3）-Ⅰ检出率分别为78．6％、57．1％、57．1％、14．3％、7．1％、0；β内酰胺酶编码基因TEM和IMP的检出率分别为92．9％和42．9％，未检出VIM、OXA、PER、GES和SHV基因，1株外膜通道蛋白oprD2基因缺失；整合酶基因及qacE△1-sulI基因检出率分别为85．7％和78．6％，整合子可变区扩增有3种片段：1000、1300和1700，测序证实分别为aadA2、aadA6．odD和dfrⅫ-orfF-aadA2，其中aadA2是首次在绿脓假单胞菌的整合子中检出，aadA6-odD是一种新型的整合子可变区基因盒组合形式。Genbank号分别为DQ091178和DQ091179。结论我院多重耐药绿脓假单胞菌对B内酰胺类抗生素的耐药主要与TEM和IMP型耐药基因有关，对氨基糖苷类抗生素的耐药主要与氨基糖苷类修饰酶基因ant（2”）-Ⅰ、8aC（3）-Ⅱ和aac（6’）-Ⅱ有关；整合子参与了绿脓假单胞菌的耐药和多重耐药。     

密度感知信号系统在铜绿假单胞菌致大鼠肺部感染中的作用

【论文特点介绍】本实验比较了PAO1和PAO—JP2的弹性蛋白酶活性、外毒素A的含量，结果显示：PAO—JP2株显示了弹性蛋白酶活性、外毒素A表达的完全缺失。结合体内实验结果，证实了密度感知信号系统通过控制铜绿假单孢菌细胞外重要毒性因子，如弹性蛋白酶活性、外毒素A的表达，在铜绿假单孢菌致病作用中扮演重要的角色。使用特定合成的AI或QS抑制剂，来达到抑制铜绿假单孢菌毒性因子的表达，减低其致病作用，是颇有前景的一种抗菌疗法。     

Bronchiektasen

Zusammenfassung Durch die Entwicklung und frühzeitige Einleitung antibiotischer Therapien und die Entwicklung von Impfstoffen sind Bronchiektasenerkrankungen seltener geworden. Betroffen sind meist die mittelgroßen Bronchien auf Segment- und Subsegmentebene. Eine chronische Entzündungsreaktion wird häufig durch Mikroorganismen wie Pseudomonas aeruginosa oder Haemophilus influenzae getriggert. Chronischer Husten mit putridem Auswurf und zeitweisen Hämoptysen sind typische klinische Beschwerden. Zur Diagnostik ist die hochauflösende CT die Methode der Wahl. Bei der Therapie steht die Behandlung der Grunderkrankung im Vordergrund. Atem- und physiotherapeutische Maßnahmen stellen nach wie vor die Basis der Therapie dar. Bei akuter Verschlechterung/Exazerbation sollte im besten Falle eine kalkulierte Antibiotikatherapie nach Antibiogramm erfolgen. Eine Erregerdiagnostik sollte vorweg erfolgen. Bei unbekanntem Erreger sollte eine empirische Therapie eingeleitet werden, die auch Pseudomonas erfasst.     

铜绿假单胞菌体外抗菌活性的检测及用药分析

目的 了解铜绿假单胞菌（PA）引起医院感染的特点以及对PA的耐药性进行分析。方法 对两家医院2003年和2004年分离出的278株铜绿假单胞菌选用15种抗生素进行药敏实验，按NCCLS标准判断。结果 PA对第三代头孢菌素的敏感率最低，对碳青酶烯类药物亚胺培南耐药率也达18．9％。结论 临床医生应对铜绿假单胞菌的耐药现状，采取有效的药物和合理的方法，保护好现有的敏感药物，减少PA对药物耐药性的上升。