广州地区淋球菌抗生素耐药性及质粒图谱研究

目的：了解广州地区淋球菌对抗生素的耐药性及质粒图谱。方法：用琼脂稀释法测定5种抗生素对167株菌的最低抑菌浓度（MIC）及用破裂解法分析160株菌的质粒图谱。结果：167株淋球菌检出PPN9株（5．4％）,TRNG16株（9．6％）,环丙沙星耐药率达78．4％,头孢三嗪,壮观霉素未发现耐药菌株。160株菌其质粒谱型2．6Md 4.5Md(24.4%),2.6Md 4.5Md 24.5Md(20.6%),2.6Md 24.5Md(20.6%),4.5Md检出率（45％）24.5Md检出率（41．3％）。结论：表明了广州地区淋球菌抗生素的耐药性和质粒图谱,有助于淋病的治疗和防治。     

细菌菌群的分布及耐药性分析

目的：了解本院临床分离致病菌菌群的分布及耐药情况,给临床经验用药提供可靠依据。方法：按《全国临床微生物检验操作规程》培养分离菌种,用美国BD公司的Sceptor半自动细菌鉴定仪或法国梅里埃公司的Vitek-60细菌鉴定仪进行鉴定及药物敏感实验,结果：临床分离 的致病菌中革兰阳性球菌占39．6％,革兰阴性杆菌占60．4％,前5位细菌分别为铜绿假单胞菌188株（17．0％）,大肠埃希菌143株（13．0％）,表皮葡萄球菌140株（12．7％）,肠球菌115株（10．7％）,金黄色葡萄球菌109株（9．9％）,药敏结果显示,苯唑西林耐药葡萄球菌（MRS）的耐药率明显高于苯唑西林敏感葡萄球菌109株（9．9％）,药敏结果显示,苯唑西林耐药葡萄球菌（MRS）的耐药率明显高于苯唑西林敏感葡萄球菌（MSS）,球菌中未分离出万古霉素耐药的菌株,但肠球菌中检测出有对万古霉素中介的菌株;肠杆菌科细菌对亚胺培南的耐药率最低,铜绿假单胞菌对头孢他啶,头孢哌酮,氨曲南,丁胺卡那霉素,亚胺培南的耐药率亦较低。结论：革兰阴性杆菌分离率高于革兰阳性球菌;革兰阳性球菌对万古霉素敏感率最高,肠杆菌科对亚胺培南敏感率最高,非发酵菌对亚胺培南,氨基糖甙类,头孢他啶,头孢哌酮药蓄亦较低。     

老年慢性阻塞性肺病患者继发革兰氏阴性杆菌感染的菌型及耐药性分析

目的：探讨老年慢性阻塞性肺疾病（COPD）继发院内革兰氏阴性杆菌感染的发病机理、菌型分布及耐药性。方法：从本院老年COPD继发医院革兰氏阴性杆菌（GNB）肺炎患者痰液中分离的213株GNB进行菌型分类,选用12种常用抗菌药物进行体外MIC药敏试验。结果：213株占COPD继发医院肺炎病原菌总数的69．6％（213／306）。老年COPD患者继发革兰氏阴性菌感染菌种分类为：铜绿假单胞菌（35．7％）、肺炎克雷伯菌（21．1％）、大肠埃希菌（17．4％）、阴沟肠杆菌（11．7％）、嗜麦芽假单胞菌（7．9％）、其他病原菌（6．1％）;药敏结果表明,所有GNB对抗菌药物耐药率均呈上升趋势。结论：铜绿假单胞菌是COPD继发GNB感染的主要致病菌,在临床治疗中必须重视菌型鉴定和药敏试验,合理使用抗生素,才能控制院内感染GNB的发生和日益增高的耐药趋势。     

苏州地区儿童感染志贺菌的耐药监测

目的调查苏州地区2000年至2006年儿童感染志贺菌的耐药情况,了解其耐药趋势,以指导临床合理用药。方法对2000年至2006年苏州大学附属儿童医院临床标本中分离的589株志贺菌（福氏志贺菌470株,宋内志贺菌119株）采用K-B法对氨苄西林、哌拉西林、头孢哌酮、头孢曲松、环丙沙星、SMZ＋TMP、亚胺培南进行药敏试验。结果于7年中470株福氏志贺菌的耐药率：氨苄西林：始终在90．0％以上;哌拉西林：从22．6％逐渐上升到63．2％;头孢哌酮：1．1％～56．1％;头孢曲松：2．2％～43．9％;环丙沙星：一直在10．0％左右;SMZ＋TMP：在70．0％～96．0％波动;对亚胺培南均敏感。119株宋内志贺菌的耐药率：氨苄西林：从7．1％逐渐上升到82．8％;哌拉西林：0～79．3％;头孢哌酮：0～55．2％;头孢曲松：0～51．7％;SMZ＋TMP：在62．5％～100．0％波动;对环丙沙星和亚胺培南均敏感。结论本地区儿童感染福氏志贺菌对氨苄西林严重耐药,同时伴有对SMZ＋TMP的高耐药率,对哌拉西林和3代头孢菌素的耐药率呈逐年上升趋势。宋内志贺菌的耐药率在2004年之前（除对复方新诺明外）,远低于福氏志贺菌,但在2005年其对氨苄西林和3代头孢菌素的耐药率突然上升,大有赶超福氏志贺菌之势。因此苏州地区儿童感染志贺菌的耐药情况不容乐观,对儿童细菌性痢疾的治疗将会面临困境。     

超广谱β-内酰胺酶菌种间分布及耐药性分析

目的：了解质粒介导超广谱β-内酰胺酶（ESBL）的产生及菌种间分布。方法：将初筛的可疑菌用E-test法确诊,对细菌进行ESBL检测,药物敏感试验用K-B纸片法进行。结果：717株革兰阴性菌中,检出ESBL61株,其中大肠埃希菌16株,肺炎克雷伯菌12株,阴沟杆菌9株,弗劳地枸椽酸杆菌6株,类产碱杆菌5株,黄杆菌,施氏假单胞菌各3株,聚团肠杆菌2株,液化沙雷菌2株,铜绿假单胞菌,不动杆菌,鼠伤寒沙门菌各1株,对亚胺培南,丁胺卡那,环内沙星,呋喃妥因和头孢他啶的敏感率分别为100%,30.2%,26.6%,71.5%和37.7%,结论：ESBL易借助耐药质粒传播,在细菌间分布广泛,具有较高的检出阳性率。     

烧伤病房患者创面金葡菌分布及耐药性分析

目的分析烧伤病房患者不同创面金葡菌的分布及耐药性,为临床合理选用抗菌药提供依据。方法对2006年1月至2013年12月间中国人民解放军第八五医院烧伤病房患者创面分离出金葡菌,采用K—B纸片扩散法进行药物敏感试验。分析金葡菌的耐药性,并对难愈性创面、非难愈性创面的耐甲氧西林金葡菌（MRSA）与甲氧西林敏感金葡菌（MSSA）的耐药性进行对比分析。结果分离出金葡菌112株,其中难愈性创面有70株MRSA和17株MSSA来自难愈性创面,16株MRSA和9株MSSA来自非难愈性创面。金葡菌对青霉素、红霉素、克林霉素的耐药率较高（分别为94．64％、81．25％和74．11％）,对复方新诺明、呋喃妥因的耐药率较低（分别为16．07％和1．79％）,对万古霉素、利奈唑烷的耐药率为0。MRSA的耐药率高于MSSA。来源于难愈性创面与非难愈性创面的MRSA仅在对利福平的耐药率上有明显差异,而来源于两创面的MSSA的耐药率无明显差异。结论创面金葡菌中MRSA的构成比高,难愈性创面MRSA耐药严重,应积极防控创面MRSA感染和扩散。     

某三甲医院2007—2011年ICU气管切开术后肺部感染患者208例临床分析

目的：探讨湖南某三甲医院重症监护病房（ICU）患者气管切开后肺部感染病原菌的类型及其耐药性,为临床经验性用药提供帮助。方法：回顾性分析ICU208例气管切开术后并发肺部感染患者的痰细菌培养及药物敏感性测定结果。结果：共分离出420株致病菌,革兰阴性菌293株,占69．76％,其中铜绿假单胞菌98株居首位;革兰阳性菌105株,占25．24％,其中金黄色葡萄球菌47株为最多;真菌占22株,占5．23％。分离出产超广谱B内酰胺酶（ESBLs）菌87株,耐甲氧西林金黄色葡萄球菌（MRSA）24株。所分离致病菌对常用抗菌药物均有不同程度的耐药,且为多重耐药。结论：ICU气管切开患者肺部感染病原菌以革兰阴性菌为主,耐药率高,临床应加强病原学监测,重视细菌的种类分布和耐药趋势,合理使用抗生素。     

深圳市儿童社区获得性肺炎细菌病原学及其耐药性

目的研究儿童社区获得性肺炎细菌病原学及其耐药性特征,指导临床合理应用抗菌药物。方法对2006年2月至2007年3月1年期间住院的5岁及5岁以下社区获得性肺炎病人,进行深部呼吸道吸引物细菌培养,并且检测分离菌株对常用抗菌药物的耐药性。结果1441例病人中,722例检出细菌共761株,分离阳性率为50．1％,分离菌依次为肺炎克雷伯菌170株（22．3％）、大肠埃希菌130株（17．1％）、肺炎链球菌89株（11．7％）、金黄色葡萄球菌63株（8．3％）及流感和副流感嗜血杆菌60株（7．9％）。耐甲氧西林金黄色葡萄球菌（MRSA）检出率为15．9％;对青霉素不敏感的肺炎链球菌（包括PISP和PRSP）检出率为84．3％;肺炎克雷伯菌、大肠埃希菌、粘质沙雷菌和阴沟肠杆菌产ESBLs的检出率分别为31．2％、46．2％、94．8％和16．8％;流感嗜血杆菌和副流感嗜血杆菌对氨苄西林的耐药率为36％和40％;铜绿假单胞菌和鲍曼复合不动杆菌对亚胺培南的耐药率分别为10．7％和13．2％。结论在深圳儿童社区获得性肺炎的分离菌中,革兰阴性菌明显多于革兰阳性菌,分离菌依次为肺炎克雷伯菌、大肠埃希菌、肺炎链球菌、金黄色葡萄球菌及流感和副流感嗜血杆菌。分离细菌对常用抗菌药物的耐药性较为严重。     

广州地区儿童血培养病原菌的分布及耐药性研究

目的探讨儿童血培养病原菌的分布特点及其耐药情况,为临床诊疗提供参考。方法对广州市儿童医院2005年至2006年临床各科室送检血液标本所分离病原菌的分布及药敏结果进行回顾性分析。结果共检出385株病原菌,其中革兰阳性菌208株,占54．0％,革兰阴性菌164株,占42．6％,真菌13株,占3．4％。分离率前6位的病原菌依次为凝固酶阴性葡萄球菌（CNS,35．8％）、肺炎克雷伯菌（8．8％）、不动杆菌（5．5％）、大肠埃希菌（4．9％）、铜绿假单胞菌（4、7％）、金黄色葡萄球菌（4．4％）。病原菌的病区分布特点：儿科重症监护病房以不动杆菌等非发酵菌为主要分离菌（占41．7％）,新生儿重症监护病房以CNS为主（40．5％）,血液病区以肠杆菌科细菌为主（35．7％）,新生儿病房及传染病房均以CNS为主要分离菌。CNS对青霉素、氨苄西林、红霉素耐药率均超过80％,但对万古霉素、替考拉宁和阿米卡星敏感,MRCNS检出率达72.5％。肠杆菌科细菌对哌拉西林、氨苄西林、头孢噻肟及头孢哌酮的耐药率为50％～100％,但对亚胺培南、阿米卡星和诺氟沙星耐药率较低。不动杆菌对广谱青霉素、第3代头孢菌素、氨曲南及庆大霉素的耐药率较高而对亚胺培南、头孢哌酮／舒巴坦较为敏感。结论凝固酶阴性葡萄球菌是广州地区儿童败血症最主要的病原菌。不同病区检出病原菌种类有较大差异。根据病原菌种类及药敏结果合理应用抗菌药是有效控制感染和减少耐药菌株产生的重要手段。     

儿童细菌性腹泻病原菌分布及耐药性分析

目的 了解小儿细菌性腹泻的病原菌分布及耐药情况。方法 对773例临床上疑为细菌性腹泻病的患儿的粪便标本进行培养、鉴定及药物敏感试验、超广谱β-内酰胺酶（ESBLs）检测。结果 773例标本中分离出阳性菌株671株,总检出率为86．6％。其中埃希菌属、志贺菌属及耶尔森菌3种致病性细菌占阳性菌株的54．5％,肠杆菌属、非发酵菌群、酵母样真菌、肠球菌属、变形杆菌属、克雷伯菌属和枸橼酸杆菌属等条件致病菌占阳性菌株的45．5％。埃希菌属及志贺菌属对头孢他啶及氨曲南耐药率低（16．4％以下）,而肠杆菌属等条件致病菌对常用抗生素耐药率均较高。G^-杆菌中ESBLs的检出率为38．3％,其中大肠埃希菌占66．7％。结论 埃希菌属是儿童细菌性腹泻病的主要致病菌（50．2％）,且耐药率及ESBLs产生率高,但肠杆菌属等条件致病菌检出率也较高（45．5％）,且耐药率高,可能与抗生素广泛使用,肠杆菌群失调有关,应引起高度重视。     

应用微卫星标记对雌核发育银鲫的遗传多样性初探

利用Crooijmans et al.(1997)分离的包含CA重复单元的普通鲤鱼（Cyprinus carpino.L)的8个微卫星DNA标记,对银鲫（Carassius auratus gibelio Bloch)的5个不同雌核发育系的24尾个体进行PCR扩增。分析电泳结果发现,除MFW28未能在银鲫中稳定地扩增出相应的同源序列,其余的7对引物扩增的重复性和稳定性都很好,随引物不同,各等位基因数为1-14个,大小在100-506bp。在MFW1、MFW4、MFW19、MFW20、MFW23和MFW246个微卫星的扩增图谱中,不同的雌核发育系扩增出各自独特的图谱,而同一系内的不同个体间具有高度的遗传同质性,但仍然在个别个体中检测到少量的多态片段。不同系间的扩增图谱呈现出高度的遗传异质性,共鉴定出23个可以用于有效区分5个不同雌核发育系的分子标记。这5个微卫星标记反映了银鲫5个雌核发育系间的相互亲缘关系,其中P和A系同属一个雌核发育系,F系起源于E系,A、D和E系可能分别独立地起源于不同的杂交事件,鉴定的微卫星分子标记为进行银鲫群体遗传学和进化遗传学研究,以及银鲫的分子标记育种和进行基因组作图提供了理想的工具。     

鹅新城疫病毒P基因的RNA编辑及其相关蛋白的分子特征

应用RT-PCR方法对鹅新城疫病毒安徽分离株WF00GP基因进行了RNA编辑分析,发现w‰GP基因在保守的编辑位点上插入一个G,使得P基因增加编码V蛋白,而且它的最大ORF的长度为1188bp,编码395个氨基酸的P蛋白。wkG株和参考毒株的P基因的同源性和系统发育分析表明,WF00G株与水禽源毒株NA．1、ZJ1、FPI／02、PX2／03、Duck／1／05和SF02等亲缘关系较近,与传统疫苗株或弱毒株HB92、LaSota和Clone30以及F48E9等的亲缘关系相对较远。进一步对其V蛋白羧基端序列分析发现,虽然编辑位点氨基酸序列（132KKG134）和羧基端的5个Cys残基位点是高度保守的,但是V蛋白c末端氨基酸存在较大变异,APMV-1毒株V蛋白羧基端氨基酸的突变呈现“基因型一致性”。     

嗜酸氧化亚铁硫杆菌亚铁氧化酶基因分子多态性研究

嗜酸氧化亚铁硫杆菌（Acidithiobacillus ferrooxidans, A.f）属于化能自养、革兰氏阴性细菌,好氧嗜酸,以氧化亚铁离子或还原态硫化物（S0&S2-）获得能量生长。在其Fe2+氧化系统中,亚铁氧化酶起重要作用。对不同硫化矿区分离得到的5株A.f进行生长动力学和对亚铁氧化活性的对比研究,结果显示不同菌株之间存在表型差异。提取A.f菌株的基因组DNA,设计引物,对亚铁氧化酶基因进行PCR扩增,同时对扩增产物直接进行测序,并同GenBank的参考序列进行比对分析,发现序列相似性在99%～97%之间,分析编码区域发现在第187～195位可能存在一个高变异区：在第187～189位,菌株YTW编码的氨基酸Thr→Pro;而在第193～195位,菌株TK是Met→Asn; 菌株BY则是Met→Ile。另外在位点第219位,所有菌株都是T→C,但编码的氨基酸未发生变化,属于同义密码子。对于编码区上游序列,比对后没有差异;而对于编码区下游序列,经比对发现存在一些差异位点。     

Comparison of extracellular proteases produced by Aeromonas salmonicida strains, isolated from various fish species

Extracellular products (ECPs) of five typical and 25 atypical Aeromonas salmonicida isolates from various fish species and geographical locations were analysed by substrate specificity, inhibition of proteolytic activity and substrate SDS-PAGE. The type strains of Aer. salmonicida subsp. salmonicida and Aer. salmonicida subsp. achromogenes were included for comparison. The results indicated that the strains formed six protease groups. The proteases produced by the two type strains were of a different nature. All the typical strains belonged to one group and showed proteolytic activities comparable to P1 and P2 proteases. Three atypical (oxidase-negative) strains secreted a protease comparable to P1. With the exception of these three, all strains produced metallo-gelatinases. A metallo-caseinase (AsaP1) was detected in the ECP of subsp. achromogenes type strain and 10 of the atypical strains. A number of proteolytic components with different apparent molecular weights (AMWs) were identified. These include caseinases with AMWs of > 100, 80, 60 and 30 kDa and gelatinolytic components with different AMWs, including some with AMW higher than P1 and lower than P2. The protease production of the isolates was not found to be host specific.     

Incidence and some characteristics of fimbriae FY and 31A of Escherichia coli isolates from calves with diarrhea in Japan

Escherichia coli isolates from calves with diarrhea (1 day to 8 weeks old, 140 individuals) were surveyed for the three immunologically distinct fimbrial adhesins FY, 31A, and K99. Of a total of 1,370 strains isolated, 96 (7.0%), 34 (2.5%), 75 (5.5%), and 13 (0.9%) were identified as FY+, 31A+, FY+.31A+, and K99+, respectively. The K99+ strains also manifested heat-stable enterotoxin production (ST+), while FY+, 31A+, and FY+.31A+ strains were ST-. Expression of FY and 31A was repressed at lower temperatures or poor aeration. The FY+ and 31A+ E. coli showed mannose-resistant hemagglutinating activity with bovine erythrocytes. Electron microscopy revealed that FY is a gently curled fimbria with a mean diameter of 4.2 nm, and 31A is a fimbria with a mean diameter of 5.1 nm. The molecular mass of protein subunits was found to be approximately 20 kilodaltons (Kd) and 19 Kd for FY and 31A, respectively. Lethal diarrhea of neonatal calves was induced by challenge with the combination of a K99+.ST+ E. coli strain and either a 31A+ E. coli strain or a 31A+ E. coli strain plus an FY+ E. coli strain under the experimental conditions in which lethal diarrhea was not induced by challenge with a K99+.ST+ E. coli strain alone.     

二斑叶螨不同抗性品系最佳内参基因的筛选及CYP392E亚家族基因的表达分析

【目的】 筛选二斑叶螨Tetranychus urticae敏感品系（SS）、 抗阿维菌素品系(Av-R)和抗螺虫乙酯品系（Sp-R）中合适的内参基因, 研究二斑叶螨不同抗性品系CYP392E亚家族基因表达水平的变化。【方法】采用实时荧光定量PCR （quantitative real time PCR, qRT-PCR）技术, 对二斑叶螨α-tubulin, β-actin, ELFn, GAPDH, 5.8S rRNA基因和SDHA共6个看家基因的表达稳定性进行分析以期筛选出合适的内参基因, 并在此基础上进一步分析二斑叶螨不同品系P450酶系CYP392E亚家族基因的表达量差异。【结果】在二斑叶螨SS, Av-R和Sp-R品系中稳定性最高的内参基因为ELFn。以ELFn为内参基因, CYP392E7基因相对表达量在二斑叶螨Av-R品系中显著高于SS品系(P<0.05), 为后者的2.18倍, 而在Sp-R品系和SS品系中差异不显著; 其余基因的相对表达量在2个抗性品系中均没有增加, Av-R品系的CYP392E4, CYP392E9和CYP392E10基因以及Sp-R品系的CYP392E1和CYP392E9基因相对表达量甚至显著下调, 分别为SS品系的48%, 74%, 65%, 63%和73%。【结论】在二斑叶螨SS, Av-R和Sp-R品系中ELFn为理想的内参基因, Av-R品系CYP392E4, CYP392E7, CYP392E9和CYP392E10基因相对表达量的显著变化可能与二斑叶螨对阿维菌素的抗性形成有关, Sp-R品系CYP392E1和CYP392E9基因也可能与二斑叶螨对螺虫乙酯的抗性形成有一定联系。     

Evaluation of the biofilm-forming ability and genetic typing for clinical isolates of Pseudomonas aeruginosa by enterobacterial repetitive intergenic consensus-based PCR

Biofilm formation is an important phenotype associated with chronic Pseudomonas aeruginosa infections. In the present study, a total of 48 P. aeruginosa strains isolated from clinical specimens were examined for their biofilm-forming ability using a microtiter plate method. The different biofilm-forming abilities were demonstrated among the strains; however, most strains formed a larger biofilm than strain PAO1, a reference strain. The genetic typing was also carried out by enterobacterial repetitive intergenic consensus-based polymerase chain reaction. Although they were divided into five groups (A to E), most of the strains showing the higher biofilm-forming ability were found to be in groups D and E, suggesting a significant relationship between the biofilm-forming ability and the genetic group.     

Isolation and characteristics of 17β-estradiol-degrading <Emphasis Type="Italic">Bacillus</Emphasis> spp. strains from activated sludge

The natural estrogen 17β-estradiol (E2) is a major endocrine disruptor, with adverse effects on wildlife and humans. The aim of this study was to isolate microorganisms able to effectively remove E2 from wastewater. Accordingly, five E2-degrading strains of bacteria were isolated from activated sludge collected from a wastewater treatment plant. Based on their 16S RNA gene sequences, these five strains belonged to the genus Bacillus. All five isolates were capable of converting E2 to estrone (E1), greatly reducing total estrogenic activities in wastewater during E2 biodegradation. However, only two strains (strain E2Y1 and E2Y4) were able to further transform E1, whereas it accumulated in the culture medium of the other isolates. Among all isolates, strain E2Y4, with 100% of the 1,400 bp 16S RNA gene matched that of B. subtilis CICC10075, exhibited the highest E2 and E1 degradation capacities, degrading 1 mg E2/l completely within 4 days and further transforming 40% of the metabolite E1. Furthermore, the E2 degradation rates of strain E2Y4 increased with increasing initial concentrations of the steroid, with a high degradation capacity maintained even at initial concentrations up to 50 mg/l. These results demonstrate the potential significance of strain E2Y4 in biological remediation applications.     

Development of a new PCR protocol for the detection of species and genotypes (strains) of Echinococcus in formalin-fixed, paraffin-embedded tissues

The aim of our study was to establish a new PCR protocol for the detection and discrimination of Echinococcus granulosus complex on one hand and Echinococcus multilocularis in formalin-fixed, paraffin-embedded tissues (FFPTs) on the other. The target sequences for all PCRs are located on a 471bp segment of the mitochondrial ND1 gene, the fragment sizes of the amplification products are 295bp (for the sheep strain of E. granulosus), 204bp (for the pig strain of E. granulosus) and 252bp (for E. multilocularis), respectively. In total, 80 FFPTs from patients with histologically confirmed echinococcosis (76 with E. granulosus and four with E. multilocularis) operated on in Austrian hospitals between 1978 and 2005 were examined. In 68 (85%) samples, we were able to detect specific DNA fragments with our newly established PCR protocols. Thirty-eight (47.5%) of 80 clinical samples were identified as the G1 strain, 26 (32.5%) as the G5, 6 or 7 strains and four (5%) as E. multilocularis. The specificity of all three PCRs was 100%; for the discrimination between G6 and G7 strains, sequencing of an additional 234bp PCR fragment was necessary and showed that three out of 26 G5, 6 or 7 PCR-positive patients were infected with E. granulosus genotype G6 (the camel strain).