青藏高原牦牛携带屎肠球菌及其毒力基因和耐药性检测

目的 了解青藏高原牦牛携带屎肠球菌(Enterococcus faecium)情况,以及耐药性和毒力基因。方法 使用链球菌培养基分离细菌;使用生化反应和16s rDNA序列分析方法鉴定;采用PCR方法检测从牦牛粪便中分离的屎肠球菌携带细胞溶血素(cytolysin,cylA)、明胶酶E(gelatinase, gelE)、表面蛋白(enterococcal surface protein, esp)、胶质蛋白粘附素collagen-binding-adhesin of Enterococcal faecium,acm)、聚集物质(aggregation substance, asa1)及透明质酸酶(hyalronidase,hyl)6种毒力基因的情况;应用K-B纸片法对屎肠球菌分离株进行16种抗生素的敏感性分析;参照PulseNet 脉冲场凝胶电泳(Pulsed-field gel electrophoresis, PFGE)实验方法对屎肠球菌分离株进行PFGE分型分析。结果 我们从320份牦牛粪便分离到33株屎肠球菌。这些菌株中毒力基因asa1的阳性率最高,为6.1%,acm和hyl次之,均为3.0%,其他毒力基因皆未检出。33株牦牛屎肠球菌中,有19株菌株具有抗生素耐药性,包括4株多重耐药菌株和15株单耐菌株。牦牛屎肠球菌对利福平耐药率最高,为48.5%;对其他抗生素的耐药率从高到低依次为青霉素G15.2%、四环素12.1%、强力霉素12.1%、红霉素9.1%、环丙沙星6.1%、氨苄西林6.1%、高浓度庆大霉素6.1%、磷霉素6.1%、左氧氟沙星6.1%。所有菌株对万古霉素、替拉考宁和氯霉素均敏感。细菌染色体DNA酶切片段的PFGE分析发现,33株屎肠球菌分离株共产生30种PFGE带型,可分为A-H 8个聚类群,耐药菌株分布在6个聚类群中。结论 青藏高原牦牛携带屎肠球菌,呈现高度遗传多态性,部分菌株携带毒力基因,对多种抗生素耐药。研究提示,屎肠球菌可能会通过牦牛相关食品传播。     

临床分离粪肠球菌和屎肠球菌菌株的检测及耐药性研究

目的分析粪肠球菌、屎肠球菌对抗菌药物的耐药性及敏感性,为合理使用抗菌药物提供证据。方法运用Phonex 100全自动微生物鉴定/药敏系统对临床分离的菌株进行鉴定和药敏试验。结果 103株肠球菌耐药率最高的药物依次为红霉素（94.2%）、环丙沙星（85.4%）、利福平（81.6%）,对万古霉素（94.2%）和替考拉宁（93.2%）的敏感性最高。发现万古霉素耐药的粪肠球菌和屎肠球菌各3株。粪肠球菌和屎肠球菌对青霉素的耐药率分别为22%和71.7%,对氨苄西林的耐药率分别为16.7%和86.8%。结论 肠球菌属总体耐药率较高,红霉素、环丙沙星、利福平对肠球菌的敏感率较低,已检测发现VRE,临床上应加强监测,并根据药敏结果、感染严重程度合理选择适当的抗菌药物。     

屎肠球菌类毒力基因hyl功能的动物实验研究

目的构建屎肠球菌透明质酸酶基因(hyaluronidase,hyl)突变株(*hyl),通过动物实验来研究hyl基因的功能。方法通过小鼠腹膜炎模型和兔心内膜炎模型来评价屎肠球菌hyl基因突变株(*hyl)的毒力下降情况。结果突变株在小鼠腹膜炎模型中的半数致死量(LD50)提高了7倍,存活率也明显高于野生株,引起的兔心内膜炎的病理改变也较野生株轻微。结论hyl基因在屎肠球菌致病中起着重要的作用,可能是屎肠球菌的毒力因子之一。     

肠球菌表面蛋白基因与生物膜形成关系的探讨

目的探讨肠球菌表面蛋白（esp）基因与生物膜形成之间的关系，以利临床采取预防措施和选药治疗。方法应用PCR方法检测145株临床分离肠球菌中esp的携带率．并用斑点杂交确证。用96孔聚苯乙烯板进行生物膜形成试验。结果96株粪肠球菌和49株屎肠球菌中esp的检出率分别为34．4％和36．7％；生物膜形成率分别为93．8％和91．8％；esp阳性的肠球菌生物膜形成率为100％。结论肠球菌普遍具有形成生物膜的能力，但生物膜的形成并不仅限于携带esp的肠球菌。     

不同动物源肠球菌耐药性及毒力基因测定与分析

目的通过测定120株不同来源肠球菌耐药性分布情况,毒力基因cylA、efaA、gelE、esp、ace和AS的携带率,旨在了解肠球菌耐药现状和毒力基因分布情况,为选择临床治疗方案和探索肠球菌致病机制提供参考。方法设计6对特异性引物,应用PCR方法对分离到的120株肠球菌进行6种毒力基因检测,同时用纸片扩散法对11种常用抗生素进行耐药性试验。结果 120个肠球菌分离株对抗生素均具有3重以上的耐药性,且不同来源菌株的多重耐药性有明显差异,其中以病死猪内脏分离株多重耐药性最为严重;总体上,上述分离株对林可霉素耐药率最高,为达96.00%(115/120),对环丙沙星、卡那霉素、四环素和达福普汀的耐药率均达到50%以上,而对替考拉宁的耐药率最低,仅为0.83%(1/120)。6种毒力基因cylA、efaA、gelE、esp、ace和AS在受试菌株中的阳性检出率依次为39.17%、56.67%、68.33%、23.33%、26.67%和13.33%;不同来源的肠球菌毒力基因携带率不同,且携带毒力基因数量也有明显差异,其中,来自病死猪内脏的肠球菌分离株携带毒力基因的比例较高,且有超过50%的菌株同时携带4种毒力基因及以上。结论肠球菌的耐药率和多重耐药性与毒力基因携带阳性率有一致性,且与菌株来源关系密切,因此对这一传统意义上的机会性病原体值得深入研究其中的因果关系。     

导尿管相关粪肠球菌生物被膜形成的分子机制探讨

目的 探讨经筛选分离的导尿管生物被膜粪肠球菌的明胶酶编码基因gelE、菌毛操纵子ebpA,毒力调控器fsr系统的fsrB基因与粪肠球菌生物被膜形成的相关性,为深入研究粪肠球菌生物被膜形成的分子机制奠定基础.方法 运用刚果红培养基筛选导尿管生物被膜阳性菌并对其进行生物被膜半定量检测;通过细菌生理生化反应进行鉴定;采用PCR技术检测分离得到的生物被膜粪肠球菌及其相应的浮游菌的gelE、ebpA和fsrB基因.结果 从导尿管中分离鉴定筛选到21株生物被膜粪肠球菌和21株浮游状态粪肠球菌.21株粪肠球菌生物被膜菌组gelE、ebpA和fsrB基因检出的阳性率分别是9.52％(2/21)、95.24％ (20/21)、9.52％(2/21);其相应的21株浮游菌组粪肠球菌检出的阳性率分别是85.71％(18/21)、9.52％ (2/21)、90.48％(19/21),P均＜0.05.结论 gelE、ebpA和fsrB基因与粪肠球菌生物被膜的形成密切相关.ebpA基因能够促进粪肠球菌生物被膜的形成,gelE和fsrB基因会抑制粪肠球菌生物被膜的形成.     

消化内科患者感染粪肠球菌的毒力基因检测及耐药性研究

[目的]检测我院消化内科患者感染粪肠球菌的毒力基因,分析粪肠球菌的耐药性。[方法]收集患者的痰液、血液及尿液标本,分离并鉴定粪肠球菌(重复的除外)158株;制备细菌DNA模板并设计引物,PCR扩增粪肠球菌的毒力基因,采用K-B法测定粪肠球菌对一般常用抗生素的耐药性。[结果]患者感染的粪肠球菌分离株8种毒力基因为ace基因(335bp)、cylA基因(327bp)、cylB基因(843bp)、cylM基因(742bp)、cylL1基因(624bp)、efaA基因(180bp)、esp基因(933bp)及gelE基因(1200bp),片段大小与理论值相符;ace、cylA、cylB、cylM、cylL1、efaA、esp及gelE基因的阳性检出率分别为15.19%、55.70%、44.94%、48.10%、53.16%、69.62%、56.96%及63.92%;粪肠球菌临床分离株对四环素、红霉素、庆大霉素、氨苄西林、环丙沙星、呋喃妥因及万古霉素的耐药率分别为77.85%、81.65%、74.05%、41.14%、53.16%、17.72%及6.96%,对替考拉宁未产生耐药性。[结论]推测本院消化内科患者感染粪肠球菌8种毒力基因的携带率为15.19%~69.62%,粪肠球菌对四环素等常用抗生素均有不同程度的耐药性,对替考拉宁无耐药性,可为临床治疗提供一定参考。     

肠球菌生物学性状及耐药性的调查

目的　了解湖北地区肠球菌的耐药性 ,指导临床用药。方法　对 9所大型综合医院感染标本中分离的肠球菌进行鉴定和药敏试验 ,并以“WHONET4”软件分析。结果　 335株肠球菌中 ,粪肠球菌检出率居首位 (77 9% ) ,屎肠球菌次之(9 2 % )。氨苄青霉素耐药肠球菌 (ARE)分离率为 17 0 % ,氨基糖苷类高水平耐药肠球菌 (HLAR)为 38 2 % ,未检出耐万古霉素肠球菌 (VRE)。结论　湖北地区肠球菌耐药性呈上升趋势 ,应加强肠球菌的鉴定及耐药监测     

肠球菌耐药性与耐万古霉素基因检测

目的了解国内肠球菌对万古霉素耐药情况与机制。方法琼脂平板法测定抗生素对肠球菌最低抑菌浓度(MIC)值,聚合酶链反应(PCR)检测MIC值上升的肠球菌耐万古霉素基因。结果340株临床分离肠球菌中,粪肠球菌对青霉素类耐药率为15%左右,对氟喹诺酮类耐药率18.8%～37.6%,屎肠球菌耐药率明显高于粪肠球菌。9株肠球菌(1株为屎肠球菌)对万古霉素MIC值为8～16mg/L,替考拉宁MIC为0.25～1mg/L,对青霉素、氨苄西林均敏感,PCR检测结果,只有粪肠球菌B144vanC2/3阳性,其余菌株van基因均为阴性。结论耐万古霉素肠球菌国内仍处于低水平,且没有发现具有临床意义的vanA、vanB型耐药,万古霉素、替考拉宁、青霉素、氨苄西林均可作为治疗粪肠球菌感染药物,但需密切注意肠球菌耐药。     

248株肠球菌对抗菌药物的敏感性分析

目的了解全身感染和伤口分泌物感染的粪肠球菌、屎肠球菌对抗菌药物的敏感性,为临床提供治疗依据。方法采用微量稀释法对248株自临床送检的血液、尿液及其他标本分离的肠球菌进行药敏试验,测定其MIC值。结果248株肠球菌属中,粪肠球菌187株、屎肠球菌61株,两者的比例为3.06∶1。粪肠球菌对氨苄西林、环丙沙星、左氧氟沙星、青霉素G的敏感率为63.2%～99.1%,高于屎肠球菌的16.7%～31.9%;屎肠球菌对四环素的敏感率为40.3%,高于粪肠球菌的19.3%;2种肠球菌对红霉素的敏感率分别是13.9%和8.3%,对利奈唑烷和万古霉素的敏感性均＆gt;95%;分离自伤口分泌物的粪肠球菌对氨苄西林、环丙沙星、左氧氟沙星、青霉素G、四环素的敏感性略高于血液分离菌,分离自伤口分泌物的屎肠球菌对氨苄西林、红霉素、高浓度庆大霉素、青霉素G、四环素的敏感性低于血液分离菌,伤口分泌物和血液分离得到屎肠球菌对利奈唑烷均100.0%敏感。结论粪肠球菌、屎肠球菌对不同的抗菌药物的敏感性差异较大,治疗时应根据耐药特点及菌种间的耐药性差异选择相应的药物和方案,目前万古霉素和利奈唑烷仍然是治疗肠球菌属感染的有效药物。     

A potential virulence gene,hylEfm, predominates in Enterococcus faecium of clinical origin

An open reading frame (hyl(Efm)) with homologies to previously described hyaluronidase genes has been identified in nonstool isolates of Enterococcus faecium. E. faecium isolates (n=577) from diverse sources were screened for the presence of hyl(Efm) and esp(Efm), a putative virulence gene associated with epidemic E. faecium strains. The presence of esp(Efm) was roughly twice that of hyl(Efm), but both were found primarily in vancomycin-resistant E. faecium isolates in nonstool cultures obtained from patients hospitalized in the United States. These data suggest that specific E. faecium strains may be enriched in determinants that make them more likely to cause clinical infections. Differences in the prevalence of these strains may help explain variations in the clinical importance of multiresistant E. faecium across different continents.     

Molecular epidemiology of Enterococcus sp. isolated in a university hospital in Algeria

Abstract Background: The aim of this study was to describe the epidemiology of enterococci isolated from infections at an Algerian university hospital, and to evaluate the prevalence of vancomycin-resistant enterococci (VRE) and the clonal cluster present in this country. Methods: Patients who presented at Annaba University Hospital with Enterococcus infections were prospectively included over a 1-y period (2010). All Enterococcus sp. isolated were characterized by antibiotic resistance, van and erm genes, repetitive sequence-based polymerase chain reaction (rep-PCR), multi-locus sequence typing (MLST), and virulence genes. Results: A total of 125 Enterococcus isolates recovered from 125 patients (59% female; median age 54 y, range 2-86y) were studied. No differences in epidemiological data were observed between infections by Enterococcus faecalis vs Enterococcus faecium. However a high proportion of E. faecium were resistant to ampicillin (95%). The prevalence of VRE, corresponding to 4 vanC1-Enterococcus gallinarum, was 3.2%. A high level of genomic diversity among strains was noted, with the importance of sequence type (ST) 78 (which belongs to clonal complex (CC) 17) in E. faecium and ST317 and CC2 in E. faecalis. Conclusions: This first study on enterococci isolated in Algeria shows the low prevalence of VRE, but the presence of clonal complexes linked to VRE and vancomycin-sensitive enterococci associated with hospital infections. Moreover the high level of macrolide resistance and/or ampicillin resistance in E. faecium suggests close monitoring of the epidemiology of these strains.     

Epidemiological study on vancomycin-resistant enterococci from fecal samples in the east area of Japan

Recently, Vancomycin-resistant enterococci (VRE) have become important nosocomial pathogens in the world. In Japan, the VRE-infection was first reported in 1996. However, an epidemiological study on VRE has not been aggressively done in Japan. We conducted a survey study to explore the incidence and antimicrobial susceptibility of vancomycin-resistant enterococci isolated from fecal samples at 45 hospitals in the east area of Japan (Kanto, Koshinetsu, Tohoku, and Hokkaido) during June 1998 to March 1999. The Enterococcosel agar containing vanocomycin (BBL) was used for screening VRE from fecal samples in each hospital. The susceptibilities of the isolates to 8 antimicrobials were determined by the broth microdilution method and the definitions of resistance were based on current standards of the NCCLS standards. The VRE genotypes (vanA, vanB, vanC1, and van C2/3) were confirmed by amplifying the respective genes by PCR. Eight hundred and ninety four strains of enterococci were tested by the microtiter plates hybridization method (WAKUNAGA SEIYAKU, Japan). One thousand five hundred eighty three strains of enterococci were collected from 6,914 patients in 45 hospitals. These strains included 72 (4.5%) strains Enterococcus faecalis, 33 (2.1%) strains Enterococcus faecium, 17 (1.1%) strains Enterococcus avium, 1,040 (65.7%) strains Enterococcus gallinarum, 386 (24.4%) strains Enterococcus cassliflavus, and 35 (2.2%) strains Enterococcus flavescens. These strains of vancomycin-resistant E. faecalis were isolated from 3 patients, two of these 3 strains had van A gene and other one had van B gene. Those 3 strains were in the Kanto area, and 2 of 3 strains were in Tokyo, Generally, though van A type VRE was highly resistaant to both vancomycin and teicoplanin. In our study, two strains of van A type E. faecalis were highly resistant to vancomycin (MICs > 128 micrograms/ml) and susceptible to teicoplanin with MICs 4 micrograms/ml. Those two strains were different in susceptibilities of minocycline and ofloxacin. The result of the analysis of PFGE had also different patterns. VanB type E. fecalis was highly resistant to vancomycin and susceptible to teicoplanin (MICs 0.25 microgram/ml). For ampicillin and imipenem, 3 strains of E. faecalis were susceptible (MIC < or = 1 microgram/ml). One of 562 strains of E. gallinarum had vanB and vanC1 genes and was moderately resistant to vancomycin and susceptible to teicoplanin. All strains of E. casseliflavus and E. flavescens had vanC2/C3 gene only. All strains of E. faecium and E. avium did not detect van genes. From this result, it was supposed that VRE were very rare in the east of Japan.     

上海地区复旦大学附属儿科医院近10年儿童泌尿道感染病原菌分布及其耐药性变迁

目的 了解泌尿道感染患儿病原菌的构成和耐药变化.方法 以复旦大学附属儿科医院2001年1月至2010年12月间临床诊断为泌尿道感染的患儿为研究对象,对尿液进行细菌培养和鉴定.药物敏感试验采用琼脂扩散法(KB).计数资料采用卡方检验,耐药率的趋势变化采用曲线回归分析.结果 10年间共分离到10 686株致病菌,致病菌构成比分析显示,大肠埃希菌的分离率最高,为38％～56％.粪肠球菌从0升至17％ (F=34.075,P=0.000),屎肠球菌从3％升至15％(F=7.139,P=0.028).大肠埃希菌对头孢唑啉、头孢呋辛钠、头孢噻肟、头孢他啶的耐药率呈上升趋势,分别从35.8％上升至62.3％(F=215.735,P=0.000)、33.1％上升至57.0％(F=70.674,P=0.000)、19.0％上升至56.5％(F=52.355,P=0.000)、2.1％上升至23.5％(F=16.807,P=0.003)；大肠埃希菌对环丙沙星的耐药率在23.4％～33.8％(F=20.989,P=0.349),对阿米卡星及头孢哌酮/舒巴坦的耐药率均不超过7％,未发现对亚胺堵南耐药.屎肠球菌对氨苄西林、环丙沙星的耐药率分别超过60.0％及50.0％,对呋喃妥因的耐药率从32.0％降到9.4％(F=34.075,P=0.000).粪肠球菌对氨苄西林的耐药率由40.0％降到9.6％(F=17.497,P=0.009),对环丙沙星的耐药率从60.0％降到21.6％(F=12.826,P=0.009),对呋喃妥因的耐药率从9.2％降到1.4％(F=7.139,P=0.028).两种肠球菌对红霉素的耐药率均超过75.0％,未发现对万古霉素耐药的肠球菌.结论 大肠埃希菌、粪肠球菌、屎肠球菌是儿童泌尿道感染的三种主要致病菌,肠球菌在泌尿道感染致病菌中的构成比呈上升趋势.氨苄西林对粪肠球菌仍敏感,而屎肠球菌可选择药物已很少.     

Variant esp gene as a marker of a distinct genetic lineage of vancomycin-resistant Enterococcus faecium spreading in hospitals

In the USA, vancomycin-resistant Enterococcus faecium (VREF) is endemic in hospitals, despite lack of carriage among healthy individuals. In Europe, however, hospital outbreaks are rare, but VREF carriage among healthy individuals and livestock is common. We used amplified fragment-length polymorphism analysis to genotype 120 VREF isolates associated with hospital outbreaks and 45 non-epidemic isolates from the USA, Europe, and Australia. We also looked for the esp virulence gene in these isolates and in 98 VREF from animals. A specific E. faecium subpopulation genetically distinct from non-epidemic VREF isolates was found to be the cause of the hospital epidemics in all three continents. This subpopulation contained a variant of the esp gene that was absent in all non-epidemic and animal isolates. Identification of the variant esp gene will be important in guiding infection-control strategies, and the Esp protein could be a new target for antibacterial therapy.     

The conjugative transfer of beta-hemolysin plasmid in Enterococcus faecalis to Enterococcus faecium

The conjugative transfer by using a filter mating method in 109 cases of combinations between 20 strains of beta-hemolytic Enterococcus faecalis and 28 strains of alpha-hemolytic Enterococcus faecium was tried. A stable transconjugation was observed in two cases: from E. faecalis No. 170 to E. faecium No. 49 and from E. faecalis No. 500 to E. faecium No. 48. From the results, it was proved that the beta-hemolysin plasmid of E. faecalis is transferred by conjugation to E. faecium. The virulence (LD50) of E. faecium No. 17049, No. 50048 (heterogeneous transconjugant) and E. faecalis No. 17067, No. 50067 (homologous transconjugant) in mice was compared with their parent strains. The virulence of E. faecalis transconjugants was more marked than that of the recipients. However, the virulence of E. faecium transconjugants was not enhanced. This fact suggests that, only in E. faecalis, does beta-hemolysin play a role in its pathogenicity in mice. beta-Hemolysin in E. faecium transconjugants does not participate in pathogenicity in mice. It may be due to the fact that it takes more time to cause beta-hemolysis on blood agar plate or beta-hemolysin production in B-YEG broth by E. faecium transconjugants than by E. faecalis transconjugants.