esp和hyl基因在不同来源屎肠球菌菌株中的分布及与万古霉素耐药性的相关性研究

目的检测屎肠球菌（Enterococcus faecium，Efm）可能的毒力基因esp和hyl在不同来源的临床分离屎肠球菌中的分布，探讨这两个基因与屎肠球菌致病性关系，以及万古霉素耐药性是否与其致病性有关。方法采用菌落杂交法检测364株不同PFGE型的屎肠球菌中esp、hyl、vanA和vanB基因的分布，同时采用NCCLS的微量稀释法测定这些细菌对万古霉素和替考拉林的MIC。结果临床分离菌株中esp（Efm）和hyl（Efm）的阳性率（59．5％和32．8％）明显高于非临床分离菌株（14．4％和5．3％）；非临床分离屎肠球菌中esp和hyl阳性的菌株，除了来自屎肠球菌感染患者粪便之外，其余绝大多数均为阴性。临床分离菌株中耐万古霉素菌株（VRE）esp的阳性率（64．8％）明显高于万古霉素敏感菌株（VSE，42．9％），且以vanA最为明显（72．7％），hyl基因在临床分离菌株中的分布与万古霉素耐药性无关。绝大多数hyl基因阳性菌株（94％）esp亦呈阳性。结论esp和hyl基因主要出现于临床感染的屎肠球菌，可能与其致病性有关。esp基因与万古霉素耐药性密切相关；hyl基因则仅与vanA型耐药有相关性。     

耐万古霉素肠球菌耐药基因及毒力因子研究

目的 研究耐万古霉素肠球菌(VRE) van基因及肠球菌毒力基因esp、hyl、cylA、gelE 的携带情况,分析其对抗菌药物的耐药特性和流行特性,为临床抗菌药物的选择和感染控制提供依据.方法 采用含有6 μg/ml万古霉素的琼脂筛选平板(ADSP)筛选VRE菌株,VITEK-60全自动微生物分析仪检测常用抗菌药物的敏感性;运用PCR方法检测万古霉素耐药基因vanA、vanB、vanC1/C2、vanM和其携带的毒力基因esp、hyl、cylA、gelE;并对VRE菌株进行多位点序列分型(MLST).结果 360株肠球菌中共筛出VRE菌株7株,均为屎肠球菌;7株VRE对万古霉素均为高水平耐药,部分菌株对替考拉宁中介和敏感.所有VRE菌株均携带vanA基因,vanB、vanC1/ C2、vanM均为阴性;毒力因子esp均为阳性,其中一株菌株同时携带4种毒力因子.7株VRE的ST型呈散在分布.结论 本研究发现基因型为vanA型而表型为vanB型的VRE菌株;VRE菌株常对多种抗菌药物耐药且毒力因子携带率高;利奈唑胺可作为治疗VRE菌株的推荐药物.     

血液分离肠球菌毒力基因及生物膜形成相关性分析

目的了解血液分离肠球菌毒力基因及其与生物膜形成能力的相关性。方法收集血培养分离粪肠球菌42株和屎肠球菌44株,采用纸片扩散法检测菌株对常规药物的敏感性;PCR法检测肠球菌6种毒力基因cyl、esp、asal、hyl、gelE和agg;采用结晶紫染色法测定肠球菌生物膜形成能力;统计分析肠球菌毒力基因与生物膜形成能力的关系。结果屎肠球菌对青霉素、氨苄西林、左旋氧氟沙星的耐药率高于粪肠球菌;除esp、hyl外,cyl、asal、gelE和agg基因在粪肠球菌检出率均显著高于屎肠球菌(P<0.05);血液分离肠球菌生物膜阳性率为32.6%(28/86),其中粪肠球菌和屎肠球菌生物膜阳性率分别为59.5%(25/42)和6.8%(3/44);肠球菌毒力基因的携带与其生物膜阳性率无相关性(P>0.05)。结论致血流感染的屎肠球菌其耐药率高于粪肠球菌,粪肠球菌生物膜的形成能力高于屎肠球菌,两者的毒力基因与生物膜形成不具相关性。     

万古霉素非敏感肠球菌的筛选和基因型分析

目的对实验室保存的325株肠球菌进行万古霉素非敏感肠球菌筛选,并对筛选出的菌株进行耐药表型、耐药基因型、菌株同源性分析。方法采用琼脂平皿二倍稀释法筛选万古霉素非敏感肠球菌,并检测菌株对替考拉宁的敏感性;采用PCR方法检测万古霉素非敏感肠球菌的耐药基因型;通过肠道细菌基因间重复一致序列(ERIC)PCR技术、脉冲场凝胶电泳(PFGE)技术、多位点序列分型(MLST)技术进行菌株的同源性分析。结果从325株临床分离肠球菌中共筛选出17株万古霉素非敏感肠球菌,包括1株粪肠球菌、11株屎肠球菌和5株鹑鸡肠球菌。其中1株粪肠球菌和11株屎肠球菌对万古霉素显示高水平耐药,对替考拉宁耐药或中介耐药,PCR检测结果显示van A型;5株鹑鸡肠球菌对万古霉素中介耐药,对替考拉宁敏感,PCR检测结果显示van C1型。ERIC同源性分析显示同基因型的大多数菌株显示相似的分型;PFGE同源性分析显示,17株临床万古霉素非敏感肠球菌分型不同,不属于单克隆流行播散;MLST同源性分析显示,1株临床万古霉素非敏感粪肠球菌属于ST4,11株临床万古霉素非敏感屎肠球菌共分为6个ST型,其中4株属于ST78,是屎肠球菌出现频率最高的ST型。结论我国万古霉素耐药菌在粪肠球菌中分离率较低,但屎肠球菌中万古霉素耐药情况较严重,并且耐药菌株携带易于传播和转移的van A基因。同源性分析结果显示万古霉素耐药存在同源性传播的可能性。     

粪肠球菌和屎肠球菌临床分离株的毒力因子与耐药性分析

目的检测临床分离肠球菌的耐药和毒力因子，比较粪肠球菌和屎肠球菌的耐药性和毒力特征。方法采用琼脂稀释法检测肠球菌对万古霉素（VAN）、四环素（TET）、环丙沙星（CIP）、红霉素（ERY）、复方新诺明（SMZ）、替考拉宁（TEC）、克林霉素（CLI）的耐药性；采用微量板测定肠球菌的生物膜形成能力；观察肠球菌的β溶血和明胶溶解结果，同时用PCR方法检测相应基因cylA和gelE。结果粪肠球菌和屎肠球菌的耐药率分别为0％-100％和3．23％-96．77％，后者对环丙氟哌酸、红霉素的耐药程度高于前者。B溶血试验阳性率为19．23％，cylA基因阳性率为35．38％；明胶表型阳性率为21．54％，gelE阳性率为40．0％，其中有46．15％（12／26）阳性者未出现相应表型；生物膜形成检出率为36．92％；粪肠球菌3种毒力因子（表型和基因型）的阳性率均高于屎肠球菌。结论屎肠球菌耐药率高于粪肠球菌，粪肠球菌毒力因子阳性率高于屎肠球菌。     

临床分离屎肠球菌和粪肠球菌敏感性对比分析

目的 了解医院临床分离的145株屎肠球菌和86株粪肠球菌的耐药性.方法 采用法国梅里埃公司的VITEK2COMPACT全自动微生物分析仪对菌株进行鉴定和抗菌药物敏感性检测.结果 屎肠球菌和粪肠球菌主要来源于尿液（48.92％）,其次为血液（12.99％）.屎肠球菌和粪肠球菌对万古霉素、利奈唑烷和替加环素的敏感率均为100％;屎肠球菌除对喹奴普汀/达福普汀和四环素的敏感率显著高于粪肠球菌外,对呋喃妥因、氨苄西林、青霉素G、莫西沙星、环丙沙星、左氧氟沙星的敏感率均显著低于粪肠球菌.结论 屎肠球菌和粪肠球菌对抗菌药物的敏感性存在差异,临床应根据抗菌药物敏感性特征结合种间耐药性差异制定治疗方案.目前万古霉素、利奈唑烷,替加环素为治疗屎肠球菌和粪肠球菌感染的最有效抗菌药物.     

血液分离肠球菌毒力基因检测及生物膜形成测定北大核心CSCD

目的：检测肠球菌的主要毒力基因,并测定其生物膜形成情况。方法收集血液来源标本中的粪肠球菌28株,屎肠球菌54株,采用多重PCR检测肠球菌的5种主要毒力基因：asa1、esp、hyl、cylA及gelE;利用微孔板法检测生物膜的形成。结果粪肠球菌中asa1、esp、cylA、gelE的同时检出率为50％,且28株菌均检测到了至少一种毒力基因,仅在粪肠球菌中检测到了asa1、cylA和gelE基因;屎肠球菌对esp的检出率分别为50％,hyl和esp的同时检出率为22．2％,hyl基因仅在屎肠球菌中存在,18．5％的屎肠球菌没有检测到5种毒力基因中的任何一种。粪肠球菌和屎肠球菌形成生物膜阳性菌株的比率分别为85．7％和63．0％。结论致血液感染粪肠球菌在主要毒力基因的种类、毒力基因检出率及生物膜形成阳性菌株数均高于屎肠球菌。     

2012～2017年北京某医院耐万古霉素屎肠球菌临床分布及分子特征变迁分析

目的 对我院6年间分离的耐万古霉素屎肠球菌(VREfm)进行临床分布和分子特征变迁分析。方法 收集2012年1月至2017年12月从我院住院患者临床标本中分离的168株VREfm,采用多重PCR法检测vanA和vanB基因。采用另一种多重PCR法检测常见5种毒力基因(esp、gelE、asa1、cylA和hyl)。同源性分析采用多位点序列分型(MLST)。结果 所有菌株均携带vanA基因,且同时对万古霉素和替考拉宁耐药,基因型和表型均属于高水平耐药。分离自尿液标本的菌株最多(58.9%),其次是肛拭子(16.1%)和血液(15.5%)。esp和hyl的检出率分别为89.2%和27.9%,且hyl在血液来源菌株的检出率显著高于肛拭子(42.3%vs 14.8%,P<0.05)。MLST分型共检出21个ST型,均属于同一个group,其中ST78占绝对优势(50.0%),在医院内广泛传播,其他ST型均处于散发状态,且呈动态变化。与全部168株VREfm相比,ST78型具有较高的esp检出率(97.6%vs 89.2%,P<0.05)和较低的hyl检出率(15.5%vs 27.9...     

1999-2006年北京朝阳医院革兰阳性球菌耐药性分析

目的 探讨北京朝阳医院1999-2006年临床分离的革兰阳性球菌的耐药变迁,以指导临床合理使用抗菌药物.方法 采用MIC法进行抗菌药物敏感性试验,以WHONET5.3软件分析数据.结果 6192株临床分离的革兰阳性球菌中,前4位病原菌依次为凝同酶阴性葡萄球菌、金黄葡萄球菌、粪肠球菌、屎肠球菌.耐甲氧西林金黄葡萄球菌(MRSA)、耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)平均检出率分别为88.4%、86.9%,金黄葡萄球菌对青霉素、氨苄西林耐药率最高,8年间始终保持在90.0%以上.未发现对万古霉素耐药或中介的金黄葡萄球菌或凝固酶阴性葡萄球菌.2003年首次分离耐万古霉素肠球菌(VRE),至2006年共发现30株VRE,其中13株为耐万古霉索粪肠球菌,均为VanB基因型;17株为耐万古霉素屎肠球菌,均为VanA基因型,耐万古霉素屎肠球菌分离率呈上升趋势.对于粪肠球菌活性最高的是万古霉素、氨苄西林、青霉素,敏感率分别为98.7%、95.7%、85.6%.但是青霉素敏感性略有下降,从94.3%降至84.6%.克林霉素的耐药率8年中始终维持在99.0%以上.屎肠球菌对红霉素、克林霉素的耐药率均达95.0%以上,对青霉素、氨苄西林、环丙沙星的耐药率均大于90.0%.屎肠球菌最为敏感的药物是万古霉素,对四环素敏感性出现上升的趋势,从27.8%升到82.6%.结论 万古霉素对临床常见的革兰阳性菌保持很高的活性,发现30株耐万古霉素的肠球菌.     

972株肠球菌耐药性分析

目的分析临床分离的肠球菌的菌型特点和对常用抗生素的耐药性.方法在VITEK32仪器上用GPI鉴定卡及API系统鉴定感染性疾病各种临床标本分离的肠球菌;在VITEK32仪器上用GPS107鉴定卡及K-B法测定肠球菌对药物敏感性.结果从痰或咽拭子、尿液、宫颈分泌物、伤口分泌物等处分离的972株肠球菌分别占总数的30.7%、29.2%、16.5%、13.0%;粪肠球菌和屎肠球菌占总数的92.4%.高水平氨基糖苷类耐药(HLAR)粪肠球菌和屎肠球菌检出率高达60%左右;屎肠球菌对青霉素等β内酰胺类抗生素耐药率明显高于粪肠球菌.未检出耐万古霉素肠球菌(VRE).结论肠球菌是本地区呼吸道、泌尿系感染主要病原菌之一.临床应重视HLAR株引起的感染,根据药敏结果合理用药.     

Clinical implications of biofilm formation by Enterococcus faecalis in the urinary tract

The potential relationships between biofilm formation and pathogenicity of Enterococcus faecalis in urinary tract infections (UTI) were investigated. Over a 12-year period from 1991 through 2002, a total of 352 E.faecalis isolates were collected from patients with complicated UTI (one isolate per patient) at the urology ward of Okayama University Hospital. We analyzed the prevalence and transferability of genes encoding virulence factors(asa1, esp, cylA, gelE /sprE )and antimicrobial resistance(aac(6') /aph(2')). The production of biofilm, hemolysin and gelatinase by these isolates was also examined and the associated medical records of patients were retrospectively reviewed. Of 352 E. faecalis isolates, 315 possessed and/or genes. Of the 63 hemolysin- and 167 gelatinase-producing isolates, 59 and 94 isolates, respectively, possessed both asa1 and esp genes. E. faecalis isolates with both asa1 and esp genes formed biofilms at significantly higher rates than those with neither gene (P=0.038). The genes encoding asa1, cylA , and aac(6') /(aph(2') were transferable and appeared to have accumulated in these isolates. The E. faecalis isolates possessing asa1 and/or esp genes were found from both catheter-related or -unrelated UTI. Our study indicates that E. faecalis isolates that have accumulated virulence genes are apt to form persistent biofilms in the urinary tracts.     

Vancomycin resistance, esp, and strain relatedness: a 1-year study of enterococcal bacteremia

The prevalence of esp, a gene associated with infection-derived and outbreak strains, in enterococcal blood isolates from 2002 was determined. Fifty-five of 137 (40.1%) Enterococcus faecalis isolates, 30 of 58 (51.7%) E. faecium isolates, 1 of 1 E. raffinosus isolate, 0 of 4 E. gallinarum isolates, and 0 of 1 E. casseliflavus isolate were positive. esp wasn't associated with vancomycin resistance (VR) or clinical service. VR E. faecium isolates were less genetically diverse than vancomycin-susceptible strains. A large cluster of VR isolates, belonging to esp-positive E. faecium, was revealed. These data support the hypothesis that esp and VR may contribute to dissemination of particular clones.     

Novel vancomycin-resistance transposon, plasmid replicon types, and virulence factors of vancomycin-resistant Enterococci in Zhejiang, China

Forty-seven vancomycin-resistant Enterococcus (VRE) strains were isolated from clinical samples in 13 Zhejiang hospitals and fecal samples from ICU patients in a large teaching hospital in China. No VRE isolates were detected in healthy human subjects. CC17 was the main clonal complex in clinical Enterococcus faecium isolates but not in isolates from healthy human subjects. Novel vancomycin-resistance transposons were detected among VRE strains. This is the first report demonstrating insertion of tnpA and fosB genes in the vanRS-vanH intergenic region of Tn1546 leading to coresistance to vancomycin and fosfomycin. The four plasmid replicon types (pRUM, pRE25, pEF418, and pB82) were more common in VRE isolates, suggesting their association with vancomycin resistance and nosocomial transmission. The prevalence rate of vancomycin-resistant Staphylococcus aureus-related Inc18-like plasmid, pIP501, in VRE was 21.3%. The prevalence of the esp gene among VRE isolates was high (76.6%). In several VRE strains, the esp and hyl genes were cotransferred with the vanA gene by conjugation. Although the frequency of VRE is low in Chinese hospitals, its association with virulence determinants, the vancomycin-resistance transposon with other resistance gene insertions or plasmids may lead to multidrug resistance and the evolution of pathogenic VRE.     

Phenotypic and genotypic characterization of clinical and intestinal enterococci isolated from inpatients and outpatients in two Brazilian hospitals

The phenotypic and genotypic characteristics of clinical and intestinal enterococcal isolates recovered from inpatients and outpatients of two Brazilian hospitals, located in Niterói city, Rio de Janeiro, Brazil, were compared. A total of 601 strains were studied, including 253 isolated from different clinical sources and 348 intestinal strains (205 isolated from inpatients and 143 from outpatients) recovered from fecal specimens. Isolates were identified by using conventional physiological tests and evaluated for high-level resistance to aminoglycosides (HLR-A) and resistance to vancomycin and ampicillin by the agar screening technique. Susceptibility to several antimicrobial agents was evaluated by the disk diffusion method. The genetic diversity of Enterococcus faecalis strains presenting HLR-A was assessed by pulsed-field gel electrophoresis of chromosomal DNA after SmaI digestion. E. faecalis was the most frequent species among clinical isolates (90.1%) and intestinal strains from inpatients (53.6%). E. casseliflavus was the prevalent species among intestinal isolates from outpatients (35.0%). Clinical isolates were shown to be resistant to erythromycin (53.0%), tetracycline (52.2%), ciprofloxacin (36.4%), gentamicin (36.4%), streptomycin (30.4%), chloramphenicol (34.4%), norfloxacin (32.0%), imipenem (3.2%), and ampicillin (2.8%). Vancomycin resistance was only detected in intrinsic vancomycin-resistant enterococcal species. The overall prevalence of HLR-A was 52.2% among clinical isolates and 40.5% among intestinal strains. However, HLR-A was significantly more frequent among intestinal strains obtained from inpatients (56.6%) than among strains from outpatients (17.5%). Three major clonal groups were found among E. faecalis strains exhibiting HLR-GE or HLR-GE/ST (clonal groups GE-A and GE-B), and strains exhibiting HLR-ST (clonal group ST-A). HLR-A, particularly HLR-GE, was most frequently associated with enterococcal strains of nosocomial origin. Isolates included in the major clonal groups were recovered from clinical and intestinal sources from patients in both hospitals, indicating both intrahospital and interhospital spread of strains.     

Influence of origin of isolates, especially endocarditis isolates, and various genes on biofilm formation by Enterococcus faecalis

Endocarditis isolates of Enterococcus faecalis produced biofilm significantly more often than nonendocarditis isolates, and 39% of 79 versus 6% of 84 isolates produced strong biofilm (P < 0.0001). esp was not required, but its presence was associated with higher amounts of biofilm (P < 0.001). Mutants disrupted in dltA, efaA, ace, lsa, and six two-component regulatory systems were largely unaltered, while disruptions in epa (encoding enterococcal polysaccharide antigen), atn (encoding an autolysin), gelE (encoding gelatinase), and fsr (encoding the E. faecalis regulator) [corrected] resulted in fewer attached bacteria, as determined using phase-contrast microscopy, and less biofilm (P < 0.0001).     

Clonal spread of a vancomycin-resistant Enterococcus faecium strain among bloodstream-infecting isolates in Italy

Recent data indicated that the rate of vancomycin resistance in bloodstream-infecting enterococcal isolates in Italy is one of the highest in Europe. The aims of this study were to characterize bloodstream-infecting vancomycin-resistant enterococci (VRE) obtained from various Italian hospitals and to establish whether the isolates were clonally related. During the years 2001 to 2003, a total of 39 VRE isolates were obtained from 19 hospital laboratories in various areas of Italy. Species identification and resistance genotypes of the isolates were obtained by multiplex PCR. Further characterization included antibiotic susceptibility testing, pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA, detection of virulence genes (esp and hyl), and multilocus sequence typing (MLST) of selected isolates. VRE were identified as 31 Enterococcus faecium (VREfm) isolates and 8 E. faecalis isolates. All but one isolate carried the vanA gene; one VREfm isolate carried the vanB gene. Analysis of the PFGE profiles showed that 28 VREfm isolates shared a similar electrophoretic profile, designed type 1, and were clonally related. All type 1 isolates were resistant to ampicillin, streptomycin, gentamicin, and rifampin and were positive for the esp gene. MLST identified an allelic profile (ST78) comprising purK allele 1, belonging to the C1 clonal lineage, characteristic of human infection and hospital outbreak isolates. The vanB-carrying VREfm isolate, of PFGE type 2, was shown to be a single-locus variant of ST78. Our data indicate that the recent increase in the number of bloodstream infections caused by VRE in Italy is due to the spread of a hospital-adapted, multidrug-resistant VREfm clone belonging to an internationally disseminated lineage.     

Genetic and phenotypic differences among Enterococcus faecalis clones from intestinal colonisation and invasive disease

This study investigated the differences among Enterococcus faecalis isolates from the intestinal compartment of healthy volunteers (n = 36), intensive care unit (ICU) patients (n = 29) and blood isolates (n = 31) from the same institution, in comparison with seven epidemic clones from other institutions. In general, isolates from colonised ICU patients and from bacteraemic patients showed higher rates of antimicrobial resistance than isolates from colonised healthy volunteers, particularly for erythromycin and aminoglycosides. The proportion of isolates/clone was 1.05 in the community, 2.63 in the ICU, and 1.47 among bacteraemic cases, suggesting low clonal variation in ICUs. Two clones, RENC1 and RENC2, were frequently found as intestinal colonisers of ICU patients, and RENC1 was also found to colonise healthy volunteers. These two clones were a cause of bacteraemia in the institution studied, and RENC2 was also detected in various other Spanish hospitals. Both RENC1 and RENC2 were esp+, bacteriocin producers, and were resistant to all antibiotics tested except vancomycin and ampicillin. RENC1 produced haemolysin whereas RENC2 produced protease. The ace, agg, cylA, esp and gelE genes were more common among colonising strains from ICU patients than among isolates from individuals in the community. In both colonised groups (ICUs and the community), 40-50% of isolates harbouring the gelE and cylA genes did not express the corresponding phenotypes. Thus, the study indicated that particular E. faecalis clones might be well-adapted to hospital environments, and that surveillance should be directed specifically towards rapid detection of these disseminating clones in order to prevent infections and clonal spread.     

Diversity of clones and genotypes among vancomycin-resistant clinical enterococcus isolates recovered in a spanish hospital

Forty-three vancomycin-resistant enterococci (VRE) from different patients were recovered in a Spanish Hospital (2003-2010), representing 0.4% of the total of enterococci recovered. Mechanisms detected were vanA (five Enterococcus faecium, two E. faecalis), vanB2 (seven E. faecium, five E. faecalis), vanB1 (one E. faecalis), and vanC1/2 (22 E. gallinarum, 1 E. casseliflavus). Four different Tn1546 structures were found among the seven vanA strains, three of them with insertions (ISEf1 or IS1542) or deletions. Most of the VRE presented a multiresistance phenotype and harbored different resistance genes [erm(B), tet(M), tet(L), ant(6)-Ia, aac(6')-aph(2'), aph(3')-IIIa, and catA]. Sixteen unrelated pulsotypes were detected among the 20 vanA/vanB E. faecalis and E. faecium isolates by pulsed-field-gel-electrophoresis and 11 unrelated pulsotypes among the 22 E. gallinarum isolates. Six different sequence types (ST) were demonstrated among the 12 vancomycin-resistant E. faecium strains (one of them new), and 5 were included into the clonal-complex (CC) CC17. Five different ST were detected among the eight E. faecalis strains. The esp gene was detected in 58% and 25% of E. faecium and E. faecalis strains, respectively, and the hyl gene in 78% and 89%, respectively. A high diversity of clones and genotypes of VRE were detected in this hospital.