肠球菌中糖肽类耐药基因及其调控

肠球菌对糖肽类耐药表分ＶａｎＡ，ＶａｎＢ和ＶａｎＣ３种，ＶａｎＡ型最常见，耐药质粒携带转座子Ｔｎ１５４６，Ｔｎ１５４６带有一系列耐药基因，它们的基因产物共同作用，导致菌株对糖肽类药物耐药。其中连接酶（ＶａｎＡ）和脱氢酶（ＶａｎＨ）催化合成Ｄ－Ａｌａ－Ｄ－Ｌａｃ，替代Ｄ－Ａｌａ－Ｄ－Ａｌａ连接入肽聚糖前体，阻止了糖肽与细胞壁的结合，Ｄ，Ｄ－二肽酶（ＶａｎＸ）通过水解Ｄ－Ａｌａ－Ｄ－Ａｌａ而抑制正常肽     

肠球菌CRISPR-Cas系统基因结构的分析及其与耐药基因的关系

CRISPR-Cas系统是一种细菌的适应性免疫系统,参与特异性防御不同类型的可移动遗传元件,如质粒、噬菌体、转座子等的入侵。旨在分析肠球菌（Enterococcus）基因组中该系统的基因结构,并探讨其与细菌耐药基因之间的关系。NCBI数据库中下载10种肠球菌的全基因组信息,利用软件对CRISPR-Cas系统的分布、cas1基因、重复序列、间隔序列等进行比对分析;查找耐药相关基因,分析其与CRISPR-Cas系统之间的关系。235株肠球菌中含完整CRISPR-cas系统的有35株（14.9%）,含确定CRISPR阵列196个和cas基因簇46个。肠球菌基因组中CRISPR系统主要为II-A型（80.4%）,其次是II-C型（15.2%）,cas1基因序列的系统发育分析结果与CRISPR-cas系统的分型基本一致。肠球菌CRISPR-Cas系统的分布在不同菌种之间差异较大;CRISPR-Cas系统可能阻碍肠球菌某些耐药基因的水平转移。     

耐万古霉素肠球菌基因分型及耐药机制的研究进展

近年来肠球菌的耐药性呈上升趋势,已引起越来越多的临床医生和微生物学家的重视。目前,肠球菌对抗生素的敏感性以糖肽类最高,但20世纪90年代以来,国内、外均报道了耐万古霉素的肠球菌。耐药菌株的增多给临床治疗带来了较大困难,因此,对耐万古霉素肠球菌耐药机制的探讨尤为重要。基于对万古霉素和替考拉宁的耐药水平、诱导性及转移性的不同,对糖肽类抗生素耐药的肠球菌已报道5类为获得性耐药肠球菌,1类为固有耐药肠球菌。本文就耐万古霉素肠球菌的基因分型与耐药机制的研究进展作一综述。     

耐万古霉素肠球菌与定植肠球菌的致病基因检测

目的 通过对耐万古霉素肠球菌（Vancomycin-resistant Enterococci,VRE）及分离非耐万古霉素肠球菌（Vancomycin-sensitive Enterococci,VSE）致病基因的筛查,了解致病基因分布情况,探索肠球菌致病机制,为日后致病机制的研究提供基线资料。方法 收集浙江省人民医院的部分临床患者标本中分离出的VRE,与ICU采集的患者肛周和手臂标本分离的肠球菌属细菌。以PCR方法筛查出ace、asa1、cylA、efaA、esp、gelE和hyl七种致病基因。结果 VRE组esp总体阳性率最高,约占71.4%,其中屎肠球菌中esp最高,约占73.5%,其致病基因携带谱以“esp-hyl”基因组合为主;粪肠球菌全部菌株均携带efaA基因,其致病基因均为三种以上基因组合,以“esp-cylA-gelE-asa1-efaA”多见。VSE组总体阳性率同样esp最高,约占40.0%;其中屎肠球菌以esp、hyl单个基因携带为主;粪肠球菌以“esp-ace-cylA-gelE-asa1-efaA”最多见。结论 两种肠球菌各种致病基因携带率差异较大,两种肠球菌致病机制存在差异,本地区致病基因发挥作用的以esp、hyl为主。     

肠球菌万古霉素耐药基因簇遗传特性

万古霉素耐药肠球菌自20世纪80年代后期被发现以来,已逐渐发展成为重要的医院感染病原菌。此类耐药肠球菌携带的万古霉素耐药基因簇编码产物可催化合成与万古霉素、替考拉宁等糖肽类抗生素亲和力极低的细胞壁前体导致耐药。目前已在肠球菌中发现的万古霉素耐药基因簇根据基因序列及构成不同分为9个型别;依据它们编码的连接酶合成产物不同又可分为D-Ala:D-Lac连接酶基因簇(VanA、VanB、VanD及VanM型)和D-Ala:D-Ser连接酶基因簇(VanC、VanE、VanG、VanL和VanN型)。这些耐药基因簇介导的耐药水平及其传播模式各有特点。文章综述了肠球菌中万古霉素耐药基因簇的类型、基因构成及传播特性。     

万古霉素耐药肠球菌基因分型及耐药性分析

目的了解本地区万古霉素耐药肠球菌(Vancomycin resistant Enterococci,VRE)耐药基因型别及耐药性,为临床治疗提供依据。方法用PCR方法检测56株VRE的耐药基因vanA、vanB、vanC、vanD、vanE和vanG;用K-B法检测其对临床常用14种抗菌药物的药敏,并用肉汤稀释法检测万古霉素和替考拉宁的药敏。结果 56株VRE中,vanA阳性的43株;vanB、vanC、vanD、vanE和vanG阳性0株;未检测到万古霉素耐药相关基因的13株。2011-2014年万古霉素和替考拉宁的MIC值呈逐年向左漂移趋势,与同期替考拉宁的使用有关。结论本研究所收集VRE对万古霉素的耐药大部分为高水平耐药,所携带的耐药基因类型主要为vanA,另有其他未知基因型以及少数vanA阳性但是表现为万古霉素敏感菌株。     

血流感染粪肠球菌和屎肠球菌的临床分布及耐药研究

回顾性分析上海市某三甲医院血培养阳性标本中粪肠球菌和屎肠球菌的临床分布及对抗菌药物的耐药特征,为临床治疗其所致感染奠定基础。收集上海市某三甲医院2012年2月—2016年9月血流感染患者血液标本中的粪肠球菌和屎肠球菌,采用法国生物梅里埃公司的VITEK 2Compact全自动细菌鉴定和药敏分析系统进行细菌鉴定及药敏测定,研究细菌临床分布特点及对常用抗菌药物的耐药特征。共分离获得30株粪肠球菌和17株屎肠球菌。粪肠球菌样本主要来自泌尿科、消化科和血液科,所占比例分别为13.33%、16.67%和10.00%。粪肠球菌对青霉素、氨苄西林、环丙沙星、左氧氟沙星、四环素和红霉素的耐药率分别为13.33%、10.00%、36.67%、33.33%、66.67%和60.00%。屎肠球菌样本主要来自消化科(29.41%),其对以上抗菌药物的耐药率分别为88.24%、82.35%、88.24%、76.47%、23.53%和70.59%。屎肠球菌对青霉素、氨苄西林、环丙沙星和左氧氟沙星的耐药率显著高于粪肠球菌,而对四环素的耐药率显著低于粪肠球菌。两者均对替加环素、利奈唑胺和万古霉素敏感,但万古霉素对屎肠球菌的最低抑菌浓度显著低于粪肠球菌。结果提示,屎肠球菌对青霉素、氨苄西林、环丙沙星、左氧氟沙星的耐药率高于屎肠球菌,对万古霉素敏感,且其万古霉素最低抑菌浓度低于粪肠球菌。本研究为治疗这两种细菌所致感染的经验性用药提供了数据支持。     

耐万古霉毒肠球菌的耐药机制研究

肠球菌是致病性低的革兰阳性人体正常菌群,具有先天耐药性,也容易被诱导产生新的耐药性,在过去十年中,临床上出现了耐万古霉素的肠球菌,常引起致死感染,治疗极为困难。本文就其耐药机制,起源,危险因素,治疗和预防方面作一综述,有利于临床工作者对VRE的感染进行预防和控制。     

粪肠球菌对泰利霉素药物敏感性与耐药基因erm的分布

目的 了解粪肠球菌对泰利霉素和其他常用抗菌药物的耐药性,以及泰利霉素耐药与红霉素耐药相关基因ermA、ermB、ermC之间的关系。方法 对本院2010‒2016年从各种临床标本收集鉴定的320株粪肠球菌,用微量肉汤稀释法测定这些菌株对泰利霉素及8种临床常用抗菌药物的最小抑菌浓度,并用PCR法检测耐药基因ermA、ermB、ermC的分布。结果 320株粪肠球菌对泰利霉素中介耐药26株,耐药138株,耐药率达51.3%;对红霉素耐药率达95.6%,泰利霉素抗粪肠球菌效果优于红霉素。对利奈唑胺、万古霉素、呋喃妥因和氨苄西林耐药率分别为15.6%、0.6%、2.2%和0.6%。共10株（3.1%）携带ermA基因,207株（64.7%）携带ermB基因,对泰利霉素中介组中有23株ermB基因阳性,耐药组有131株ermB基因阳性,仅1株（0.3%）ermC基因阳性,该菌同时携带ermB基因。结论 粪肠球菌对泰利霉素已有较高耐药率。粪肠球菌对泰利霉素MIC值改变与ermB基因密切相关,与ermA、ermC基因无明显相关性。     

Detection of antibiotic resistance profiles and aminoglycoside-modifying enzyme (AME) genes in high-level aminoglycoside-resistant (HLAR) enterococci isolated from raw milk and traditional cheeses in Turkey

Molecular Biology Reports - The aim of this study was isolation and identification of the high-level aminoglycoside-resistant (HLAR) enterococci in raw milk and dairy products and to analyze their...     

vanA-mediated high-level glycopeptide resistance in Enterococcus faecium from animal husbandry

Abstract Glycopeptide-resistant Enterococcus faecium strains were isolated from a pig farm and a poultry farm both using avoparcin as a food additive. Such organisms were not isolated in a hen's eggs-producing farm not using avoparcin. Glycopeptide-resistant enterococci were also detected in broiler chicken carcasses that were delivered to a hospital's kitchen. The resistance was determined by the vanA gene as indicated by the detection of the inducible 39-kDa cytoplasmic membrane protein and of a vanA -specific DNA sequence amplified by polymerase chain reaction. Genomic DNA fragment patterns of strains from animal sources were different from each other and also from those of strains isolated in hospitals and from sewage treatment plants. This findings suggest the dissemination of the vanA determinant among different enterococcal strains of distinct ecological origin.     

Detection of virulence factors in high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium isolates from a Tunisian hospital

Phenotypic and genotypic determination of virulence factors were carried out in 46 high-level gentamicin-resistant (HLGR) clinical Enterococcus faecalis (n=34) and Enterococcus faecium (n=12) isolates recovered from different patients in La Rabta Hospital in Tunis, Tunisia, between 2000 and 2003 (all these isolates harboured the aac(6')-aph(2") gene). The genes encoding virulence factors (agg, gelE, ace, cylLLS, esp, cpd, and fsrB) were analysed by PCR and sequencing. The production of gelatinase and hemolysin, the adherence to caco-2 and hep-2 cells, and the capacity for biofilm formation were investigated in all 46 HLGR enterococci. The percentages of E. faecalis isolates harbouring virulence genes were as follows: gelE, cpd, and ace (100%); fsrB (62%); agg (56%); cylLLS (41.2%); and esp (26.5%). The only virulence gene detected among the 12 HLGR E. faecium isolates was esp (58%). Gelatinase activity was detected in 22 of the 34 E. faecalis isolates (65%, most of them with the gelE+-fsrB+ genotype); the remaining 12 isolates were gelatinase-negative (with the gelE+-fsrB- genotype and the deletion of a 23.9 kb fragment of the fsr locus). Overall, 64% of the cylLLS-containing E. faecalis isolates showed beta-hemolysis. A high proportion of our HLGR E. faecalis isolates, in contrast to E. faecium, showed moderate or strong biofilm formation or adherence to caco-2 and hep-2 cells.     

屎肠球菌的临床分布与耐药性分析

目的了解医院屎肠球菌的临床分布和耐药情况,为临床抗感染的预防与治疗提供参考。方法回顾性分析1999年1月至2011年12月临床标本中分离的1161株屎肠球菌;用WHONET5．6软件分析耐药率变迁。结果临床分离的1161株屎肠球菌,在同期分离的1944株肠球菌属中占59．72％。主要分离自尿液和血液,分别占40．91％和26．87％;主要分离自外科病区、内科病区、ICU和儿科病区的菌株,分别占29．37％、25．15％、13．95％和13．53％;屎肠球菌对多种抗菌药物耐药,对万古霉素、替考拉宁和利奈唑胺的耐药率较低,分别为1．04％、0．94％和1．85％。结论屎肠球菌在临床的分离率逐年增加,已成为医院内感染的主要病原菌之一,其多药耐药和高耐药现象相当严重,目前万古霉素、替考拉宁和利奈唑胺仍然是治疗肠球菌属引起感染的有效药物。     

临床分离肠球菌的耐药性研究

目的了解近3年来粤东地区临床分离的肠球菌的耐药特征,为预防耐万古霉素肠球菌（VRE）的产生和控制VRE播散提供理论依据和实验基础。方法收集临床分离的215株肠球菌,细菌鉴定及药敏试验采用VITEK-60全自动细菌鉴定仪。结果215株肠球菌中,尿标本中分离肠球菌75株（35％）;痰液中分离出34株（16％）。粪肠球菌141株（65．5％）,屎肠球菌74株（34．5％）。粪肠球菌对万古霉素、呋喃妥因、青霉素G和莫西沙星的敏感率较高,70％～100％;对红霉素和四环素的敏感性较差,11％～33％。屎肠球菌对万古霉素敏感性较好为100％,四环素为62％;而对呋喃妥因、高链霉素、青霉素和左氧氟沙星等敏感性较差（5％～47％）。未检出耐万古霉素肠球菌。结论粤东地区近3年来未发现耐万古霉素肠球菌。肠球菌对各种常见抗生素敏感程度呈下降趋势。屎肠球菌较粪肠球菌耐药性更高。应根据细菌学培养结果合理用药,减少耐药菌株的发生率。     

Erythromycin resistance and virulence genes in Enterococcus faecalis from swine in China

This study aims to describe the erythromycin resistance phenotypes and genotypes, and the prevalence of virulence genes of Enterococcus faecalis isolated from swine in China. A total of 117 nonreplicate E. faecalis isolates, obtained from 502 clinical samples taken from different pig farms between 2007 and 2009 were included in the study. Minimum inhibitory concentrations were determined using the broth microdilution method. All of the isolates were screened for the presence of seven virulence genes (ace, asa1, cylA, efaA, esp, gelE, and hyl). In addition, the DNA from rythromycin-resistant isolates were amplified with primers specific for erythromycin resistance erm(A), erm(B), erm(C), mef(A/E), and msr(C) genes. Results show that erythromycin, tylosin, and ciprofloxacin resistance rates in E. faecalis were 66.67% (n=78), 66.67% (n=78), and 64.10% (n=75), respectively. About 69.23% of isolates (n=81) were positive for gelE, 48.72% (n=57) for ace, 15.38% (n=18) for efa, 7.69% (n=9) for asa1, and 6.84% (n=8) for esp. Among the erythromycin-resistant isolates, erm(B) (n=54) was the most prevalent resistance gene, followed by erm(A) (n=37). A significant correlation was found between the presence of the gelE virulence gene and erythromycin resistance (P<0.05). These findings suggest that enterococci from swine should be regarded with caution because they can be reservoirs for antimicrobial resistance and virulence genes.     

血培养标本中病原菌的分布特征、耐药性变迁和耐药基因分型

目的 分析嘉兴市中医医院血培养标本中病原菌的分布特征、耐药性变迁和耐药基因分型。方法 将本院2013年1月至2016年12月送检的血培养标本进行培养、转种、分离鉴定和药敏试验,采用聚合酶链反应（PCR）技术进行产超广谱β-内酰胺酶（ESBLs）革兰阴性菌和革兰阳性菌的耐药基因检测,并对阳性菌株、科室分布、耐药性等相关数据进行分析。结果 2013年1月至2016年12月共送检血培养标本27 003份,共分离出病原菌（剔除重复菌株）978株,阳性率为3.62%,2015‒2016年阳性率显著低于2013‒2014年,差异有统计学意义（P<0.01）。血培养阳性株数前三位为ICU、儿科和肾内科,2015‒2016年与2013‒2014年相比,ICU的阳性株数构成比显著下降,儿科、呼吸内科和其他科室显著上升,差异有统计学意义（P<0.01）。血培养致病菌检出率最高的为大肠埃希菌,占18.51%,其次为表皮葡萄球菌,占10.53%,不可忽视的是真菌,占4.91%。2015‒2016年与2013‒2014年相比,金黄色葡萄球菌检出率显著上升,粪肠球菌和近平滑假丝酵母菌的检出率显著下降,差异有统计学意义（P<0.05）。血培养分离的大肠埃希菌对氨苄西林的耐药率最高,肺炎克雷伯菌对呋喃妥因的耐药率最高。2015‒2016年与2013‒2014年相比,大肠埃希菌对氨苄西林/舒巴坦和庆大霉素的耐药率显著下降（P<0.01）,肺炎克雷伯菌对庆大霉素的耐药率显著上升（P<0.01）。分离的表皮葡萄球菌和金黄色葡萄球菌对青霉素的耐药率最高,2015‒2016年与2013‒2014年相比,表皮葡萄球菌对红霉素和氯洁霉素的耐药率显著下降（P<0.01）,金黄色葡萄球菌对环丙沙星和左氧氟沙星的耐药率显著下降（P<0.01）。大肠埃希菌ESBLs（+）菌株对氨苄西林、氨苄西林/舒巴坦、头孢唑啉等的耐药率以及所有基因型表达率均显著高于ESBLs（‒）菌株（P<0.05或P<0.01）;肺炎克雷伯菌ESBLs（+）菌株对头孢唑啉、头孢曲松、庆大霉素等的耐药率以及TEM、SHV、CTX-MⅡ基因型表达率均显著高于ESBLs（‒）菌株（P<0.05或P<0.01）。表皮葡萄球菌检出mecA基因（+）72株,金黄色葡萄球菌检出mecA基因（+）53株。屎肠球菌和粪肠球菌分离株中,未发现有vanA（+）、vanB（+）和vanM（+）万古霉素耐药株。结论 了解本院血培养致病菌整体变化趋势、病区分布特点及耐药性变迁和耐药基因分型,对临床合理使用抗生素和院内感染的控制有重要意义。     

Amino acid substitutions in the VanS sensor of the VanA-type vancomycin-resistant Enterococcus strains result in high-level vancomycin resistance and low-level teicoplanin resistance

The vancomycin-resistant enterococci GV1, GV2 and GV3, which were isolated from droppings from broiler farms in Japan have been characterized as VanA-type VRE, which express high-level vancomycin resistance (256 or 512 microg ml(-1), MIC) and low-level teicoplanin resistance (1 or 2 microg ml(-1), MIC). The vancomycin resistances were encoded on plasmids. The vancomycin resistance conjugative plasmid pMG2 was isolated from the GV2 strain. The VanA determinant of pMG2 showed the same genetic organization as that of the VanA genes encoded on the representative transposon Tn1546, which comprises vanRSHAXYZ. The nucleotide sequences of all the genes, except the gene related to the vanS gene on Tn1546, were completely identical to the genes encoded on Tn1546. Three amino acid substitutions in the N-terminal region of the deduced VanS were detected in the nucleotide sequence of vanS encoded on pMG2. There were also three amino acid substitutions in the vanS gene of the GV1 and GV3 strains in the same positions as in the vanS gene of pMG2. Vancomycin induced the increased teicoplanin resistance in these strains.     

肝硬化患者肠球菌感染及体外药敏监测

目的研究肝硬化患者肠球菌感染的现状及药敏特点,以加强对肝硬化肠球菌感染的认识,指导用药。方法留取标本进行细菌培养、鉴定及药敏试验。结果所有纳入研究范围的肝硬化患者共检出肠球菌112株,其中粪肠球菌89株,占79.5%,比例最高,屎肠球菌占14.3%,居第2位。腹水中检出肠球菌64株,占57.1%,其次为痰和尿液,分别为38.1%和14.4%。肠球菌对红霉素、奈替米星的耐药率超过80%,对青霉素G、氨苄西林、呋喃妥因的敏感率超过70%,对左氧氟沙星的敏感率为58.9%,对头孢唑啉、头孢派酮的敏感率分别只有25.0%和33.0%,检出高耐氨基糖苷类肠球菌(HLAR)58株,占51.8%,未检出耐万古霉素和替考拉宁肠球菌,但检出万古霉素中介肠球菌4株,占3.6%。结论肠球菌是肝硬化患者医院感染的重要致病菌,尤以粪肠球菌和屎肠球菌为主,屎肠球菌的耐药率高于粪肠球菌,万古霉素和替考拉宁是治疗肠球菌感染的首选药物。     

Detection of methicillin- and aminoglycoside-resistant genes and simultaneous identification of S. aureus using triplex real-time PCR Taqman assay

In this study we describe a triplex real-time PCR assay that enables the identification of S. aureus and detection of two important antibiotic resistant genes simultaneously using real-time PCR technology in a single assay. In this triplex real-time PCR assay, the mecA (methicillin resistant), femA (species specific S. aureus) and aacA-aphD (aminoglycoside resistant) genes were detected in a single test using dual-labeled Taqman probes. The assay gives simultaneous information for the identification of S. aureus and detection of methicillin and aminoglycoside resistance in staphylococcal isolates. 152 clinical isolates were subjected to this triplex real-time PCR assay. The results of the triplex real-time PCR assay correlated with the results of the phenotypic antibiotic susceptibility testing. The results obtained from triplex real-time PCR assay shows that the primer and probe sets were specific for the identification of S. aureus and were able to detect methicillin- and aminoglycoside-resistant genes. The entire assay can be performed within 3 h which is a very rapid method that can give simultaneous information for the identification of S. aureus and antibiotic resistance pattern of a staphylococcal isolate. The application of this rapid method in microbiology laboratories would be a valuable tool for the rapid identification of the S. aureus isolates and determination of their antibiotic resistance pattern with regards to methicillin and aminoglycosides.