杭州市五家医院万古霉素耐药肠球菌耐药转座子结构及分子分型

目的 明确万古霉素耐药肠球菌(VRE)耐药转座子结构及分子分型.方法 收集2006年4月至2007年4月杭州市5家医院21株VRE菌株,用Etest法进行抗菌药物的药敏试验,并通过PCR、接合试验、质粒提取、耐药转座子结构、脉冲凝胶电泳(PFGE)、多位点序列分型(MIST)及多位点串联重复序列分型(MLVA)进行研究.结果 21株VRE基因型及表型均符合vanA.属于10个不同的PFGE型,7个不同的MLST分型,4个不同的MLVA分型,其中18株属于克隆复合体CC17,另外3株为ST343与CC17接近.所有VRE菌株均对利奈唑胺及替加环素敏感.多对引物对转座子的不同区域的PCR扩增并进行序列拼接、比对,发现其中2株VRE菌株携带典型的耐药转座子Tn1546,其余19株VRE菌株均携带一种新的与Tn1546相似耐药转座子,均在vanXY之间反向插入IS1485.18株VRE菌株均可通过滤膜接合试验进行万古霉素耐药转移,接合菌均含有约54 000 bp大小的质粒.结论 杭州市5家医院21株VRE菌株均为vanA表型及基因型,发现了一种新的万古霉素耐药转座子结构.21株VRE菌株经MLST分型属于7个不同的序列分型,属于或接近易在医院环境里生存并在近年来迅速造成了全球播散的克隆复合体CC17.     

万古霉素耐药肠球菌的检测和分子流行病学分析

目的为了解本院万古霉素耐药肠球菌（VRE）的流行情况，对VRE株进行实验室检测和分子流行病学检测分析。方法采用多重PCR及测序检测万古霉素耐药基因van，Etest法测定VRE株对万古霉素、替考拉宁的耐药表型；应用脉冲场凝胶电泳（PFGE）对11株VRE进行检测流行病学分析。结果检出1株VanA、4株VanB、5株vanC1，1株VanC2，对万古霉素、替考拉宁的药敏表型与基因型一致；11株VRE中2号vanB株与5号vanB株显示出相同片段，其他VRE株则分别有多于5个区带不同。结论实验室准确检测VRE对防止VRE感染和流行是非常重要的；PFGE电泳分析结果表明了本院11株VRE医院感染为散发。     

万古霉素耐药肠球菌耐药基因检测及分子流行病学调查

目的了解我院分离的万古霉素耐药肠球菌的耐药相关基因及流行情况。方法收集2012~2013年间分离的7株经E-test确认的万古霉素耐药肠球菌(VRE),提取基因组DNA,PCR检测van耐药基因型,多重PCR检测asal,gelE,cylA,esp,hyl5个毒力基因;多位点序列分型(MLST)及脉冲场凝胶电泳(PFGE)进行流行病学调查。结果 7株VRE耐药基因型van均为A型;有6株检测到毒力基因esp、hyl,其余为阴性;MLST及PFGE均显示菌株2、4、5、7为主要型别(ST 17),其余3株各不相同。结论我院分离的VRE耐药基因型为vanA型,毒力基因esp、hyl检出率较高,2013上半年有小规模VRE流行,应加强对于VRE的管控工作。     

滤膜法万古霉素耐药肠球菌转移方式的研究

目的 研究万古霉素耐药肠球菌转座子的转移方式,为临床预防感染和流行病学调查提供依据.方法 滤膜法进行质粒接合转移试验;依据2009年美国临床实验室标准化研究所(CLSI)推荐的纸片扩散法对万古霉素耐药的屎肠球菌进行药敏试验;PCR方法检测接合子耐药基因.结果 40株VRE供体菌均将VanA基因型转移给受体菌粪肠球菌JH2-2,并伴有其他耐药性的同时转移.40株接合子均检测出VanA基因.证实40株VRE全部转移成功.结论 肠球菌携带VanA基因的转座子Tn1546可以通过质粒接合转移试验在种间转移,引起肠球菌耐药播散.接合转移试验是研究肠球菌耐药性转移的重要方法.     

耐万古霉素肠球菌耐药基因、转座子结构和多位点序列分析

目的分析耐万古霉素肠球菌(VRE)耐药基因和多位点序列分型(MLST)情况。方法收集三亚中心医院临床分离的耐万古霉素的80株屎肠球菌和12株粪肠球菌,采用VITEK MS微生物鉴定系统进行菌种鉴定。采用VITEK 2 Compact自动化鉴定药敏仪测定VRE对14种常用抗菌药物的敏感性。采用微量肉汤稀释法测定VRE对万古霉素和替考拉宁的最小抑菌浓度(MIC),并确定耐药表型;采用聚合酶链反应(PCR)筛选VRE的毒力基因(esp、hyl、gelE、asal、cy1A、efaA、ace)和转座子结构;对VRE进行MLST。结果质谱鉴定结果为80株屎肠球菌和12株粪肠球菌。体外药物敏感性试验结果表明,屎肠球菌和粪肠球菌对万方霉素的耐药率均为100%,对氨苄西林的耐药率分别为97.5%和50.0%,但对呋喃妥因的耐药率较低。92株VRE对万古霉素和替考拉宁的MIC分别为192～256和32～256μg/mL,均为VanA表型;92株VRE基因型均为vanA,以esp毒力基因所占比例最高(82.5%);屎肠球菌以esp-hyl基因组合最常见,粪肠球菌以多基因组合为主;通过Tn1546基因转座子结构分析可分为A～E 5个型别;92株VRE的MLST包括7个ST型别,分别为ST17(43.5%)、ST78(32.6%)、ST203(6.5%)、ST363(5.4%)、ST555(4.3%)、ST1392(4.3%)和ST1394(3.4%),其中ST17和ST78均属于CC17克隆复合体。结论 92株VRE均为多重耐药株、vanA基因型和CC17克隆复合体,耐药基因和毒力基因携带率均较高,临床应引起重视。     

万古霉素耐药肠球菌的表型及基因型检测

目的了解我院耐万古霉素肠球菌(VRE)的耐药表型、基因型及流行情况。方法用K-B纸片扩散法检测临床分离肠球菌的药物敏感性,E-test法检测VRE对万古霉素的最低抑菌浓度(MIC);PCR检测vanA、vanB、vanC1和vanC2-3基因型;脉冲场凝胶电泳(PFGE)分析VRE同源性。结果 73株肠球菌中检出3株万古霉素耐药屎肠球菌,检出率为4.1%;3株屎肠球菌对万古霉素和替考拉宁均耐药,但对利奈唑胺敏感;基因型检测显示3株屎肠球菌均为vanA型,PFGE结果显示该3株VRE不属于同一型别。结论我院住院患者中已出现VRE,应加强医院感染控制,以阻止VRE菌株在院内的传播和流行。     

武汉地区门诊患者喹诺酮耐药鼠伤寒沙门菌耐药机制分析

目的 研究分析腹泻门诊患者中分离的喹诺酮耐药鼠伤寒沙门菌的耐药机制和遗传关系.方法 对2002-2005年问武汉同济医院腹泻门诊患者中分离的36株喹诺酮耐药鼠伤寒沙门菌进行了耐药谱测定,并通过PCR方法和序列测定对整合子、β内酰胺酶基因、喹诺酮耐药决定区的突变、qnr基因和aac(6')-Ib-cr基因进行了分析,运用脉冲场电泳方法(pulsed-field gel electrophoresis,PFGE)对所收集的菌株进行了分子分型,分析喹诺酮耐药鼠伤寒沙门菌的耐药机制和遗传关系.结果 喹诺酮耐药鼠伤寒沙门菌均为多重耐药株,普遍携带有Ⅰ类整合子,环丙沙星耐药菌株与敏感菌株在PFGE谱型上存在显著性差异,31株环丙沙星耐药菌株在喹诺酮耐药决定区中至少携带GyrA和ParC上的3个点突变,且在这些菌株中均检出了OXA-30基因,这些菌株对头孢吡肟的敏感性出现了不同程度的下降.结论 对环丙沙星耐药的鼠伤寒沙门菌在武汉地区已普遍存在,且这些菌株具有独特的遗传背景,建议在今后的细菌耐药性监测工作中应对这类细菌的耐药谱变化进行重点监测,尤其应加强对氟喹诺酮-三代头孢类抗菌药物均耐药菌株的针对性预警监测.     

碳青霉烯类耐药的肠杆菌科细菌耐药机制研究

目的 探讨碳青霉烯类耐药肠杆菌科细菌(CRE)的耐药机制,建立获得性碳青霉烯酶流行监测体系.方法 收集华中科技大学同济医学院附属同济医院2007年1月至2010年6月临床分离非重复肠杆菌科细菌5604株,其中美罗培南抑菌环直径≤21 mm的肠杆菌科细菌100株.药物敏感性试验筛选出碳青霉烯类耐药菌株,采用聚合酶链反应(PCR)对碳青霉烯酶基因和基因附属结构进行分析;采用脉冲场凝胶电泳(PFGE)和Southern印迹杂交方法分析耐药菌质粒;采用多位点序列分型(MLST)方法对菌株进行分型及分析同源性;采用十二烷基磺酸钠-聚丙烯凝胶电泳(SDS-PAGE)方法对菌株的外膜孔道蛋白进行分析.结果 共检出CRE 11株,其中克雷伯菌属细菌7株.抗菌药物敏感性试验显示所有菌株对大多数抗菌药物耐药,最低抑菌浓度美罗培南8～ 64 mg/L,亚胺培南4 ～ 64 mg/L,厄他培南4～64 mg/L,对氨基糖苷类和氟喹诺酮类抗菌药物的敏感性则变化较大.PCR检出 IMP-4阳性菌株6株,KPC-2阳性菌株3株,其中1株ST476型肺炎克雷伯菌同时携带blaIMP-4和blaKPC-2.对基因附属结构进行分析,结果显示blaKpC-2基因位于由Tn3转座子和Tn4401部分片段构成转座子上.PFGE显示大多数CRE含有3个或3个以上质粒.MLST分型发现2株肺炎克雷伯菌同属于ST476型.SDS-PAGE提示1株产酸克雷伯菌(Kox656)存在外膜孔道蛋白(OmpK35和OmpK36)双缺失.结论 本院CRE主要是肺炎克雷伯菌,产生碳青霉烯酶是细菌耐药的主要原因,IMP-4是其主要酶型.碳青霉烯酶基因的出现和传播对治疗和感染控制造成巨大威胁,应重视医院细菌耐药性特点及耐药菌感染的临床流行病学资料,从而为临床合理用药提供参考.     

耐万古霉素肠球菌的基因检测

目的 调查上海华山医院2007-2009年间VRE的分离率,并对耐药菌株的分子特性进行研究,为本地区VRE的预防和控制提供有价值的信息.方法 通过琼脂平板稀释法(ADSP)从临床鉴定的890株肠球菌中筛选VRE菌株,并通过微量肉汤稀释实验测其对万古霉素及替考拉宁的MIC;采用PCR及测序方法检测万古霉素耐药基因以及可能毒力基因esp,hyl;运用MLST对VRE进行克隆分型并通过PFGE分型技术进行验证.结果 ADSP法及微量肉汤稀释实验共筛选出13株VRE菌株.其中6株VRE(2007-2008年)只对万古霉素耐药,但对于替考拉宁敏感(万古霉素MIC64～256μg/ml);耐药基因PCR产物测序结果提示这6株VRE均携带同一种可能导致万古霉素耐药的新型基因,该基因序列与已报道的耐药基因均不同;MLST及PFGE分型提示其属于不同型别.另外7株VRE(2009年1-7月)对万古霉素和替考拉宁的MIC结果分别为32～64μg/ml和16～32 μg/ml,耐药基因检测均为vanA.MLST分型结果提示13株VRE共分为4个不同的ST型,其中11株均属克隆复合型CC-17.13株VRE毒力基因esp和hyl的阳性率分别为69.2%和30.8%.结论 上海华山医院VRE克隆分型以CC17为主,同时发现上海华山医院存在一种可能导致万古霉素耐药的新型基因介导的VRE,该新基因的功能和定位尚待进一步研究.     

耐万古霉素肠球菌基因分型及耐药性分析

目的明确万古霉素耐药肠球菌(vancomycin resistant enterococci,VRE)耐药基因型别及耐药性,指导临床用药。方法收集北京中日友好医院2016年10月至2017年10月临床分离的17株VRE,Vitek 2 Compact全自动细菌鉴定及药敏分析系统检测其对临床常用14种抗菌药物的敏感性,E-test法确证其对万古霉素和替考拉宁的药物敏感性;PCR法检测17株VRE的耐药基因vanA、vanB、vanC1、vanC2/3,并经测序验证。结果 17株VRE均为耐万古霉素屎肠球菌,均对环丙沙星、左氧氟沙星、莫西沙星、青霉素100%耐药,对利奈唑胺、替加环素、奎奴普丁/达福普汀等敏感;其耐药基因型均为vanA型,其中有4株vanB表型-vanA基因型。结论所收集的VRE均对万古霉素高水平耐药,耐药型均为vanA型,应加强临床分离株的耐药监控,合理使用抗菌药物。     

High frequency of vancomycin-resistant Enterococcus faecium isolates with VanB phenotype and vanA genotype in Korean hospitals

A total of 59 vancomycin-resistant Enterococcus faecium (VREF) clinical isolates were collected from 8 Korean hospitals for 2 months in 2004. They were investigated by genotyping for glycopeptide resistance, multilocus sequence typing (MLST), esp repeat profiling, and structural analysis of Tn1546-like element. Nine of 59 VREF isolates (15.3%) from 5 hospitals in Korea showed VanB phenotype, but they contained vanA gene. MLST and esp repeat profiling indicated that E. faecium isolates with VanB phenotype and vanA genotype occurred from independent genetic background except 3 isolates from 1 hospital. Structural analysis of Tn1546 also showed that these isolates were not clonally related. Data showed a relatively high frequency of VREF isolates with incongruence between phenotype and genotype for glycopeptide resistance in Korean hospitals.     

Loss of vancomycin resistance not completely dependent on the Tn1546 element in Enterococcus faecium isolates

We investigated characteristics of 3 Enterococcus faecium strains (SHY-1, SHY-2, and SHY-3) isolated successively from 1 patient. In vitro susceptibility testing was performed using broth microdilution method. Change of vancomycin MIC was monitored during incubation with vancomycin for SHY-3 strain. Genetic backgrounds were determined both by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). In addition, the genetic variations among Tn1546 element were investigated by polymerase chain reaction (PCR) assay and sequencing. vanA and vanX expression of 3 strains was evaluated using quantitative real-time (qRT)-PCR method. Although all the strains possessed the vanA gene, SHY-3 was susceptible to glycopeptides, while SHY-1 and SHY-2 were resistant to glycopeptides. Judged by MLST and PFGE, 3 strains have the same genetic background. The vancomycin resistance of SHY-3 was not recovered after exposure to vancomycin. The vanA and vanX genes were expressed in strains SHY-1 and SHY-2 but not in strain SHY-3, although the SHY-2 and SHY-3 strains shared the same arrangement of the van gene cluster, a common 88-bp deletion in vanS gene. Our results indicate that vancomycin resistance might not be completely dependent on the Tn1546 element.     

Molecular analysis of Tn1546-like elements mediating high-level vancomycin resistance in Enterococcus gallinarum

OBJECTIVES: Four Enterococcus gallinarum isolates, all highly resistant to vancomycin, were studied in order to investigate their relationship and to gain insight into the molecular events responsible for their acquired resistance. METHODS: Extensive molecular analysis was performed to compare the four E. gallinarum isolates and their Tn1546-like elements. RESULTS: The four strains had very similar random amplified polymorphic DNA (RAPD) patterns and different but related PFGE profiles. Genotypic analysis demonstrated that all carried both vanC-1 and vanA genes. Using a vanA probe, no hybridization was detected to plasmid DNA, whereas hybridization to different SmaI fragments of the four strains was obtained with total DNA. Amplification and sequencing experiments showed that all four strains carried a Tn1546-like element that contained the orf2, vanR, vanS, vanH, vanA and vanX genes and was flanked at both ends by oppositely oriented IS1216V sequences. On the left side of the vanA cluster, all lacked IRL, and all had, upstream from orf2, 1029 bp of the 3' end of orf1. On the right side, one of the strains lacked vanY, vanZ and IRR, whereas in one of the other three there was an IS1542 element inserted within the vanZ gene. In one strain, an additional IS1216V element was inserted in the intergenic region vanX-vanY. CONCLUSIONS: This is the first study providing a molecular analysis of chromosomal Tn1546-like elements (possibly composite transposons) associated with high-level vancomycin resistance in human and animal strains of E. gallinarum. These molecular findings, together with those from PFGE and RAPD, suggest that the four E. gallinarum isolates are related and might have a common ancestor.     

Comparison of Tn1546-like elements in vancomycin-resistant Staphylococcus aureus isolates from Michigan and Pennsylvania

In 2002, the first two clinical isolates of vancomycin-resistant Staphylococcus aureus (VRSA) containing vanA were recovered in Michigan and Pennsylvania. Tn1546, a mobile genetic element that encodes high-level vancomycin resistance in enterococci, was present in both isolates. With PCR and DNA sequence analysis, we compared the Tn1546 elements from each isolate to the prototype Tn1546 element. The Michigan VRSA element was identical to the prototype Tn1546 element. The Pennsylvania VRSA element showed three distinct modifications: a deletion of nucleotides 1 to 3098 at the 5' end, which eliminated the orf1 region; an 809-bp IS1216V-like element inserted before nucleotide 3099 of Tn1546; and an inverted 1,499-bp IS1251-like element inserted into the vanSH intergenic region. These differences in the Tn1546-like elements indicate that the first two VRSA isolates were the result of independent genetic events.     

Molecular diversity and evolutionary relationships of Tn1546-like elements in enterococci from humans and animals

We report on a detailed study on the molecular diversity and evolutionary relationships of Tn1546-like elements in vancomycin-resistant enterococci (VRE) from humans and animals. Restriction fragment length polymorphism (RFLP) analysis of the VanA transposon of 97 VRE revealed seven different Tn1546 types. Subsequent sequencing of the complete VanA transposons of 13 VRE isolates representing the seven RFLP types followed by sequencing of the identified polymorphic regions in 84 other VanA transposons resulted in the identification of 22 different Tn1546 derivatives. Differences between the Tn1546 types included point mutations in orf1, vanS, vanA, vanX, and vanY. Moreover, insertions of an IS1216V-IS3-like element in orf1, of IS1251 in the vanS-vanH intergenic region, and of IS1216V in the vanX-vanY intergenic region were found. The presence of insertion sequence elements was often associated with deletions in Tn1546. Identical Tn1546 types were found among isolates from humans and farm animals in The Netherlands, suggesting the sharing of a common vancomycin resistance gene pool. Application of the genetic analysis of Tn1546 to VRE isolates causing infections in Hospitals in Oxford, United Kingdom, and Chicago, Ill., suggested the possibility of the horizontal transmission of the vancomycin resistance transposon. The genetic diversity in Tn1546 combined with epidemiological data suggest that the DNA polymorphism among Tn1546 variants can successfully be exploited for the tracing of the routes of transmission of vancomycin resistance genes.     

A new Tn1546 type of VanB phenotype–vanA genotype vancomycin-resistant Enterococcus faecium isolates in mainland China

VanB phenotype–vanA genotype vancomycin-resistant Enterococcus faecium (VREF) has never been reported in mainland China. We investigated the frequency and molecular characteristics of this strain in a Beijing tertiary hospital. Of 23 vanA genotype VREF clinical isolates, 12 (54.3%) were VanB phenotype–vanA genotype. Mutilocus sequence typing (MLST) analysis revealed that all isolates belong to a single clonal complex (CC78), which has been disseminated worldwide. Based on MLST and pulsed-field gel electrophoresis, 23 isolates were polyclonal dissemination in our hospital. Tn1546-like element structure analysis showed that of 12 VanB phenotype–vanA genotype isolates, 5 had complete deletion of vanY and vanZ accompanying insertion of IS1216V in vanX–vanY intergenic region, 5 had ISEfa4 insertion in orf2–vanR intergenic region, a new Tn1546 structure type, and 2 were identical to VanA phenotype–vanA genotype VREF. Data showed that the deletion of vanY and vanZ genes or ISEfa4 insertion in orf2–vanR intergenic region can partly explain the causes of difference between phenotype and genotype.     

Molecular study of vancomycin-resistant enterococci isolated from humans and from food in a cattle-rearing area of France

OBJECTIVES: Study possible links between vancomycin-resistant enterococci strains isolated from human stool samples and from pork or poultry food products. METHODS: One hundred and eleven vancomycin-resistant enterococci strains (15 VanA and 96 VanC) were isolated from human stool samples and from pork or poultry food products. Characterization of the Tn1546-like element of the 15 VanA strains was realized by restriction analysis of PCR products and polymorphism study. The 96 strains of VanC phenotype (75 Enterococcus gallinarum and 21 Enterococcus casseliflavus) were analysed by pulsed-field gel electrophoresis (PFGE). RESULTS: In the study described here, polymorphism of the Tn1546-like element enabled the establishment of five groups. Groups III, IV and V were found only in human strains. Groups I and II were found to occur in strains isolated from humans and from food, suggesting a possible transfer of the Tn1546-like element. The isolates from Group I harboured the whole Tn1546 element. In Group II, the five strains possessed a novel Tn1546-like element, characterized by a single-nucleotide difference in the vanX gene and a deletion upstream from the nucleotide 164. Analysis by PFGE of the 75 E. gallinarum strains revealed 20 different patterns. One pattern was shared by isolates from pork food and human samples. None of the 21 E. casseliflavus strains tested was found to share similar PFGE patterns. CONCLUSIONS: Results tend to support the possible transfer of the Tn1546-like element between strains of VanA phenotype. Concerning VanC phenotype strains, the transfer was not demonstrated.     

Diversity of Tn1546 and its role in the dissemination of vancomycin-resistant enterococci in Portugal

We characterized the molecular diversity of vanA vancomycin-resistant enterococci (VRE; 176 isolates/87 pulsed-field gel electrophoresis types) from different sources and cities in Portugal (1996 to 2004): (i) food animals (FA; n = 38 isolates out of 31 samples), hospitalized humans (HH; n = 101/101), healthy human volunteers (HV; n = 7/4), and environmental sources (n = 30/10). Some strains were isolated from different hosts and persistently recovered for years. Twenty-four Tn1546 variants were identified, all located on plasmids (30 to 250 kb). Some Tn1546 variants were associated with specific sources such as FA (3 types), HH (11 types), or HV (1 type), while others were recovered from isolates of different origins (8 types). Polymorphisms in the central vanRSHA region of Tn1546 were scarcely detected, while alterations upstream of vanR and downstream of vanA were frequently identified involving mutations (vanS and vanX), deletions (vanY), insertions (IS1216V, ISEf1, and IS19; sequences with or without homology with others available in GenBank databases), and different genetic rearrangements. Most Tn1546 variants contained IS1216V (14 types) or ISEf1 (6 types). IS1216V was found alone or associated with an IS3-like element at different orientations and positions in Tn1546 from human, animal, and environmental samples. ISEf1 was located within vanX-vanY region at nucleotide 9044 of Tn1546 variants mostly associated with clinical isolates, suggesting a common genetic platform. IS19 was observed within the vanX-vanY region in one Tn1546 variant from poultry. Recent spread of VRE in Portugal reflects a complex epidemiology involving both clonal spread and plasmid dissemination containing a variety of Tn1546 types. Apparent Tn1546 heterogeneity among enterococci from human, animal, and environmental sources might reflect frequent genetic exchange events and evolution of particular widely disseminated genetic elements.     

Vancomycin-Resistant Enterococci from Humans and Retail Chickens in Taiwan with Unique VanB Phenotype-vanA Genotype Incongruence

Vancomycin resistant enterococci (VRE) with VanB phenotype-vanA genotype incongruence were found in all 39 VRE isolated from chicken carcasses and four human VRE isolates in Taiwan. Three identical mutations in the vanS gene were found in the VanB phenotype-vanA genotype VRE sequenced. This finding indicates possible transmission of glycopeptide resistance among different hosts.