VanB表型-vanA基因型VRE分子特征及遗传背景研究

目的 探讨VanB表型-vanA基因型VRE耐药转座子结构、分子特征及遗传背景,并与VanA表型-vanA基因型VRE进行比较分析,以确定基因型与表型不一致的形成机制.方法 收集2008年3月至2009年1月卫生部北京医院临床标本中21株VRE菌株,用Etest法对10种抗生紊进行MIC测定,并通过PCR、序列测定、接合试验、耐药转座子结构、PFGE及MLST进行分子特征和遗传背景研究.结果 21株VRE均为vanA基因型,其中3株菌呈现VanB表型(万古霉素耐药,替考拉宁敏感);21株菌属于9个不同PFGE型,6个不同MIST型;多对引物对转座子的不同区域PCR扩增并进行序列拼接、比对,发现Tn1546结构中vanX、vanY的缺失及ISEfa4的插入与VanB表型-vanA基因型VRE菌株形成相关.结论 VanB表型-vanA基因型VRE菌株在国内较为罕见,Tn1546结构的改变与菌株的基因型与表型不一致相关.     

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。     

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.     

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。     

临床分离万古霉素耐药肠球菌van基因型及分子分型

目的明确临床分离万古霉素耐药肠球菌(VRE)基因型及分子分型。方法收集临床分离VRE 17株;采用16S r RNA测序对临床株进行菌种确认,微量稀释法和琼脂稀释法测定常用抗菌药物最低抑菌浓度(MIC)。多重PCR进行van基因分型;采用多位点序列分型(MLST)进行分子分型。结果 17株VRE经16S rRNA测序确认均为屎肠球菌;其中12株对替考拉宁耐药。13株检出vanA基因,9株检出vanM基因,5株同时检出vanA及van M基因;17株VRE分属6个MLST型,其中ST78型8株、ST80型4株、ST555型2株,其余3株分别为ST117型、ST262型和ST341型。结论 VRE的临床分离株中van基因型主要为vanA(76.5%),其次为vanM(52.9%),首次发现同时携带vanA及vanM的VRE菌株。     

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.     

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。     

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.     

新型肠球菌万古霉素耐药基因簇——vanM基因簇

目的从屎肠球菌临床分离株中克隆一种介导屎肠球菌对万古霉素耐药的新基因簇。方法采用16SrRNA测序对临床株Efm-HS0661进行菌种确认,并行多位点序列分型（MLST）;通过药敏试验及接合试验,了解菌株耐药性及其可转移性;通过PCR扩增、步移法测序、限制性内切酶片段克隆获取万古霉素耐药基因及其周边序列,并将获得序列与已知万古霉素耐药基因簇进行比对。结果临床株经16SrRNA测序确认为屎肠球菌,MLST分型属ST78型;该菌株对万古霉素及替考拉宁耐药,且可通过接合试验发生转移;测序获得一6592bp核苷酸序列,为含6个万古霉素耐药相关基因的基因簇,其基因结构与已知耐药基因簇不同,命名为vanM型基因簇。其中vanM全长1032bp,其编码蛋白VanM与VanA、VanB、VanD、VanF基因氨基酸序列同源性分别为79．7％、69．7％、66．0％和78．5％。结论从万古霉素耐药屎肠球菌临床株中发现了一种新型万古霉素耐药基因簇——vanM型基因簇。     

耐亚胺培南肺炎克雷伯菌分子流行病学及耐药传播机制研 究简

目的：探讨临床收集亚胺培南耐药肺炎克雷伯菌的耐药分子机制和克隆流行情况。方法采用K-B纸片法进行药敏实验,多位点序列分型（MLST）进行临床菌株克隆分型,滤膜接合法进行质粒接合实验,PCR扩增筛选菌株的常见耐药基因和其周围序列结构。结果亚胺培南耐药肺炎克雷伯菌对青霉素类、头孢菌素类、含抑制剂β-内酰胺类、氨基糖苷类等药物均显示出很强的多重耐药性,MLST分型显示耐药克隆具有明显的流行特征,13株携带blaKPC-2基因的流行克隆均为ST11型。这13株细菌亚胺培南耐药性均由可接合性质粒介导,并且同时介导和转移了编码CTX-M型、TEM型或DHA型的β-内酰胺酶基因。 Tn4401-Tn3复合转座子或其变体2介导了blaKPC-2基因的转移。结论临床肺炎克雷伯菌亚胺培南耐药的主要原因是产KPC-2酶菌株的克隆播散,同时可接合质粒造成的耐药基因快速水平转移也起到了重要作用。     

Van genotype and molecular typing of clinical isolates of vancomycin-resistant Enterococcus北大核心CSCD

Objective To determine the genotype and molecular typing of vancomycin-resistant Enterococcus (VRE). Methods Seventeen clinical isolates of VRE were collected in 2016. The strains were identified to species and confirmed by 16S rRNA sequencing. The minimum inhibitory concentration of antimicrobial agents was determined by microdilution method and agar dilution method. Multilocus sequence typing (MLST) was used for molecular typing. Results The VRE strains were confirmed as Enterococcus faecium by 16S rRNA sequencing. All strains were resistant to vancomycin, but only 12 strains were resistant to teicoplanin. The vanA gene was identified in 13 of the 17 strains. The vanM gene was detected in 9 strains. Both vanA and vanM genes were identified in five of the 17 strains. Six MLST types were identified in the 17 strains, including ST78 (n=8), ST80 (n=4), ST555 (n=2), and one each for ST117, ST262 and ST341. Conclusions The van genotype was primarily vanA (76.5%) and vanM (52.9%) in clinical isolates of VRE. The VRE strains carrying both vanA and vanM were found for the first time. © 2018, Editorial Department of Chinese Journal of Infection. All rights reserved.     

上海地区大环内酯类抗生素耐药肺炎链球菌的遗传学分析

目的 了解上海地区肺炎链球菌中大环内酯类抗生素高耐药菌株的同源性.方法 采用脉冲场凝胶电泳(PFGE)方法对47株临床分离大环内酯类抗生素耐药肺炎链球菌(MRSP)进行同源性分析,并运用多位点序列分型(MIST)对其中6株多重耐药MRSP进行分子遗传学分析,结合血清分型推测其与国际流行克隆株的关系.结果 上海地区ermB和mefE基因阳性大环内酯类抗生素高耐肺炎链球菌中存在流行克隆A型(45%)、B型(17%).PFGE图谱A型的6株多重耐药MRSP菌株都属于亚洲流行克隆CC236(Taiwan19F-14 clone).上海地区发现一个新的MIST型别ST2116(15-16-19-15-6-20-125),推测其由CC236经基因水平交换进化而来.结论上海地区大环内酯类高耐肺炎链球菌的快速增加部分是由于耐药克隆株的播散所致.     

Antibiotic resistance of faecal enterococci in poultry, poultry farmers and poultry slaughterers

The prevalence of resistance in enterococci to antibiotics, commonly used for therapy in poultry or as antimicrobial growth promoters (AMGPs), was determined in faecal samples of two chicken populations: broilers in which antibiotic and AMGP use is common and laying-hens with a low antibiotic usage. In addition faecal samples were examined from three human populations: broiler farmers, laying-hen farmers and poultry slaughterers. MICs of an extended panel of antibiotics for a randomly chosen gentamicin- or vancomycin-resistant enterococcal isolate from each faecal specimen were also determined. The prevalence of resistance for all antibiotics tested was higher in broilers than in laying-hens. Resistance in faecal enterococci of broiler farmers was for nearly all antibiotics higher than those observed in laying-hen farmers and poultry slaughterers. The overall resistance in broilers was correlated with the resistance in broiler farmers and in poultry slaughterers. No correlation between the results obtained in the laying-hens with any of the other populations was found. The 27 gentamicin-resistant isolates all showed high-level resistance to gentamicin and two of these isolates, both Enterococcus faecium, were resistant to all antibiotics tested, except vancomycin. The 73 vancomycin-resistant enterococci (VRE) isolated from the five populations belonged to four different species and in all isolates the vanA gene cluster was detected by blot hybridization. The pulsed-field gel electrophoresis (PFGE) patterns of these vancomycin-resistant enterococci were quite heterogeneous, but Enterococcus hirae isolates with the same or a closely related PFGE pattern were isolated at two farms from the broiler farmer and from broilers. Molecular characterization of vanA-containing transposons of these isolates showed that similar transposon types, predominantly found in poultry, were present. Moreover, similar vanA elements were not only found in isolates with the same PFGE pattern but also in other VRE isolated from both humans and chickens. The results of this study suggest transmission of resistance in enterococci from animals to man. For VRE this might be clonal transmission of animal strains, but transposon transfer seems to occur more commonly.     

Molecular analysis of vancomycin-resistant Enterococcus faecalis from Michigan hospitals during a 10 year period

We evaluated the molecular relatedness of 47 clinical isolates of vancomycin-resistant Enterococcus faecalis collected from 15 Michigan hospitals from 1991 to 2000. There were 17 PFGE strain types for the 47 isolates. Ten of 15 hospitals demonstrated interhospital, and three of 15 intrahospital, dissemination of some isolates. Forty-two isolates (89.4%) demonstrated vanB resistance. All vanA isolates comprised unique PFGE groups, suggesting transposon dissemination or the presence of a similar plasmid. The results of this study suggest inter- and intrahospital dissemination of strains of vancomycin-resistant E. faecalis during a 10 year period in Michigan.     

Intra-hospital dissemination of quinupristin/dalfopristin- and vancomycin-resistant Enterococcus faecium in a paediatric ward of a German hospital

OBJECTIVES: To demonstrate nosocomial transmission of Enterococcus faecium resistant to quinupristin/dalfopristin and vancomycin/teicoplanin among paediatric patients in a German hospital ward. MATERIALS AND METHODS: Multiply-resistant E. faecium were isolated from three female patients aged 9 months, 2 and 15 years during a 10 day time span. Antibiotic susceptibilities were determined by microbroth dilution. Clonal relatedness among the isolates was investigated via SmaI-macrorestriction analysis by PFGE, multilocus sequence typing (MLST), and plasmid profiling. Presence of virulence and resistance determinants was tested by polymerase chain reaction (PCR). Selected resistance genes were localized by Southern hybridizations. RESULTS: A single E. faecium isolate per patient was investigated. All exhibited resistances to quinupristin/dalfopristin, vancomycin/teicoplanin, streptomycin (high-level), penicillin/ampicillin, erythromycin, oxytetracycline, chloramphenicol, rifampicin and fusidic acid. The isolates were susceptible to linezolid only and intermediately resistant to fluoroquinolones including moxifloxacin. PFGE revealed identical patterns for all three isolates. PCRs for virulence determinants hyaluronidase and enterococcal surface protein, esp, were negative, whereas PCR for the enterocin A gene was positive. MLST identified clonal type [8-5-1-1-1-1-1] belonging to a clonal subgroup C1 of hospital- and outbreak-related E. faecium. Southern hybridizations located several resistance genes (erm(B), vat(D), vanA) on a large plasmid, which was transferable in mating experiments with an E. faecium recipient. CONCLUSIONS: These data show routes of dissemination of resistance to multiple antibiotics including streptogramins and glycopeptides in E. faecium via vertical and/or horizontal gene transfer. The isolates spread in the absence of a direct selective pressure, as none of the patients had received earlier streptogramin or glycopeptide therapy.     

A clonal lineage of VanA-type Enterococcus faecalis predominates in vancomycin-resistant Enterococci isolated in New Zealand

Avoparcin was used as a feed additive in New Zealand broiler production from 1977 until June 2000. We report here on the effects of the usage and discontinuation of avoparcin on the prevalence of vancomycin-resistant enterococci (VRE) in broilers. Eighty-two VRE isolates were recovered from poultry fecal samples between 2000 and mid-2001. VRE isolates were only obtained from broiler farms that were using, or had previously used, avoparcin as a dietary supplement. Of these VRE isolates, 73 (89%) were VanA-type Enterococcus faecalis and nine (11%) were VanA-type Enterococcus faecium. All E. faecalis isolates were found to have an identical or closely related pulsed-field gel electrophoresis (PFGE) pattern of SmaI-digested DNA and were susceptible to both ampicillin and gentamicin. The PFGE patterns of the nine E. faecium isolates were heterogeneous. All VRE contained both the vanA and ermB genes, which, regardless of species or PFGE pattern, resided on the same plasmid. Eighty-seven percent of the VRE isolates also harbored the tet(M) gene, while for 63 and 100%, respectively, of these isolates, the avilamycin and bacitracin MICs were high (>or=256 microg/ml). Five of eight vancomycin-resistant E. faecalis isolates recovered from humans in New Zealand revealed a PFGE pattern identical or closely related to that of the E. faecalis poultry VRE isolates. Molecular characterization of Tn1546-like elements from the VRE showed that identical transposons were present in isolates from poultry and humans. Based on the findings presented here, a clonal lineage of VanA-type E. faecalis dominates in VRE isolated from poultry and humans in New Zealand.     

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.     

多位点串联重复序列分析应用于中国肠炎沙门菌分型能力的评价

目的 比较脉冲场凝胶电泳（PFGE）和多位点串联重复序列分析（MLVA）分型方法用于我国肠炎沙门菌分子分型的能力,建立我国肠炎沙门菌MLVA分型标准操作方法及数据库。方法 根据国际PulseNet公布的肠炎沙门菌PFGE和MLVA分型方案,对来自我国6个省（直辖市）的289株肠炎沙门菌进行分子分型分析,并结合流行病学资料,评价这两种分型方法对我国肠炎沙门菌分离株的分型能力。结果 289株肠炎沙门菌经XbaⅠ酶切,PFGE后获得55种带型,其分辨能力（D值）为0.8433。PFGE优势带型为JEGX01.CN0003及JEGX01.CN0001,带型频率分别为35.6%、25.6%,但二者仅表现两个条带的差异,其余型别均低于5%。采用MLVA分析,获得63种型别,分为2个群,D值为0.8608,说明MLVA分辨能力高于单酶切PFGE,但分型能力仍然有限。MLVA主要型别ST1包含了97株菌株,占37.5%,分布于各省及各年份。若联合PFGE及MLVA分型,则产生104种型别,D值为0.9058。对流行病学调查显示为肠炎沙门菌暴发病例菌株进行PFGE双酶切及MLVA分型,结果均显示这些菌株具有明显的聚集性,但不能与同期散发菌株完全区分开。结论 MLVA与PFGE分型方法的分辨能力在肠炎沙门菌中较低,在确认肠炎沙门菌引起的暴发事件时,需紧密结合流行病学调查资料,采用双酶切PFGE或MLVA进行分型分析。     

Vancomycin-resistant Enterococcus faecium strains isolated from community wastewater from a semiclosed agri-food system in Texas

Vancomycin-resistant Enterococcus faecium strains (VRE) were isolated from human wastewater but not swine fecal waste from a semiclosed agri-food system in Texas. Forty-nine VRE isolates possessed vanA, and one possessed vanB. Twenty-one pulsed-field gel electrophoresis types were identified and segregated into three groups. There was evidence of clonal dissemination among geographically separated sites.