粪肠球菌和屎肠球菌毒力基因、PAI相关基因及耐药性分析
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摘要
研究背景
肠球菌属(Enterococcus)细菌广泛分布于土壤、水和食物中,既往认为肠球菌是对人类无害的共栖菌,现已证实肠球菌可引起多种危及生命的感染。近年来,由肠球菌引起的医院感染逐年增加,已成为医院内感染的重要病原菌之一。临床感染以粪肠球菌占优势,约占85%～95%;其次为屎肠球菌,约占5%～10%,屎肠球菌引起的感染有增加趋势:其余少数为坚韧肠球菌和其它肠球菌,仅占5%以下。肠球菌对现有的大多数抗菌药物天然耐药(如青霉素类、头孢类抗菌药物),使其不易从感染部位清除;此外,肠球菌还可通过基因突变、质粒转移或转座子等方式获得耐药性。致病力的增强是近年来肠球菌发病率上升的另外一个原因。有研究认为肠球菌也可通过水平转移等方式获得外源性的毒力基因,使其毒力增强,致病能力提高,这种致病基因还可进一步在种属之间进行传播。致病岛(pathogenicity island,PAI)编码多种毒力因子,一般只存在于致病株中,而不存在于非致病菌。致病岛的发现,极大地改变了人们对细菌毒力进化的认识。2002年,Shankar等在临床致病粪肠球菌中鉴定出第一个肠球菌致病岛,该岛大约150kb大小,携带致病所需基因,以及一些可能与致病有关、不存在于非感染菌株的潜在毒力基因。目前,国外对肠球菌致病性的研究较多,而国内则较少。由于细菌的进化与所处环境有着非常密切的关系,所以不同地区的菌株会有着自身的特点,只有掌握本土菌株的致病性、耐药性以及其种群特点等方面的信息,才能有效地控制肠球菌的感染、传播以及毒力和耐药基因的扩散。
研究目的
了解广州地区粪肠球菌和屎肠球菌的耐药特征、可能携带的毒力基因和致病岛相关基因及其频率;比较临床菌株和健康人肠道携带的粪肠球菌和屎肠球菌在上述方面的差异;通过10株粪肠球菌的多位点序列分型(multilocus sequencetyping,MLST)分析结果,初步了解广州分离菌株的遗传背景,为进一步开展粪肠球菌的种群研究奠定基础。
研究方法
1.菌株本研究的肠球菌共155株(粪肠球菌103株,屎肠球菌52株)。155株菌株中,医院来源的菌株为100株(粪肠球菌55株,屎肠球菌45株),来自于广州三家医院;健康人肠球菌55株(粪肠球菌48株,屎肠球菌7株)。
2.方法
(1)采用细菌学方法分离、鉴定广州地区医院及健康人所携带的肠球菌;
(2)按照NCCLS推荐的K-B法检测粪肠球菌和屎肠球菌对红霉素(ERY)、氨苄西林(AMP)、氯霉素(CHL)、四环素(TCY)、高单位庆大霉素(GEH)、万古霉素(VAN)、替考拉宁(TEC)的敏感性;
(3)采用PCR方法检测所收集菌株的常见毒力基因efaA、gelE、esp、cylA、ace和agg,了解各基因的频率及分布特征;
(4)采用PCR和斑点杂交方法检测粪肠球菌PAI相关基因nuc、cylB、esp、hyd、psaA和gls24-like,比较不同类型和不同来源菌株所携带的PAI相关基因情况;
(5)采用多位点序列分型法(MIST)测定10株粪肠球菌7种看家基因gdh、gyd、pstS、gki、xpt、aroE、yqiL的基因序列,将其结果按MLST方法进行分类,与相应数据库进行比较,获得广州地区菌株的序列型(ST),并与国际相应资料进行比较。
结果
1.肠球菌的耐药性检测
药敏试验表明,两种肠球菌对7菌抗菌药物具有不同的耐药谱。屎肠球菌中有88.37%对青霉素素产生耐药性,粪肠球菌中该比例只有2.63%。屎肠球菌对高单位庆大霉素、红霉素、四环素和氯霉素的耐药率依次为69.77%,93.02%,37.21%,9.30%,粪肠球菌的耐药率则分别为36.84%,71.05%,77.63%和40.79%。对万古霉素和替考拉宁的耐药率则非常低,仅有4株(9.30%)耐万古霉素屎肠球菌(VRE),其中2株(4.65%)同时耐替考拉宁;在临床来源和健康人来源的粪肠球菌中未发现耐药性之间的差异。肠球菌的多重耐药情况较为严重,超过一半的菌株对3种及以上抗菌药物耐药,屎肠球菌多药耐药率高于粪肠球菌。同时耐受红霉素、四环素和氯霉素是粪肠球菌中最常见的耐药谱型,屎肠球菌中则大多同时耐受红霉素、氨苄西林和高单位庆大霉素。
2.粪肠球菌和屎肠球菌常见毒力因子的检测
毒力基因检测结果显示,6个基因广泛分布于肠球菌中,各基因阳性率由高到低依次为esp(49.68%)、agg(45.16%)、cylA(37.42%)、ace(32.26%)、gelE(27.74%)和efaA(27.74%)。155株肠球菌分离株中有113株至少携带一种毒力基因,所占比例为72.90%。粪肠球菌和屎肠球菌携带的毒力基因数目和种类不同,前者上述基因的阳性率分别为48.54%、61.17%、49.51%、46.60%、39.81%、37.86%,后者则为51.92%、13.46%、13.46%、3.85%、3.85%、7.69%;除esp基因外,其它5个基因的阳性率在两种菌中的差异均具有统计学意义(P=0.000):此外,携带2种以上毒力基因的粪肠球菌占65.05%,屎肠球菌比例仅为17.31%。对不同来源粪肠球菌进行比较,发现临床分离株的毒力基因阳性率高于健康人分离株,除了cylA和efaA基因,其余4个基因的阳性率均具有统计学差异(P=0.000);两种来源粪肠球菌携带的毒力基因数目也不同,85.45%的临床分离株同时含2种以上毒力基因,其中4或5个基因最为常见;健康人分离株中只有41.67%携带2种以上毒力基因。
3.粪肠球菌PAI基因在粪肠球菌和屎肠球菌中的分布特征
选择了粪肠球菌致病岛上6个功能上不相关、结构上散在分布的基因作为研究致病岛的标志,检测结果显示各基因阳性率由高至低依次为hyd(81.94%),psaA(78.06%)、esp(53.55%)、cylB(52.90%)、nuc(45.81%)和gls24-like(38.06%):esp基因在两种肠球菌中检出率相同,其它5个基因则是粪肠球菌高于屎肠球菌,其中nuc、cylB、gls24-like具有统计学差异。在粪肠球菌中,6种基因的阳性率均是临床分离株高于健康人分离株,结果如下:nuc(83.64%vs 39.56%);cylB(87.27%vs 45.83%);esp(61.82%vs 39.58%);hyd(94.55%vs 75.0%);psaS(92.73%vs 70.83%);gls24-like(74.55%vs 25.0%),差异均具有统计学意义。完整结构的致病岛大多出现在临床分离的粪肠球菌中,占该部分菌株的52.83%(28/53),在屎肠球菌和健康人粪肠球菌分离株中的比例分别为4.79%(2/42)和28.05%(11/39)。
4.10株粪肠球菌的多位点序列分型研究
在各种细菌分型方法中,MLST技术具有客观、准确的特点,是目前最为常用的方法之一。采用该方法分析了10株不同来源粪肠球菌的遗传背景。根据获得的数据,发现这10株菌可被分成6个序列型(sequence typing,ST),有4株菌同属于ST16,2株同属于ST4,3株菌分别为ST59,ST67,ST116,1株不能与库中数据匹配,可能为新的ST。
结论
1.粪肠球菌和屎肠球菌对7种常用抗菌药物具有不同程度的耐药,许多菌株耐受多种抗菌药物,粪肠球菌和屎肠球菌的耐药谱不同,后者耐药率和多重耐药率均高于前者,此结果与其它类似研究的结果相同。提示在临床治疗时进行菌种鉴定、有针对性地使用抗菌药物是非常必要的,万古霉素和替考拉宁是治疗该类感染的首选药。值得注意的是此次研究中也检出了耐万古霉素和替考拉宁的菌株,提醒我们要加强此类菌株的监测,以便尽可能早地发现耐药菌株,防止其传播和扩散。
2.粪肠球菌和屎肠球菌携带的毒力基因存在较大差异,前者的毒力基因阳性率明显高于后者,并且表现为多基因携带,含2种及以上基因的菌株很普遍。说明两种肠球菌在环境适应性和致病能力方面存在较大差异,可能具有不同的致病机制;屎肠球菌引起的感染近年呈上升趋势,毒力基因的获得是否使其致病性发生某种变化尚需进一步研究。
3.大小不同、结构不一的致病岛元件分布于93.55%的肠球菌中,说明PAI的可塑性非常强,至于其基因丢失的频率和可能的丢失机制,以及是否存在特定的丢失区域目前尚不清楚,需进一步研究。从结构上来说,临床分离粪肠球菌致病岛结构相对完整,健康人粪肠球菌和屎肠球菌则缺失较多,提示致病岛基因可能参与了粪肠球菌的致病过程。
4.不同来源(健康人分离株、临床分离株)粪肠球菌对7种抗菌药物的耐药性未发现明显差异,但是二者所携带的毒力基因和PAI相关基因具有较大差异,一方面可能受到样本量的影响,另一方面也提示影响粪肠球菌临床感染率的主要因素可能是毒力大小。
5.10株粪肠球菌的MLST研究表明各菌株之间的亲缘关系较远,该结果只是初步了解了广州地区粪肠球菌分离株的ST型,相关研究数据还需进一步积累。
Background
Enterococcus,widely distributed in soil,water and food and initially considered as a harmless commensal of the gastrointestinal tract,are now acknowledged to be organisms capable of causing life-threatening infections in humans.With the increasing incidence of enterococcal infections in recent years,Enterococcus species now rank among the leading causes of nosocomial infections.The two most common species found in clinical samples are Enterococcus faecalis and Enterococcus faecium, accounts for about 85%to 95%and 5%to 10%,respectively.Other species are quite rare,less than 5%.One feature of enterococcal infections that makes them particularly difficult to be treated is the increasing incidence of resistance to multiple antibiotics.Besides being intrinsically resistant to various different classes of antibiotics,enterococci are able to acquire high-level drug resistance to antibiotics, either by mutation or by horizontal gene transfer via transduction,conjugation and transformation.Another potential feature of enterococcal infections is that the virulence of enterococci can be still further enhanced by the presence of additional virulence characteristics which can be horizontally transferred among different organisms.Pathogenicity island(PAI) encode various virulence factors and normally absent from non-pathogenic strains of the same or closely related species. Enterococcal PAI,first identified in clinical isolates in 2002 by Shankar,was approximately 150 kb in size,encoded 129 ORFs and possed virtually all of the hall marks of a PAI.In addition to coding for most known auxiliary traits that enhance virulence of the organism,the enterococcal PAI includes a number of additional, previously unstudied genes that are rare in non-infection-derived isolates.Despite the increasing understanding about the enterococcal virulence determinants in many countries,little is clearly understood about isolates from China.Thus,it is important. to know the antibiotic resistance,virulence,and population structure characteristics of enterococci in guiding infection control practices and preventing the spread of enterococci.
Objective
This thesis aimed to determine the antibiotic resistance patterns,virulence gent profiles,and occurrence of PAI-associted genes among E.faecalis and E.faecium strains in hospitals of Guangzhou,and to compare these isolates with those from healthy individuals isolates.Mutilocus sequence typing(MLST) was performed for 10 E.faecalis isolates to obtain insights into the genetic relationships.
Mothods
Enterococci were collected from clinical samples and healthy individuals isolates,and were identified by conventional methods and by polymerase chain reaction(PCR).The antibiotic resistance of isolates against erythromycin(ERY), ampicillin(AMP),chloramphenicol(CHL),tetracycline(TCY),high-level gentamicin(GEH),vancomycin(VAN),and teicoplanin(TEC) were determined by Kirby-Bauer method,and interpreted as recommended by NCCLS.Presence of virlence determinants encoding aggregation substances agg,cytolysin activator cylA, gelatinase gelE,adherence factors esp and ace,and endocarditis antigen efaA was tested by PCR.Selected pathogenicity-associated genes(nuc、cylB、esp、hyd、psaA and gls24-like) were detected by PCR and dot blot hybridizations under high-stringency conditions.10 E.faecalis strains were analysed by a standard set of E. faecalis multilocus sequence typing(MLST) primers as described (http://efaecalis.mlst.net).The seven genes evaluated are gdh、gyd、pstS、gki、xpt、 aroE、yqiL.Sequence types of isolates are defined by the allelic profile at these seven loci,with each unique combination of alleles assigned a distinct sequence type number.Isolates with the same allelic profile,and therefore the same sequence type, are regarded as members of a single clone.
Results
A total of 103 E.faecalis and 52 E.faecium isolates were collected,including strains origin from clinical samples(55 E.faecalis strains and 45 E.faecium) in three hospitals and from healthy individuals(48 E.faecalis and 7 E.faecium strains).A different pattern of resistance to 7 antibiotics was found between E.faecalis and E. faecium isolates.Ampicillin resistance was common in E.faecium(88.37%),but rare still in E.faecalis(2.63%).The prevalence of E.faecium resistance to high-level gentamicin,erythromycin,tetracycline and chloramphenicol were 69.77%,93.02%, 37.21%,9.30%,respectively,whereas 36.84%,71.05%,77.63%and 40.79%of the E. faecalis isolates were resistant to high-level gentamicin,erythromycin,tetracycline and chloramphenicol,respectively.However,resistance to vancomycin and teicoplanin was only found in 4.65%and 9.30%E.faecium isolates.No differences were found between E.faecalis strains isolated from clinical samples and healthy individuals origins.Resistance to three or more antibiotics was observed in over 50% of enterococci isolates,and E.faecium isolates showed higher percentages of multidrug resistance than E.faecalis strains.In this study,resistance to erythromycin, tetracycline and chloramphenicol concomitantly was the most common pattern in E. faecalis strains,while resistance to erythromycin,ampicillin and high-level gentamicin was the most frequently in E.faecium strains.
All isolates in this study were screened for the presence of six known virulence determinants.The data showed a relatively wide distribution of the virulence genes among the enterococci,with the occurrence as follows:esp 49.68%,agg 45.16%, cylA 37.42%,ace 32.26%,gelE 27.74%and efaA 27.74%.Of 155 enterococcal isolates,there were 113 strains carrying at least one virulence genes and concomitantly up to as many as six virulence genes,accounting for 72.90%.Different and distinct patterns of incidence of virulence determinants were found for the E. faecalis and E.faecium strains.A wide variety of virulence genes were detected in most of E.faecalis isolates but was rarely found in E.faecium(agg 61.17%versus 13.46%,cylA 49.51%versus 13.46%,ace 46.60%versus 3.85%,efaA 39.81%versus 3.85%,gelE 37.86%versus 7.69%,respectively),with the exception of esp,which was found in both species(48.54%versus 51.92%,respectively).In addition,65.05% E.faecalis strains contained two or more of the virulence genes tested while the figure in E.faceium is only 17.31%.When the results of distribution of virulence genes in the different origins of E.faecalis were carefully studied,it was observed that the majority of the genes were more frequently found among clinical isolates than in healthy individuals source.Apart from cylA and efaA genes,the distribution of 4 genes,esp、agg、ace、gelE,was significantly different between the two groups. (P=0.000).Furthermore,85.45%of the clinical strains possessed two or more virulence genes,with four or five being the most common pattern,but the percentage in healthy individuals isolates is only 41.67%.
The E.faecalis PAI region is of particular interest since it was found and it was confirmed that the PAI exhibits a high variability and is largely disseminated among isolates.In this study,the data showed that the PAI-associated genes are a common trait in the genus Enterococcus.The hyd gene was the most frequently detected genes (81.94%) followed by psaA(78.06%),esp(53.55%),cylB(52.90%),nuc(45.81%)and gls24-like(38.06%).The PAI-associated genes were present in different proportion of isolates of E.faecalis and E.faccium.The E.facialis isolates tested carried multiple PAI genes,whereas the E.faceium isolates tested carried few of these genes,except for esp.The distribution of nuc、cylB、gls24-like was found significantly different in the two species of isolates.Significant difference was also found in the distribution of six PAI-associated genes in E.faecalis isolates from different origins.Incidence of these genes in clinical isolates was higher(nuc 83.64%,cylB 87.27%,esp 61.82%, hyd 94.55%,psaS 92.73%and gls24-like 74.55%,respectively) than healthy individual isolates(nuc 39.56%,cylB 45.83%,esp 39.58%,hyd 75.0%,psaS 70.83% and gls24-like 25.0%,respectively).The most complete PAI region was found in 52.83%(28/53) clinical isolates of E.faecalis,4.79%(2/42) E.faecium isolates,and 28.05%(11/39) healthy individual isolates of E.faecalis,and a large PAI region was missing from most of the two latter groups of isolates.
Among typing methods for examining relatedness of bacterial genetic backgrounds,MLST is objective and less prone to human error,has gained recognition as one of the best approaches.Using MLST,10 E.faecalis isolates selected from different human and sample sources were analysed can be classified into six different STs(ST14,ST16,ST4,ST59,ST116,ST67,ST?),with one ST not connecting to any ST identified previously in the database.Of 10 isolates,4 belong to ST16,2 belong to ST4 and each of the other 4 types were found in only one strain.
Analysis
Enterococci are important nosocomial pathogens and are one of the major causes of infection within hospitals.The ability to acquire genes encoding antibiotic resistance combined with a natural resistance to various antimicrobial agents makes many of the enterococcal isolates be resistant to a wide range of antibiotic.This is a serious problem,as it reduces the number of possible treatments available for entericoccal infections.In this study,the majority of identified entericocci were phenotypically resistant to at least one antibodies,and many isolates were multidrug resistant and show a different resistance pattern between E.faecalis and E.faecium isolates.These findings are in agreement with the findings of other studies;hence,the identification to the species level of genus Enterococcus is necessaryce for the choice of possible antimicrobial therapies.From our study,vancomycin and teicoplanin are the drugs of first choice to treat those infections.However,resistance to vancomycin and teicoplanin was also detected in isolates tested in this study,indicating that early reinforcement of precautions combined with surveillance were very important for recognising the occurrence and spread of vancomycin-resistant isolates as early as possible.
The study on virulence genes of enterococcal isolates revealed a high diversity with varying frequency and distribution of each single gene among enterococcal strains.The 155 isolates carried different virulence gene combinations as well as each of the 6 virulence gene alone.Among isolates tested,most carried esp(49.68%) and agg(45.16%) genes,whereas the remaining virulence genes were detected in variable percentages ranging from 27.74%to 37.42%,and E.faecalis strains have a higher occurrence of virulence genes than E.faecium strains.Furthermore,isolates with more than two or more genes were quite common in E.faecalis strain,whereas it was rare in E.faecium.These findings suggested that there were different pathogenic proterties existing in the two group isolates,and indicated that the E.faecalis was the dominant enterococcal isolates causing serious hospitals infections.Of particular concern is the emergence of the new E.faecium clone by acquisition of new virulence genes,causing more infections in recent years.
Pathogenicity islands are distinct genetic elements on the chromosomes of a large number of bacterial pathogens,typically carry genes associated with virulence, such as toxins,adhesins,other factors that promote colonization,and mechanisms for the delivery of bacterial products directly into host cells.Selected six functionally unrelated genes,located across the PAI of E.faecalis,were surveyed in collected isolates in this study.The results revealed a high degree of plasticity within the PAI region of the genomes of the enterococci isolates.Among the 155 isolates tested, 93.55%were found containing fragments of the PAI,and the distribution of six PAI-associated genes varied with the source and species of the strains.E.faecalis strains possessed the higher occurrence of genes and more of the PAI regions tested than did E.faeciurn strains,and the comparison of clinical isolates and healthy individual isolates of E.faecalis revealed a high rate of gene deletions in the latter. These findings support the hypothesis that this genomic region may be helpful during some stage of human infection.MLST is useful for characterizing genetic background of E.faecalis isolates and more data should be further obtained.
Conclusions
Enterococci are opportunistic pathogens.Little is known about the mechanism of virulence of these bacteria and genes involved in the pathogenesis of disease have only recently been identified.In the present,We observed differences among E. faecalis and E.faecium isolates from different origins for antibiotic resistance, pathogenicity island content,and virulence genes.In general,enterococci have been main associated with antibiotic resistance,virulence factors and pathogenicity island associated genes,although the occurrence varied with the source and species of the strains.A high prevalence of antibiotic resistance was demonstrated in both species; Prevalences and patterns of virulence genes and PAI genes were quite different between two species of enterococci,as well as E.faeealis isolates from different origins.These findings suggested that this factors may play a role in human infections and that surveillance should be directed specifically towards these disseminating clones in order to prevent infections and clonal spread.The results of this study also indicate that enterococci from healthy individual could be a reservoirs of resistance determinants and virulence traits for transmission to humans.Further efforts must continue to focus on prevention of the emergence and dissemination of these clone.
肠球菌属(Enterococcus)细菌广泛分布于土壤、水和食物中,既往认为肠球菌是对人类无害的共栖菌,现已证实肠球菌可引起多种危及生命的感染。近年来,由肠球菌引起的医院感染逐年增加,已成为医院内感染的重要病原菌之一。临床感染以粪肠球菌占优势,约占85%～95%;其次为屎肠球菌,约占5%～10%,屎肠球菌引起的感染有增加趋势:其余少数为坚韧肠球菌和其它肠球菌,仅占5%以下。肠球菌对现有的大多数抗菌药物天然耐药(如青霉素类、头孢类抗菌药物),使其不易从感染部位清除;此外,肠球菌还可通过基因突变、质粒转移或转座子等方式获得耐药性。致病力的增强是近年来肠球菌发病率上升的另外一个原因。有研究认为肠球菌也可通过水平转移等方式获得外源性的毒力基因,使其毒力增强,致病能力提高,这种致病基因还可进一步在种属之间进行传播。致病岛(pathogenicity island,PAI)编码多种毒力因子,一般只存在于致病株中,而不存在于非致病菌。致病岛的发现,极大地改变了人们对细菌毒力进化的认识。2002年,Shankar等在临床致病粪肠球菌中鉴定出第一个肠球菌致病岛,该岛大约150kb大小,携带致病所需基因,以及一些可能与致病有关、不存在于非感染菌株的潜在毒力基因。目前,国外对肠球菌致病性的研究较多,而国内则较少。由于细菌的进化与所处环境有着非常密切的关系,所以不同地区的菌株会有着自身的特点,只有掌握本土菌株的致病性、耐药性以及其种群特点等方面的信息,才能有效地控制肠球菌的感染、传播以及毒力和耐药基因的扩散。
研究目的
了解广州地区粪肠球菌和屎肠球菌的耐药特征、可能携带的毒力基因和致病岛相关基因及其频率;比较临床菌株和健康人肠道携带的粪肠球菌和屎肠球菌在上述方面的差异;通过10株粪肠球菌的多位点序列分型(multilocus sequencetyping,MLST)分析结果,初步了解广州分离菌株的遗传背景,为进一步开展粪肠球菌的种群研究奠定基础。
研究方法
1.菌株本研究的肠球菌共155株(粪肠球菌103株,屎肠球菌52株)。155株菌株中,医院来源的菌株为100株(粪肠球菌55株,屎肠球菌45株),来自于广州三家医院;健康人肠球菌55株(粪肠球菌48株,屎肠球菌7株)。
2.方法
(1)采用细菌学方法分离、鉴定广州地区医院及健康人所携带的肠球菌;
(2)按照NCCLS推荐的K-B法检测粪肠球菌和屎肠球菌对红霉素(ERY)、氨苄西林(AMP)、氯霉素(CHL)、四环素(TCY)、高单位庆大霉素(GEH)、万古霉素(VAN)、替考拉宁(TEC)的敏感性;
(3)采用PCR方法检测所收集菌株的常见毒力基因efaA、gelE、esp、cylA、ace和agg,了解各基因的频率及分布特征;
(4)采用PCR和斑点杂交方法检测粪肠球菌PAI相关基因nuc、cylB、esp、hyd、psaA和gls24-like,比较不同类型和不同来源菌株所携带的PAI相关基因情况;
(5)采用多位点序列分型法(MIST)测定10株粪肠球菌7种看家基因gdh、gyd、pstS、gki、xpt、aroE、yqiL的基因序列,将其结果按MLST方法进行分类,与相应数据库进行比较,获得广州地区菌株的序列型(ST),并与国际相应资料进行比较。
结果
1.肠球菌的耐药性检测
药敏试验表明,两种肠球菌对7菌抗菌药物具有不同的耐药谱。屎肠球菌中有88.37%对青霉素素产生耐药性,粪肠球菌中该比例只有2.63%。屎肠球菌对高单位庆大霉素、红霉素、四环素和氯霉素的耐药率依次为69.77%,93.02%,37.21%,9.30%,粪肠球菌的耐药率则分别为36.84%,71.05%,77.63%和40.79%。对万古霉素和替考拉宁的耐药率则非常低,仅有4株(9.30%)耐万古霉素屎肠球菌(VRE),其中2株(4.65%)同时耐替考拉宁;在临床来源和健康人来源的粪肠球菌中未发现耐药性之间的差异。肠球菌的多重耐药情况较为严重,超过一半的菌株对3种及以上抗菌药物耐药,屎肠球菌多药耐药率高于粪肠球菌。同时耐受红霉素、四环素和氯霉素是粪肠球菌中最常见的耐药谱型,屎肠球菌中则大多同时耐受红霉素、氨苄西林和高单位庆大霉素。
2.粪肠球菌和屎肠球菌常见毒力因子的检测
毒力基因检测结果显示,6个基因广泛分布于肠球菌中,各基因阳性率由高到低依次为esp(49.68%)、agg(45.16%)、cylA(37.42%)、ace(32.26%)、gelE(27.74%)和efaA(27.74%)。155株肠球菌分离株中有113株至少携带一种毒力基因,所占比例为72.90%。粪肠球菌和屎肠球菌携带的毒力基因数目和种类不同,前者上述基因的阳性率分别为48.54%、61.17%、49.51%、46.60%、39.81%、37.86%,后者则为51.92%、13.46%、13.46%、3.85%、3.85%、7.69%;除esp基因外,其它5个基因的阳性率在两种菌中的差异均具有统计学意义(P=0.000):此外,携带2种以上毒力基因的粪肠球菌占65.05%,屎肠球菌比例仅为17.31%。对不同来源粪肠球菌进行比较,发现临床分离株的毒力基因阳性率高于健康人分离株,除了cylA和efaA基因,其余4个基因的阳性率均具有统计学差异(P=0.000);两种来源粪肠球菌携带的毒力基因数目也不同,85.45%的临床分离株同时含2种以上毒力基因,其中4或5个基因最为常见;健康人分离株中只有41.67%携带2种以上毒力基因。
3.粪肠球菌PAI基因在粪肠球菌和屎肠球菌中的分布特征
选择了粪肠球菌致病岛上6个功能上不相关、结构上散在分布的基因作为研究致病岛的标志,检测结果显示各基因阳性率由高至低依次为hyd(81.94%),psaA(78.06%)、esp(53.55%)、cylB(52.90%)、nuc(45.81%)和gls24-like(38.06%):esp基因在两种肠球菌中检出率相同,其它5个基因则是粪肠球菌高于屎肠球菌,其中nuc、cylB、gls24-like具有统计学差异。在粪肠球菌中,6种基因的阳性率均是临床分离株高于健康人分离株,结果如下:nuc(83.64%vs 39.56%);cylB(87.27%vs 45.83%);esp(61.82%vs 39.58%);hyd(94.55%vs 75.0%);psaS(92.73%vs 70.83%);gls24-like(74.55%vs 25.0%),差异均具有统计学意义。完整结构的致病岛大多出现在临床分离的粪肠球菌中,占该部分菌株的52.83%(28/53),在屎肠球菌和健康人粪肠球菌分离株中的比例分别为4.79%(2/42)和28.05%(11/39)。
4.10株粪肠球菌的多位点序列分型研究
在各种细菌分型方法中,MLST技术具有客观、准确的特点,是目前最为常用的方法之一。采用该方法分析了10株不同来源粪肠球菌的遗传背景。根据获得的数据,发现这10株菌可被分成6个序列型(sequence typing,ST),有4株菌同属于ST16,2株同属于ST4,3株菌分别为ST59,ST67,ST116,1株不能与库中数据匹配,可能为新的ST。
结论
1.粪肠球菌和屎肠球菌对7种常用抗菌药物具有不同程度的耐药,许多菌株耐受多种抗菌药物,粪肠球菌和屎肠球菌的耐药谱不同,后者耐药率和多重耐药率均高于前者,此结果与其它类似研究的结果相同。提示在临床治疗时进行菌种鉴定、有针对性地使用抗菌药物是非常必要的,万古霉素和替考拉宁是治疗该类感染的首选药。值得注意的是此次研究中也检出了耐万古霉素和替考拉宁的菌株,提醒我们要加强此类菌株的监测,以便尽可能早地发现耐药菌株,防止其传播和扩散。
2.粪肠球菌和屎肠球菌携带的毒力基因存在较大差异,前者的毒力基因阳性率明显高于后者,并且表现为多基因携带,含2种及以上基因的菌株很普遍。说明两种肠球菌在环境适应性和致病能力方面存在较大差异,可能具有不同的致病机制;屎肠球菌引起的感染近年呈上升趋势,毒力基因的获得是否使其致病性发生某种变化尚需进一步研究。
3.大小不同、结构不一的致病岛元件分布于93.55%的肠球菌中,说明PAI的可塑性非常强,至于其基因丢失的频率和可能的丢失机制,以及是否存在特定的丢失区域目前尚不清楚,需进一步研究。从结构上来说,临床分离粪肠球菌致病岛结构相对完整,健康人粪肠球菌和屎肠球菌则缺失较多,提示致病岛基因可能参与了粪肠球菌的致病过程。
4.不同来源(健康人分离株、临床分离株)粪肠球菌对7种抗菌药物的耐药性未发现明显差异,但是二者所携带的毒力基因和PAI相关基因具有较大差异,一方面可能受到样本量的影响,另一方面也提示影响粪肠球菌临床感染率的主要因素可能是毒力大小。
5.10株粪肠球菌的MLST研究表明各菌株之间的亲缘关系较远,该结果只是初步了解了广州地区粪肠球菌分离株的ST型,相关研究数据还需进一步积累。
Background
Enterococcus,widely distributed in soil,water and food and initially considered as a harmless commensal of the gastrointestinal tract,are now acknowledged to be organisms capable of causing life-threatening infections in humans.With the increasing incidence of enterococcal infections in recent years,Enterococcus species now rank among the leading causes of nosocomial infections.The two most common species found in clinical samples are Enterococcus faecalis and Enterococcus faecium, accounts for about 85%to 95%and 5%to 10%,respectively.Other species are quite rare,less than 5%.One feature of enterococcal infections that makes them particularly difficult to be treated is the increasing incidence of resistance to multiple antibiotics.Besides being intrinsically resistant to various different classes of antibiotics,enterococci are able to acquire high-level drug resistance to antibiotics, either by mutation or by horizontal gene transfer via transduction,conjugation and transformation.Another potential feature of enterococcal infections is that the virulence of enterococci can be still further enhanced by the presence of additional virulence characteristics which can be horizontally transferred among different organisms.Pathogenicity island(PAI) encode various virulence factors and normally absent from non-pathogenic strains of the same or closely related species. Enterococcal PAI,first identified in clinical isolates in 2002 by Shankar,was approximately 150 kb in size,encoded 129 ORFs and possed virtually all of the hall marks of a PAI.In addition to coding for most known auxiliary traits that enhance virulence of the organism,the enterococcal PAI includes a number of additional, previously unstudied genes that are rare in non-infection-derived isolates.Despite the increasing understanding about the enterococcal virulence determinants in many countries,little is clearly understood about isolates from China.Thus,it is important. to know the antibiotic resistance,virulence,and population structure characteristics of enterococci in guiding infection control practices and preventing the spread of enterococci.
Objective
This thesis aimed to determine the antibiotic resistance patterns,virulence gent profiles,and occurrence of PAI-associted genes among E.faecalis and E.faecium strains in hospitals of Guangzhou,and to compare these isolates with those from healthy individuals isolates.Mutilocus sequence typing(MLST) was performed for 10 E.faecalis isolates to obtain insights into the genetic relationships.
Mothods
Enterococci were collected from clinical samples and healthy individuals isolates,and were identified by conventional methods and by polymerase chain reaction(PCR).The antibiotic resistance of isolates against erythromycin(ERY), ampicillin(AMP),chloramphenicol(CHL),tetracycline(TCY),high-level gentamicin(GEH),vancomycin(VAN),and teicoplanin(TEC) were determined by Kirby-Bauer method,and interpreted as recommended by NCCLS.Presence of virlence determinants encoding aggregation substances agg,cytolysin activator cylA, gelatinase gelE,adherence factors esp and ace,and endocarditis antigen efaA was tested by PCR.Selected pathogenicity-associated genes(nuc、cylB、esp、hyd、psaA and gls24-like) were detected by PCR and dot blot hybridizations under high-stringency conditions.10 E.faecalis strains were analysed by a standard set of E. faecalis multilocus sequence typing(MLST) primers as described (http://efaecalis.mlst.net).The seven genes evaluated are gdh、gyd、pstS、gki、xpt、 aroE、yqiL.Sequence types of isolates are defined by the allelic profile at these seven loci,with each unique combination of alleles assigned a distinct sequence type number.Isolates with the same allelic profile,and therefore the same sequence type, are regarded as members of a single clone.
Results
A total of 103 E.faecalis and 52 E.faecium isolates were collected,including strains origin from clinical samples(55 E.faecalis strains and 45 E.faecium) in three hospitals and from healthy individuals(48 E.faecalis and 7 E.faecium strains).A different pattern of resistance to 7 antibiotics was found between E.faecalis and E. faecium isolates.Ampicillin resistance was common in E.faecium(88.37%),but rare still in E.faecalis(2.63%).The prevalence of E.faecium resistance to high-level gentamicin,erythromycin,tetracycline and chloramphenicol were 69.77%,93.02%, 37.21%,9.30%,respectively,whereas 36.84%,71.05%,77.63%and 40.79%of the E. faecalis isolates were resistant to high-level gentamicin,erythromycin,tetracycline and chloramphenicol,respectively.However,resistance to vancomycin and teicoplanin was only found in 4.65%and 9.30%E.faecium isolates.No differences were found between E.faecalis strains isolated from clinical samples and healthy individuals origins.Resistance to three or more antibiotics was observed in over 50% of enterococci isolates,and E.faecium isolates showed higher percentages of multidrug resistance than E.faecalis strains.In this study,resistance to erythromycin, tetracycline and chloramphenicol concomitantly was the most common pattern in E. faecalis strains,while resistance to erythromycin,ampicillin and high-level gentamicin was the most frequently in E.faecium strains.
All isolates in this study were screened for the presence of six known virulence determinants.The data showed a relatively wide distribution of the virulence genes among the enterococci,with the occurrence as follows:esp 49.68%,agg 45.16%, cylA 37.42%,ace 32.26%,gelE 27.74%and efaA 27.74%.Of 155 enterococcal isolates,there were 113 strains carrying at least one virulence genes and concomitantly up to as many as six virulence genes,accounting for 72.90%.Different and distinct patterns of incidence of virulence determinants were found for the E. faecalis and E.faecium strains.A wide variety of virulence genes were detected in most of E.faecalis isolates but was rarely found in E.faecium(agg 61.17%versus 13.46%,cylA 49.51%versus 13.46%,ace 46.60%versus 3.85%,efaA 39.81%versus 3.85%,gelE 37.86%versus 7.69%,respectively),with the exception of esp,which was found in both species(48.54%versus 51.92%,respectively).In addition,65.05% E.faecalis strains contained two or more of the virulence genes tested while the figure in E.faceium is only 17.31%.When the results of distribution of virulence genes in the different origins of E.faecalis were carefully studied,it was observed that the majority of the genes were more frequently found among clinical isolates than in healthy individuals source.Apart from cylA and efaA genes,the distribution of 4 genes,esp、agg、ace、gelE,was significantly different between the two groups. (P=0.000).Furthermore,85.45%of the clinical strains possessed two or more virulence genes,with four or five being the most common pattern,but the percentage in healthy individuals isolates is only 41.67%.
The E.faecalis PAI region is of particular interest since it was found and it was confirmed that the PAI exhibits a high variability and is largely disseminated among isolates.In this study,the data showed that the PAI-associated genes are a common trait in the genus Enterococcus.The hyd gene was the most frequently detected genes (81.94%) followed by psaA(78.06%),esp(53.55%),cylB(52.90%),nuc(45.81%)and gls24-like(38.06%).The PAI-associated genes were present in different proportion of isolates of E.faecalis and E.faccium.The E.facialis isolates tested carried multiple PAI genes,whereas the E.faceium isolates tested carried few of these genes,except for esp.The distribution of nuc、cylB、gls24-like was found significantly different in the two species of isolates.Significant difference was also found in the distribution of six PAI-associated genes in E.faecalis isolates from different origins.Incidence of these genes in clinical isolates was higher(nuc 83.64%,cylB 87.27%,esp 61.82%, hyd 94.55%,psaS 92.73%and gls24-like 74.55%,respectively) than healthy individual isolates(nuc 39.56%,cylB 45.83%,esp 39.58%,hyd 75.0%,psaS 70.83% and gls24-like 25.0%,respectively).The most complete PAI region was found in 52.83%(28/53) clinical isolates of E.faecalis,4.79%(2/42) E.faecium isolates,and 28.05%(11/39) healthy individual isolates of E.faecalis,and a large PAI region was missing from most of the two latter groups of isolates.
Among typing methods for examining relatedness of bacterial genetic backgrounds,MLST is objective and less prone to human error,has gained recognition as one of the best approaches.Using MLST,10 E.faecalis isolates selected from different human and sample sources were analysed can be classified into six different STs(ST14,ST16,ST4,ST59,ST116,ST67,ST?),with one ST not connecting to any ST identified previously in the database.Of 10 isolates,4 belong to ST16,2 belong to ST4 and each of the other 4 types were found in only one strain.
Analysis
Enterococci are important nosocomial pathogens and are one of the major causes of infection within hospitals.The ability to acquire genes encoding antibiotic resistance combined with a natural resistance to various antimicrobial agents makes many of the enterococcal isolates be resistant to a wide range of antibiotic.This is a serious problem,as it reduces the number of possible treatments available for entericoccal infections.In this study,the majority of identified entericocci were phenotypically resistant to at least one antibodies,and many isolates were multidrug resistant and show a different resistance pattern between E.faecalis and E.faecium isolates.These findings are in agreement with the findings of other studies;hence,the identification to the species level of genus Enterococcus is necessaryce for the choice of possible antimicrobial therapies.From our study,vancomycin and teicoplanin are the drugs of first choice to treat those infections.However,resistance to vancomycin and teicoplanin was also detected in isolates tested in this study,indicating that early reinforcement of precautions combined with surveillance were very important for recognising the occurrence and spread of vancomycin-resistant isolates as early as possible.
The study on virulence genes of enterococcal isolates revealed a high diversity with varying frequency and distribution of each single gene among enterococcal strains.The 155 isolates carried different virulence gene combinations as well as each of the 6 virulence gene alone.Among isolates tested,most carried esp(49.68%) and agg(45.16%) genes,whereas the remaining virulence genes were detected in variable percentages ranging from 27.74%to 37.42%,and E.faecalis strains have a higher occurrence of virulence genes than E.faecium strains.Furthermore,isolates with more than two or more genes were quite common in E.faecalis strain,whereas it was rare in E.faecium.These findings suggested that there were different pathogenic proterties existing in the two group isolates,and indicated that the E.faecalis was the dominant enterococcal isolates causing serious hospitals infections.Of particular concern is the emergence of the new E.faecium clone by acquisition of new virulence genes,causing more infections in recent years.
Pathogenicity islands are distinct genetic elements on the chromosomes of a large number of bacterial pathogens,typically carry genes associated with virulence, such as toxins,adhesins,other factors that promote colonization,and mechanisms for the delivery of bacterial products directly into host cells.Selected six functionally unrelated genes,located across the PAI of E.faecalis,were surveyed in collected isolates in this study.The results revealed a high degree of plasticity within the PAI region of the genomes of the enterococci isolates.Among the 155 isolates tested, 93.55%were found containing fragments of the PAI,and the distribution of six PAI-associated genes varied with the source and species of the strains.E.faecalis strains possessed the higher occurrence of genes and more of the PAI regions tested than did E.faeciurn strains,and the comparison of clinical isolates and healthy individual isolates of E.faecalis revealed a high rate of gene deletions in the latter. These findings support the hypothesis that this genomic region may be helpful during some stage of human infection.MLST is useful for characterizing genetic background of E.faecalis isolates and more data should be further obtained.
Conclusions
Enterococci are opportunistic pathogens.Little is known about the mechanism of virulence of these bacteria and genes involved in the pathogenesis of disease have only recently been identified.In the present,We observed differences among E. faecalis and E.faecium isolates from different origins for antibiotic resistance, pathogenicity island content,and virulence genes.In general,enterococci have been main associated with antibiotic resistance,virulence factors and pathogenicity island associated genes,although the occurrence varied with the source and species of the strains.A high prevalence of antibiotic resistance was demonstrated in both species; Prevalences and patterns of virulence genes and PAI genes were quite different between two species of enterococci,as well as E.faeealis isolates from different origins.These findings suggested that this factors may play a role in human infections and that surveillance should be directed specifically towards these disseminating clones in order to prevent infections and clonal spread.The results of this study also indicate that enterococci from healthy individual could be a reservoirs of resistance determinants and virulence traits for transmission to humans.Further efforts must continue to focus on prevention of the emergence and dissemination of these clone.
引文
1.Treitman AN,Yarnold PR,Warren J,et al.Emerging incidence of Enterococcus faeeium among hospital isolates(1993 to 2002)[J].J Clin Microbiol,2005,43(1):462-463.
2.Seetulsingh PS,Tomayko JF,Condrom PE,et al.Chromosomal DNA restriction endonuclease digestion patterns of β-lactamase-producing Enterococcus faecalis isolates collected from a single hospital over a 7-year period[J].J Clin Microbiol,1996,34(8):1892-1896.
3.Ma XH,Kudo M,Takahashi A,et al.Evidence of nosocomial infection in Japan caused by high-level gentamicin-resistant Enterococcus faecalis and identification of the pheromone-responsive conjugative plasmid encoding gentamcin resistance[J].J Clin Microbiol,1998,36(9):2460-2464.
4.Titze-de-Almeida R,Rollo Filho M,Nogueira CA,et al.Molecular epidemiology and antimicrobial susceptibility of Enterococci recovered from Brazilian intensive care units[J].Braz J Infect Dis,2004,8(3):197-205.
5.Richards MJ,Edwards JR,Culver DH,et al.Nosocomial infections in combined medical-surgical intensive care units in the United States.Infect Control Hosp Epidemiol,2000,21:510-515.
6.Donald EL,Keller N,Barth A,et al.Clinical prevalence antimicrobial susceptibility and geographic resistance patterns of Enterococci:results from the SENTRY antimicrobial surveillance program,1997-1999.Clin Infecti Dis,2001,32(12):133-145.
7.Uttley AH,Collins CH,Naidoo J,et al.Vancomycin-resistant enterococci,Lancet.1988,1:57-58.
8.李爽,张正.万古霉素耐药肠球菌的检测和分子流行病学分析[J].中国实验诊断学,2006,10(4):372-375.
9.陈知行,陈慧莉,康梅,等.906株肠球菌属细菌的耐药性分析[J].华西医学,2004,19(2):227-228.
10.朱红军,费选文,林祥伟,等.临床分离肠球菌分布及其耐药性监测[J].中国感染控制杂志,2004,3(2):154-156.
11.申正义,王洪波,孙自镛.湖北地区外科感染常见致病菌1314株耐药性监测分析[J].中华普通外科杂志,2001,28(4):215-216.
12.应春妹,项明洁,倪语星.耐万古霉素肠球菌表型检测及基因分型[J].临床检验杂志,2001,19(5):277-279.
13.刘业霞,赵安成.临床常见肠球菌分布及耐药率分析[J].国际检验医学杂志,2007,28(8):689-692.
14.Sanchez-Silos RM,Perez-Giraldo C,Martin P,et al.Pathogenicity of Enterococcus spp.Characteristics of 169 hospital isolates[J].Enferm Infect Microbial Clin,2000,18(4):165-169.
15.Shankar N,Baghdayan AS,Gilmore MS.Modulation of virulence within a pathogenicity island in vancomycinresistant Enterococcus faecalis[J].NATURE,2002,417(6890):746-750.
16.强华,刘光英,李能.肠球菌溶血素的表型及基因型检测[J].福建医科大学学报,2007,41(3):197-199.
17.Cox CR,Coburn PS,Gilmore MS.Enterococcal cytolysin:a novel two component peptide system that serves as a bacterial defense against eukaryotic and prokaryotic cells.Curt Protein Pept Sci,2005;6(1):77-84.
18.Hickey RM,Twomey DP,Ross RP,et al.Production of enterolysin a by a raw milk enterococcal isolate exhibiting multiple virulence factors[J].Microbiology,2003,149(3):655-664.
19.Shankar N,Lockatell CV,Baghdayan AS.Role of Enterococcus faecalis surface protein Esp in the pathogenesis of ascending urinary tract infection.Infect Immun,2001,69(7):4366-4372.
20.Lund B,Edlund C.Bloodstream isolates of Enterococcus faecium enriched with the enterococcal surface protein gene,esp,show increased adhesion to eukaryotic cells.J Clin Microbiol.2003,41(11):5183-5185.
21.Coque TM,Willems R,Canton R,et al.High occurrence of esp among ampicillin-resistant and vancomycin-susceptible Enterococcus faecium clones from hospitalized patients.J Antimicrob Chemother.2002,50(6):1035-1038.
22.程梅,高良,梅亚林.肠球菌Esp基因检测及其致病作用[J].江西医学检验,2006,(3):217-218
23.Shankar V,Baghdayan AS,Huycke MM,et al.Infection-Derived Enterococcus faecalis Strains Are Enriched in esp,a Gene Encoding a Novel Surface Protein[J].Infect Immun,1999,67(1):193-200.
24.Ramadhan AA,Hegedus E.Biofilm formation and esp gene carriage in enterococci[J].J Clin Pathol,2005,58(7):685-686.
25.Tendolkar PM,Baghdayan AS,Gilmore MS,et al.Enterococcal surface protein,Esp,enhances biofilm formation by Enterococcus faecalis[J].Infect Immun.2004,72(10):6032-6039.
26.Harada T,Tsuji N,Otsuki K,Murase T.Detection of the esp gene in high-level gentamicin resistant Enterococcus faecalis strains from pet animals in Japan[J].Vet Microbiol,2005,106(1-2):139-143.
27.Nallapareddy SR,Qin X,Weinstock GM,et al.Enterococcus faecalis adhesin,Ace,mediates attachment to extracellular matrix proteins collagen type Ⅳ and laminin as well as collagen type Ⅰ[J].Infect Immun,2000,68(9):5218-5224.
28.Low YL,Jakubovics NS,Flatman JC,et al.Manganese-dependent regulation of the endocarditis-associated virulence factor EfaA of Enterococcus faecalis[J].J Med Microbiol.2003,52(Pt 2):113-119.
29.Rozdzinski E,Marre R,Susa M,et al.Aggregation substance-mediated adherence of Enterococcus faecalis to immobilized extracellular matrix proteins[J].Microb Pathog,2001,30(4):211-220.
30.Leavis H,Top J,Shankar N,et al.A Novel Putative Enterococcal Pathogenicity Island Linked to the esp Virulence Gene of Enterococcus faecium and Associated with Epidemicity[J].J Bacteriol,2004,186(3):672-682.
31.Sahm DF,Kissinger J,Gilmore MS,et al.In vitro susceptibility studies of vancomycin-resistant Enterococcus faecalis[J].Antimicrob Agents Chemother,1989,33(9):1588-1591.
32. Shankar N, Coburn P, Pillar C, et al. Enterococcal cytolysin: activities and association with other virulence traits in a pathogenicity island[J]. Int J Med Microbiol, 2004,293(7-8):609-618.
33. Heikens E, Bonten MJ, Willems RJ. Enterococcal surface protein Esp is important for biofilm formation of Enterococcus faecium E1162[J]. J Bacteriol,2007,189(22):8233-8240.
34. Van Wamel WJ, Hendrickx AP, Bonten MJ, et al. Growth condition-dependent Esp expression by Enterococcus faecium affects initial adherence and biofilm formation[J]. Infect Immun. 2007, 75(2):924-931.
35. Raad II, Hanna HA, Boktour M, et al. Relationship between biofilm formation,the enterococcal surface protein (Esp) and gelatinase in clinical isolates of Enterococcus faecalis and Enterococcus faecium[J]. FEMS Microbiol Lett,2006,256(1):145-150.
36. Tendolkar PM, Baghdayan AS, Shankar N. The N-terminal domain of enterococcal surface protein, Esp, is sufficient for Esp-mediated biofilm enhancement in Enterococcus faecalis[J]. J Bacteriol, 2005,187(17):6213-6222.
37. Waters CM, Dunny GM. Analysis of functional domains of the Enterococcus faecalis pheromone-induced surface protein aggregation substance[J]. J Bacteriol,2001,183(19):5659-5667.
38. Sussmuth SD, Muscholl-Silberhorn A, Wirth R, et al. Aggregation substance promotes adherence, phagocytosis and intracellular survival of Enterococcus faecalis within human macrophages and suppresses respiratory burst[J]. Infect Immun, 2000,68(9): 4900-4906.
39. Coburn PS, Baghdayan AS, Dolan GT, et al. Horizontal transfer of virulence genes encoded on the Enterococcus faecalis pathogenicity island[J]. Molecular Microb, 2007,63(2):530-544.
40. Shankar N, Baghdayan AS, Willems R, et al. Presence of pathogenicity island genes in Enterococcus faecalis isolates from pigs in Denmark[J]. J Clinical Microbiol, 2006,144(11): 4200-4203
41. Nallapareddy SR, Wenxiang H, Weinstock GM, et al. Molecular characterization of a widespread,pathogenic,and antibiotic resistance-receptive Enterococcus faecalis lineage and dissemination of its putative pathogenicity island[J].J Bacteriol,2005,187(16):5709-5718
42.McBridel SM,Fischetti VA,LeBlanc DJ,et al.Genetic Diversity among Enterococcus faecalis[J].PloS ONE,2007,2(7):e582
43.Macovei L,Zurek L.Ecology of Antibiotic Resistance Genes:Characterization of Enterococci from Houseflies Collected in Food Settings[J].Appl Environ Microbiol,2006,72(6):4028-4035.
44.林红燕,沈怀亮,魏衍超.临床检出肠球菌耐药谱4年的变迁[J].中华医院感染学杂志,2005,15(1):115-117.
45.罗燕萍,白立彦,李俊玲,等.211株肠球菌属细菌对18种抗菌药物的耐药性特征[J].中华医院感染学杂志,2004,14(1):81-82.
46.夏克蓉,张敏华.粪肠球菌与屎肠球菌耐药性比较分析[J].江西医学,2005,23(5):483.
47.Reviriego C,Eaton T,Martin R,et al.Screening of virulence determinants in Enterococcus faecium strains isolated from breast milk[J].J Hum Lact,2005,21(2):131-137.
48.Dupre I,Zanetti S,Schito AM,et al.Incidence of virulence determinants in clinical Enterococcus faecium and Enterococcus faecalis isolates collected in Sardinia(Italy)[J].Med Microbiol,2003,52:491-498.
49.Lopes Mde F,Simoes AP,Tenreiro R,et al.Activity and expression of a virulence factor,gelatinase,in dairy enterococci[J].Int J Food Microbiol,2006,112(3):208-214.
50.Creti R,Imperi M,Bertuccini L,et al.Survey for virulence determinants among Enterococcus faecalis isolated from different sources[J].Med Microbio,2004,53:13-20.
51.Eaton TJ,Gasson MJ.Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates[J].Appl Environ Microbiol,2001,67:1628-1635.
52.Bittencourt de Marques E,Suzart S.Occurrence of virulence associated genes in clinical Enterococcus faecalis strains isolated in Londrina,Brazil[J].Med Microbiol,2004,53:1069-1073.
53.马立艳,许淑珍,马纪平.肠球菌部分致病基因和表型的检测[J].中华检验医学杂志,2005,28(5):529-532.
54.Abriouel H,Omar NB,Molinos AC,et al.Comparative analysis of genetic diversity and incidence of virulence factors and antibiotic resistance among enterococcal populations from raw fruit and vegetable foods,water and soil,and clinical samples[J].Int J Food Microbiol,2008,123(1-2):38-49.
55.Mannu L,Paba A,Daga E,et al.Comparison of the incidence of virulence determinants and antibiotic resistance between Enterococeus faecium strains of dairy,animal and clinical origin[J].Int J Food Microbiol.2003,88(2-3):291-304
56.Dupont H,Vael C,Muller-Serieys C,et al.Prospective evaluation of virulence factors of enterococci isolated from patients with peritonitis:impact on outcome[J].Diagn Microbiol Infect Dis,2008,60(3):247-253.
57.Eisner HA,Sobottka I,Mack D,et al.Virulence Factors of Enterococcus faecium and Enteroeoccus faecalis Strains Isolated from Humans and Pets[J].Eur J Clin Microbiol Infect Dis.2000,19(1):39-42.
58.Lepage E,Brinster S,Caron C,et al.Comparative Genomic Hybridization Analysis of Enteroeoccus faecalis:Identification of Genes Absent from Food Strains[J].J Bacteriol.2006,188(19):6858-6868.
59.Evaluation of Virulence Factors and Plasmid-Related Transmissibility among Different Isolates of Enteroeocci.Iranian Biomedical Journal,2005,9(2):51-55.
60.马立艳,许淑珍.健康成人与住院病人肠球菌致病基因及表型和耐药性的比较研究[J].临床和实验医学杂志,2006,58(1):5-7.
61.Qin X,Singh KV,Weinstock GM,et al.Effects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence[J].Infect Immune,2000,68(5):2579-2586.
62.www.mlst.net
2.Seetulsingh PS,Tomayko JF,Condrom PE,et al.Chromosomal DNA restriction endonuclease digestion patterns of β-lactamase-producing Enterococcus faecalis isolates collected from a single hospital over a 7-year period[J].J Clin Microbiol,1996,34(8):1892-1896.
3.Ma XH,Kudo M,Takahashi A,et al.Evidence of nosocomial infection in Japan caused by high-level gentamicin-resistant Enterococcus faecalis and identification of the pheromone-responsive conjugative plasmid encoding gentamcin resistance[J].J Clin Microbiol,1998,36(9):2460-2464.
4.Titze-de-Almeida R,Rollo Filho M,Nogueira CA,et al.Molecular epidemiology and antimicrobial susceptibility of Enterococci recovered from Brazilian intensive care units[J].Braz J Infect Dis,2004,8(3):197-205.
5.Richards MJ,Edwards JR,Culver DH,et al.Nosocomial infections in combined medical-surgical intensive care units in the United States.Infect Control Hosp Epidemiol,2000,21:510-515.
6.Donald EL,Keller N,Barth A,et al.Clinical prevalence antimicrobial susceptibility and geographic resistance patterns of Enterococci:results from the SENTRY antimicrobial surveillance program,1997-1999.Clin Infecti Dis,2001,32(12):133-145.
7.Uttley AH,Collins CH,Naidoo J,et al.Vancomycin-resistant enterococci,Lancet.1988,1:57-58.
8.李爽,张正.万古霉素耐药肠球菌的检测和分子流行病学分析[J].中国实验诊断学,2006,10(4):372-375.
9.陈知行,陈慧莉,康梅,等.906株肠球菌属细菌的耐药性分析[J].华西医学,2004,19(2):227-228.
10.朱红军,费选文,林祥伟,等.临床分离肠球菌分布及其耐药性监测[J].中国感染控制杂志,2004,3(2):154-156.
11.申正义,王洪波,孙自镛.湖北地区外科感染常见致病菌1314株耐药性监测分析[J].中华普通外科杂志,2001,28(4):215-216.
12.应春妹,项明洁,倪语星.耐万古霉素肠球菌表型检测及基因分型[J].临床检验杂志,2001,19(5):277-279.
13.刘业霞,赵安成.临床常见肠球菌分布及耐药率分析[J].国际检验医学杂志,2007,28(8):689-692.
14.Sanchez-Silos RM,Perez-Giraldo C,Martin P,et al.Pathogenicity of Enterococcus spp.Characteristics of 169 hospital isolates[J].Enferm Infect Microbial Clin,2000,18(4):165-169.
15.Shankar N,Baghdayan AS,Gilmore MS.Modulation of virulence within a pathogenicity island in vancomycinresistant Enterococcus faecalis[J].NATURE,2002,417(6890):746-750.
16.强华,刘光英,李能.肠球菌溶血素的表型及基因型检测[J].福建医科大学学报,2007,41(3):197-199.
17.Cox CR,Coburn PS,Gilmore MS.Enterococcal cytolysin:a novel two component peptide system that serves as a bacterial defense against eukaryotic and prokaryotic cells.Curt Protein Pept Sci,2005;6(1):77-84.
18.Hickey RM,Twomey DP,Ross RP,et al.Production of enterolysin a by a raw milk enterococcal isolate exhibiting multiple virulence factors[J].Microbiology,2003,149(3):655-664.
19.Shankar N,Lockatell CV,Baghdayan AS.Role of Enterococcus faecalis surface protein Esp in the pathogenesis of ascending urinary tract infection.Infect Immun,2001,69(7):4366-4372.
20.Lund B,Edlund C.Bloodstream isolates of Enterococcus faecium enriched with the enterococcal surface protein gene,esp,show increased adhesion to eukaryotic cells.J Clin Microbiol.2003,41(11):5183-5185.
21.Coque TM,Willems R,Canton R,et al.High occurrence of esp among ampicillin-resistant and vancomycin-susceptible Enterococcus faecium clones from hospitalized patients.J Antimicrob Chemother.2002,50(6):1035-1038.
22.程梅,高良,梅亚林.肠球菌Esp基因检测及其致病作用[J].江西医学检验,2006,(3):217-218
23.Shankar V,Baghdayan AS,Huycke MM,et al.Infection-Derived Enterococcus faecalis Strains Are Enriched in esp,a Gene Encoding a Novel Surface Protein[J].Infect Immun,1999,67(1):193-200.
24.Ramadhan AA,Hegedus E.Biofilm formation and esp gene carriage in enterococci[J].J Clin Pathol,2005,58(7):685-686.
25.Tendolkar PM,Baghdayan AS,Gilmore MS,et al.Enterococcal surface protein,Esp,enhances biofilm formation by Enterococcus faecalis[J].Infect Immun.2004,72(10):6032-6039.
26.Harada T,Tsuji N,Otsuki K,Murase T.Detection of the esp gene in high-level gentamicin resistant Enterococcus faecalis strains from pet animals in Japan[J].Vet Microbiol,2005,106(1-2):139-143.
27.Nallapareddy SR,Qin X,Weinstock GM,et al.Enterococcus faecalis adhesin,Ace,mediates attachment to extracellular matrix proteins collagen type Ⅳ and laminin as well as collagen type Ⅰ[J].Infect Immun,2000,68(9):5218-5224.
28.Low YL,Jakubovics NS,Flatman JC,et al.Manganese-dependent regulation of the endocarditis-associated virulence factor EfaA of Enterococcus faecalis[J].J Med Microbiol.2003,52(Pt 2):113-119.
29.Rozdzinski E,Marre R,Susa M,et al.Aggregation substance-mediated adherence of Enterococcus faecalis to immobilized extracellular matrix proteins[J].Microb Pathog,2001,30(4):211-220.
30.Leavis H,Top J,Shankar N,et al.A Novel Putative Enterococcal Pathogenicity Island Linked to the esp Virulence Gene of Enterococcus faecium and Associated with Epidemicity[J].J Bacteriol,2004,186(3):672-682.
31.Sahm DF,Kissinger J,Gilmore MS,et al.In vitro susceptibility studies of vancomycin-resistant Enterococcus faecalis[J].Antimicrob Agents Chemother,1989,33(9):1588-1591.
32. Shankar N, Coburn P, Pillar C, et al. Enterococcal cytolysin: activities and association with other virulence traits in a pathogenicity island[J]. Int J Med Microbiol, 2004,293(7-8):609-618.
33. Heikens E, Bonten MJ, Willems RJ. Enterococcal surface protein Esp is important for biofilm formation of Enterococcus faecium E1162[J]. J Bacteriol,2007,189(22):8233-8240.
34. Van Wamel WJ, Hendrickx AP, Bonten MJ, et al. Growth condition-dependent Esp expression by Enterococcus faecium affects initial adherence and biofilm formation[J]. Infect Immun. 2007, 75(2):924-931.
35. Raad II, Hanna HA, Boktour M, et al. Relationship between biofilm formation,the enterococcal surface protein (Esp) and gelatinase in clinical isolates of Enterococcus faecalis and Enterococcus faecium[J]. FEMS Microbiol Lett,2006,256(1):145-150.
36. Tendolkar PM, Baghdayan AS, Shankar N. The N-terminal domain of enterococcal surface protein, Esp, is sufficient for Esp-mediated biofilm enhancement in Enterococcus faecalis[J]. J Bacteriol, 2005,187(17):6213-6222.
37. Waters CM, Dunny GM. Analysis of functional domains of the Enterococcus faecalis pheromone-induced surface protein aggregation substance[J]. J Bacteriol,2001,183(19):5659-5667.
38. Sussmuth SD, Muscholl-Silberhorn A, Wirth R, et al. Aggregation substance promotes adherence, phagocytosis and intracellular survival of Enterococcus faecalis within human macrophages and suppresses respiratory burst[J]. Infect Immun, 2000,68(9): 4900-4906.
39. Coburn PS, Baghdayan AS, Dolan GT, et al. Horizontal transfer of virulence genes encoded on the Enterococcus faecalis pathogenicity island[J]. Molecular Microb, 2007,63(2):530-544.
40. Shankar N, Baghdayan AS, Willems R, et al. Presence of pathogenicity island genes in Enterococcus faecalis isolates from pigs in Denmark[J]. J Clinical Microbiol, 2006,144(11): 4200-4203
41. Nallapareddy SR, Wenxiang H, Weinstock GM, et al. Molecular characterization of a widespread,pathogenic,and antibiotic resistance-receptive Enterococcus faecalis lineage and dissemination of its putative pathogenicity island[J].J Bacteriol,2005,187(16):5709-5718
42.McBridel SM,Fischetti VA,LeBlanc DJ,et al.Genetic Diversity among Enterococcus faecalis[J].PloS ONE,2007,2(7):e582
43.Macovei L,Zurek L.Ecology of Antibiotic Resistance Genes:Characterization of Enterococci from Houseflies Collected in Food Settings[J].Appl Environ Microbiol,2006,72(6):4028-4035.
44.林红燕,沈怀亮,魏衍超.临床检出肠球菌耐药谱4年的变迁[J].中华医院感染学杂志,2005,15(1):115-117.
45.罗燕萍,白立彦,李俊玲,等.211株肠球菌属细菌对18种抗菌药物的耐药性特征[J].中华医院感染学杂志,2004,14(1):81-82.
46.夏克蓉,张敏华.粪肠球菌与屎肠球菌耐药性比较分析[J].江西医学,2005,23(5):483.
47.Reviriego C,Eaton T,Martin R,et al.Screening of virulence determinants in Enterococcus faecium strains isolated from breast milk[J].J Hum Lact,2005,21(2):131-137.
48.Dupre I,Zanetti S,Schito AM,et al.Incidence of virulence determinants in clinical Enterococcus faecium and Enterococcus faecalis isolates collected in Sardinia(Italy)[J].Med Microbiol,2003,52:491-498.
49.Lopes Mde F,Simoes AP,Tenreiro R,et al.Activity and expression of a virulence factor,gelatinase,in dairy enterococci[J].Int J Food Microbiol,2006,112(3):208-214.
50.Creti R,Imperi M,Bertuccini L,et al.Survey for virulence determinants among Enterococcus faecalis isolated from different sources[J].Med Microbio,2004,53:13-20.
51.Eaton TJ,Gasson MJ.Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates[J].Appl Environ Microbiol,2001,67:1628-1635.
52.Bittencourt de Marques E,Suzart S.Occurrence of virulence associated genes in clinical Enterococcus faecalis strains isolated in Londrina,Brazil[J].Med Microbiol,2004,53:1069-1073.
53.马立艳,许淑珍,马纪平.肠球菌部分致病基因和表型的检测[J].中华检验医学杂志,2005,28(5):529-532.
54.Abriouel H,Omar NB,Molinos AC,et al.Comparative analysis of genetic diversity and incidence of virulence factors and antibiotic resistance among enterococcal populations from raw fruit and vegetable foods,water and soil,and clinical samples[J].Int J Food Microbiol,2008,123(1-2):38-49.
55.Mannu L,Paba A,Daga E,et al.Comparison of the incidence of virulence determinants and antibiotic resistance between Enterococeus faecium strains of dairy,animal and clinical origin[J].Int J Food Microbiol.2003,88(2-3):291-304
56.Dupont H,Vael C,Muller-Serieys C,et al.Prospective evaluation of virulence factors of enterococci isolated from patients with peritonitis:impact on outcome[J].Diagn Microbiol Infect Dis,2008,60(3):247-253.
57.Eisner HA,Sobottka I,Mack D,et al.Virulence Factors of Enterococcus faecium and Enteroeoccus faecalis Strains Isolated from Humans and Pets[J].Eur J Clin Microbiol Infect Dis.2000,19(1):39-42.
58.Lepage E,Brinster S,Caron C,et al.Comparative Genomic Hybridization Analysis of Enteroeoccus faecalis:Identification of Genes Absent from Food Strains[J].J Bacteriol.2006,188(19):6858-6868.
59.Evaluation of Virulence Factors and Plasmid-Related Transmissibility among Different Isolates of Enteroeocci.Iranian Biomedical Journal,2005,9(2):51-55.
60.马立艳,许淑珍.健康成人与住院病人肠球菌致病基因及表型和耐药性的比较研究[J].临床和实验医学杂志,2006,58(1):5-7.
61.Qin X,Singh KV,Weinstock GM,et al.Effects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence[J].Infect Immune,2000,68(5):2579-2586.
62.www.mlst.net