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

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

血液分离肠球菌毒力基因检测及生物膜形成测定北大核心CSCD

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

2012～2017年北京某医院耐万古霉素屎肠球菌临床分布及分子特征变迁分析

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

粪肠球菌毒力基因及耐药性分析

目的:了解临床标本与健康人群肠道来源粪肠球菌(Enterococcus faecalis,Efa)的毒力基因分布,探讨毒力基因与细菌致病性及抗生素耐药性之间关系。方法:收集89株分离临床标本和24株健康人群的粪肠球菌,采用PCR方法检测aggA、cylA、cylB、cylM、eep、efaA、enlA、esp和gelE基因,K-B法测定临床分离株对万古霉素、四环素、环丙沙星、高浓度链霉素、青霉素、替考拉宁、呋喃妥因的敏感性。结果:86.7%的粪肠球菌携带有毒力基因,以eep(76.1%)、efaA(74.3%)、esp(52.2%)和aggA(52.2%)基因为多,其他毒力基因的阳性率为31.9%～47.8%。除了毒力基因enlA以外,临床分离菌株其他毒力基因的阳性率明显高于健康人分离菌株。所有菌株中携带8种毒力基因的菌株最为多见,占总菌株的23.9%。粪肠球菌对四环素、环丙沙星和青霉素耐药率较高,对呋喃妥因耐药率低,未检出对万古霉素和替考拉宁耐药株。除了高浓度庆大霉素,携带毒力基因数的多少与抗菌药物的耐药性之间不存在关联。结论:粪肠球菌毒力因子的携带与致病性密切相关,与耐药性之间的关系需进一步研究。     

血液分离肠球菌毒力基因及生物膜形成相关性分析

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

粪肠球菌和屎肠球菌临床分离株的毒力因子与耐药性分析

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

1999-2006年北京朝阳医院革兰阳性球菌耐药性分析

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

22株肠球菌耐药性及8种耐药基因研究

我们收集了2003年12月至2004年5月分离自解放军98医院(湖州)的22株肠球菌，对其进行了耐药性和8内酰胺类耐药相关基因(TEM)、氨基糖苷类耐药相关基因[aac(6′／aph(2″)，aph(3′)Ⅲ，ant(6Ⅰ)]、四环素耐药相关基因(terM)、红霉素耐药相关基因(ermB)、万古霉素耐药相关基因(vanA、vanB)检测。     

临床分离屎肠球菌和粪肠球菌敏感性对比分析

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

对氨基糖甙类高水平耐药肠球菌的检测及药敏结果分析

目的了解肠球菌对高水平庆大霉素的耐药情况及用于耐万古霉素肠球菌(VRE)治疗的氯霉素(C)、红霉素(E)、四环素(TE)、利福平(RA)的耐药情况. 方法应用纸片扩散法药敏试验检测从临床标本中分离出的40株肠球菌.分析其药敏结果. 结果庆大霉素高水平耐药株(HLGR)16株(40.0%)、氯霉素耐药株17株(42.5%)、红霉素耐药株26株(65.0%)、四环素耐药株28株(70.0%)、利福平耐药株22株(55.0%),未检出对万古霉素耐药肠球菌. 结论治疗肠球菌感染时,应根据分离株的耐药特点选择不同的治疗方案.     

Genetic characterization of vancomycin‐resistant enterococci isolates from wild rabbits

The presence of van A‐containing E. faecium isolates was demonstrated in three of 77 faecal samples (3.9%) of wild rabbits recovered in Portugal. Enterococcal strains with intrinsic vancomycin resistance (van C‐1 or van C‐2/3 gene) were found in five (6.5%) and three (3.9%) faecal samples, respectively. The mechanisms of resistance for other antibiotics were studied in these vancomycin‐resistant isolates. All van A strains showed resistance for tetracycline [with the presence of tet (L) gene, associated or not with tet (M) gene] and for erythromycin [with the presence of the erm (B) gene]. Two isolates were resistant to ciprofloxacin and one to ampicillin. Two van C‐1 strains and one van C‐2/3 strain were tetracycline resistant [containing the tet (M) gene associated with tet (L) gene] and erythromycin resistant [with erm (B) gene]. Two van C‐1 and two van C‐2/3 strains were also ciprofloxacin resistant and one van C‐1 strain was, additionally, resistant to quinupristin‐dalfopristin. The two remaining isolates (van C‐1, van C‐2/3) did not show resistance for any additional antibiotic. The intestinal tract of wild rabbits could be a reservoir of van A‐containing enterococci. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

动物源性和人源性的肠球菌对抗生素耐药性及ermB基因相关性研究

目的 了解大环内酯类耐药基因在人源和动物源性肠球菌之间水平转移的可能性. 方法 采用KB法和琼脂稀释法检测52株动物源性和55株儿科临床分离肠球菌对8种抗生素的敏感性;PCR检测肠球菌的ermB、mefA、tetM、aac(6')/aph(2")基因以及Tn1545的整合酶int基因;PCR扩增联合测序对比人源性和动物源性ermB等位基因的同源性;接合实验研究ermB基因在不同种属、不同来源的肠球菌之间水平转移. 结果 两种来源肠球菌对红霉素的耐药率高达80%以上;ermB是肠球菌对红霉素耐药的主要基因,在人源性和动物源性肠球菌的检出率分别为61.82%(34/55)和53.85%(28/52);已测序菌株中53.0%的北京临床分离株与69.5%的动物分离株携带同一ermB等位基因EF525477;通过接合实验,ermB基因可以在不同来源、不同种属分离株之间进行传递. 结论 动物源性和人源性肠球菌对红霉素和四环素都有高耐药率;滤膜实验证实ermB基因在体外条件下可以在动物源性肠球菌和人源性肠球菌之间传递,但传递频率比较低.动物源性和人源性肠球菌及不同种属的肠球菌有相同ermB等位基因,提示其ermB耐药基因在动物源性和人源性肠球菌水平传递存在的可能性.     

REVIEW OF VIRULENCE FACTORS OF ENTEROCOCCUS: AN EMERGING NOSOCOMIAL PATHOGEN

Enterococcus, considered a normal commensal of intestinal tract, is fast emerging as a pathogen causing serious and life threatening hospital borne infections. This is attributed to acquisition of multi drug resistance and virulence factors of the organisms. The sequencing of Enterococcus faecalis has given a lot of insight into its genetic makeup. The E. faecalis strain V583, which has been sequenced, contains a total of 3182 open reading frames (ORFs) with 1760 of these showing similarity to known proteins and 221 of unknown functions. Strikingly unique to this genome is the fact that over 25% of the genome is made up of mobile and exogenously acquired DNA which includes a number of conjugative and composite transposons, a pathogenicity island, integrated plasmid genes and phage regions, and a high number of insertion sequence (IS) elements. This review addresses the genomic arrangement and the study of virulence factors that have occurred since the sequencing of the genome.     

Vancomycin‐resistant enterococci from Portuguese wastewater treatment plants

The objective of this study was to evaluate the incidence of vancomycin resistant enterococci in sludge and sewage of urban and poultry‐slaughterhouse wastewater treatment plants. A total of 17 vancomycin resistant enterococci (eight vanA ‐containing Enterococcus faecium and nine vanC1/vanC2 ‐containing Enterococcus gallinarum/casseliflavus) were found among 499 isolates of sewage and sludge samples of 14 urban and nine poultry‐slaughterhouse wastewater treatment plants. These seventeen VRE isolates showed resistance to kanamycin (n = 8), tetracycline (n = 7), erythromycin (n = 7), ciprofloxacin (n = 7), ampicillin (n = 7), streptomycin (n = 6), and gentamicin (n = 2). The tetM gene, related with tetracycline resistance, was found in six of eight van A‐containing isolates, in all seven vanC‐1 isolates and in one of two vanC‐2 isolates. The ermB gene in seven erythromycin‐resistant isolates; and the aac6 ′‐aph2 ″ gene in the two high‐level‐gentamicin‐resistant isolates. Moreover, two vanA ‐containing E. faecium isolates harbored the hyl virulence gene, and three isolates the entA bacteriocin gene. The purK‐1 allele was detected in our urban vanA ‐containing E. faecium isolate, and we found as well the purK‐6 allele in one poultry‐slaughterhouse vanA ‐containing E. faecium isolate. This study suggests that the wastewater treatment plants might be an important source of dissemination of antibiotic‐resistant enterococci in Portugal. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Pathogenicity of enterococci outside of urinary tract and blood stream

Summary It is evident at this time that enterococci by themselves are able to cause infections outside the bloodstream and the urinary tract only rarely and under very special circumstances in which local defense mechanisms are severely compromised (e.g., by plastic devices). In most instances, they have been found in mixed culture and probably act synergistically with other bacteria to cause damage to the host. They could, however, be carried from their habitat into the bloodstream and eventually cause septicemia. Such a danger is probably heightened if super-colonization is fostered through antibiotics that are ineffective against them, e.g., cephalosporins.     

Identification of enterococci and determination of their glycopeptide resistance in German and Austrian clinical microbiology laboratories

Objectives: To determine exactly how German and Austrian routine laboratories perform tests for the identification of enterococci and determination of their glycopoptide resistance.
Methods: Six enterococcal test strains with different types of glycopeptide resistance ( Enterococcus faecium , VanA; E. faecalis , VanA; E. faecium , VanB; E. faecalis , VanB; E. gallinarum , VanC₁; E. casseliflavus , VanC₂) were sent as anonymous isolates to 73 clinical microbiology laboratories (65 in Germany; eight in Austria). The participating laboratories had to identify the strains up to the species level and to determine their antibiotic susceptibilities to the glycopeptides vancomycin and teicoplanin by the test method(s) that are used daily in the corresponding laboratories.
Results: The analysis of the results received from 62 laboratories (56 from Germany, six from Austria) demonstrated that the most used routine method in susceptibility testing was the agar diffusion test, followed by the Etest, and the microbroth dilution procedure. The majority of participants had no difficulties in susceptibility testing with the VanA-type strains. However, the agar diffusion test was often not able to recognize clearly the VanB and VanC strains; some problems also arose with VanB isolates in the Etest. With the microbroth dilution method, the corresponding type of glycopeptide resistance was correctly determined in the majority of enterococcal test strains. Difficulties also arose in identification, especially with the VanC strains ( E. gallinarum and E. casseliflavus ), which were often falsely identified as E. faecium. The reasons for these errors are obviously based on the lack of important tests (such as motility and presence of a yellow pigment) in some commercially available identification test kits.     

Virulence factors and bacteriocins in faecal enterococci of wild boars

The production of antimicrobial, haemolytic and gelatinase activities was tested in 67 enterococci (39 E. faecium, 24 E. hirae, 2 E. faecalis, and 2 Enterococcus spp.), recovered from faecal samples of wild boars. In addition, the presence of genes encoding bacteriocin and virulence factors was also analysed by PCR and sequencing. Production of antimicrobial activity was checked in all enterococci against 9 indicator bacteria and it was detected in 11 E. faecium isolates (16.5%); eight and two of them harboured the genes encoding enterocin A + enterocin B and enterocin L50A/B, respectively. Sixty-seven per cent of our enterococci harboured different combinations of genes of the cyl operon, but none of them contained the complete cyl L(L)L(S)ABM operon, necessary for cytolysin expression. The presence of gel E gene, associated with the fsr ABC locus, was identified in 4 E. faecium and two E. faecalis isolates, exhibiting all of them gelatinase activity. beta -hemolytic activity was not found in our isolates. Both cpd and ace genes, encoding respectively the accessory colonisation factor and pheromone, were detected in two E. faecalis isolates, and the hyl gene, encoding hyalorunidase, in two E. faecium isolates, one of them gelatinase-positive. Genes encoding bacteriocins and virulence factors are widely disseminated among faecal enterococci of wild boars and more studies should be carried out to know the global distribution of these determinants in enterococci of different ecosystems.