表皮葡萄球菌纤维蛋白原结合基因分布的研究

目的：研究不同来源的表皮葡萄球菌(表葡菌)以及其他葡萄球菌中纤维蛋白原结合基因(fbe基因)的分布情况。方法：采用聚合酶链反应(PCR)方法检测表葡菌临床分离株或健康人群寄植株以及其他类型葡萄球菌的fbe基因，并对fbe全基因序列进行了测序。结果：临床分离的表葡菌中fbe基因阳性率达到84．2％，健康人群来源的表葡菌中fbe基因阳性率达92．6％，而其他类型葡萄球菌中均未扩增出fbe基因；表葡菌337#fbe基因全长序列为3457bp，与国外文献报道对比有95％同源性。结论：fbe基因在表葡菌中广泛存在，fbe全基因序列与国外报道的结果相似。     

同源重组构建表皮葡萄球菌纤维蛋白原结合基因缺失突变菌株

目的构建表皮葡萄球菌（表葡菌）纤维蛋白原结合基因（fbe基因）缺失突变菌株，为fbe基因功能研究提供实验工具。方法采用同源基因重组技术。构建质粒△pBT2（含fbe：：ermB基因），将其电转化进入金葡菌RN4220，再次抽提后电转化进入fbe基因阳性表葡菌（HB）中。将含有质粒△pBT2的表葡菌HB在5mg／L红霉素平板42℃经多次传代，使质粒ApBT2裂解，fbe：：ermB基因同源重组到表葡菌HB的染色体上，通过红霉素耐药、氯霉素敏感等特性筛选出含有fbe：：ermB基因的新表葡菌HB-ermB。结果经酶切鉴定后确认质粒△pBT2构建成功以及成功转导入金葡菌RN4220和表葡菌HB中，经PCR方法以及测序确认表葡菌HB的fbe基因缺失突变菌株HB-ermB构建成功。结论采用同源重组的方法完成了表葡菌HB的fbe基因缺失突变菌株的构建。     

重组溶葡萄球菌酶体外诱导的金黄色葡萄球菌耐药株生物学特征

目的探究重组溶葡萄球菌酶体外诱导金黄色葡萄球菌(金葡菌)耐药发生规律和机制。方法用3株临床分离金葡菌,体外以亚抑菌浓度重组溶葡萄球菌酶一步法诱导耐药,观察耐药株对多种抗菌药物的药敏改变情况,测序分析肽聚糖甘氨酸五肽桥联的关键合成酶编码基因femABX。并观察其中1株耐甲氧西林金葡菌(MRSA)和1株甲氧西林敏感金葡菌(MSSA)的生长曲线、小鼠感染毒力情况。结果金葡菌耐药发生频率为10-4~10-8水平,与原代株比较,耐药株体外生长速率和小鼠体内毒力显著降低,对β内酰胺类敏感性提高2~4 000倍。测序结果显示耐药株femA编码基因存在突变,可致终止密码子提前。结论重组溶葡萄球菌酶体外可诱导金葡菌耐药性产生,但耐药株生长繁殖力、致病力及抵抗β内酰胺类抗生素的能力显著降低。     

fbe基因与表皮葡萄球菌感染

表皮葡萄球菌(表葡菌)为人体皮肤和黏膜表面的正常寄生菌群,是致导管、人工瓣膜等植入物相关感染的重要病原菌。以表葡菌为首的凝固酶阴性葡萄球菌也是医院感染的主要病原菌,甲氧西林耐药表葡菌(MRSE)的出现使治疗更复杂[1-2]。研究显示表葡菌致病力的强弱与其进入人体后对植入     

儿科院内感染细菌分布及耐药性监测

目的:了解儿科院内感染常见细菌分布并监测其耐药性.方法:收集惠儿标本,进行常规细菌培养及K-B法药敏试验.结果:分离出238株痛原菌,以克雷伯菌属为最多123株(51.7%).其次为凝固酶阴性葡萄球菌(coagulase negative staphylococci,CNS)50株(21.0%);123株克雷伯菌属中产ESBLs的菌株96株(78.0%);19株大肠埃希菌中产ESBLs的菌株12株(63.2%).产ESBLs克雷伯菌属和大肠埃希菌对抗菌药物的耐药率明显高于非产ESBLs的相应细菌.检出41株(80.2%)凝固酶阴性耐甲氧西林葡萄球菌(meahicillin resistant coagulase negative staphylococci,MRCNS),未检出耐甲氧西林的金黄色葡萄球菌(meahicillin resistant staphylococcus aureus,MRSA).凝固酶阴性葡萄球菌和金黄色葡萄球菌对青霉素全部耐药.结论:儿科的院内感染革兰阴性茵以克雷伯菌属最多见,革兰阳性菌以凝固酶阴性葡萄球菌居多,两类痛原菌多重耐药现象比较严重.     

表皮葡萄球菌感染的防治

表皮葡萄球菌 (表葡菌 )是人体皮肤和黏膜上定居的正常菌群之一 ,通常情况下致病力很低。近几年来 ,随着留置静脉导管等侵袭性操作的增多 ,表葡菌已成为医院感染的重要致病菌。本文将该领域的有关研究进展作一综述。一、病原学表葡菌是凝固酶阴性的葡萄球菌 (CNS)。其营养要求不高 ,在普通培养基上生长良好 ,为需氧菌。其无α溶血素和A蛋白 ,壁酸类型为甘油 ,耐药核糖醇内切酶阴性 ,以上特点可与金黄色葡萄球菌 (金葡菌 )相鉴别[1] 。表葡菌是一种条件致病菌 ,其毒力较低 ,但在表葡菌引起的植入物相关性感染中 ,生物膜是一个非常重要的致…     

表皮葡萄球菌附属基因调节子对生物膜形成的调节作用

目的研究表皮葡萄球菌agr对其生物膜形成的调节作用，并探讨其调节机制，以期发现新的生物膜形成调节途径，完善agr调节网络。方法用同源重组方法构建表皮葡萄球菌agr阴性突变株，从体外、体内不同角度观察agr阴性突变株与其agr阳性野生株生物膜形成能力和致病力的差异。结果表皮葡萄球菌agr阴性突变株与其agr阳性野生株相比，黏附能力、致病力均明显增强；在agr阴性突变株中许多蛋白表达是增加的，如ClpP（ATP—dependent Clp protease）、DadL（D—alanine—D—alanine ligase）等；在agr阴性突变株中基因clpP、atlE和dadL mRNA的表达分别是agr阳性野生株的6．4、7．6和3．9倍。而fbe、ica mRNA的表达在agr阴性突变株和agr阳性野生株中差异无统计学意义。结论agr基因可以下调表皮葡萄球菌生物膜的形成，是生物膜形成的抑制因子。agr基因对生物膜的下调作用可能不但通过下调atlE的表达，还可能同时下调clpP的表达；agr对生物膜的下调作用并不通过调节fbe和ica途径；首次发现agr可以通过调节dadL来调节细菌细胞壁的合成。     

金黄色葡萄球菌致病毒素基因分布特性及与致病性的相关性

目的研究金黄色葡萄球菌(简称金葡菌)致病毒素基因的分布特性及与致病性的关系。方法对临床收集的80株金葡菌,采用头孢西丁纸片法检测耐甲氧西林金葡菌(MRSA),多重聚合酶链反应法检测致病毒素基因中毒休克综合征毒素-1(TSST-1)、杀白细胞毒素(PVL),分析TSST-1、PVL基因阳性金葡菌的临床分布特性及与致病性的相关性。结果 80株金葡菌中MRSA总检出率为58.8%,PVL基因阳性金葡菌在医院的分布范围较广,主要引起呼吸系统感染、化脓性感染,也可引起其他感染性疾病,严重的可导致败血症;TSST-1基因阳性的MRSA均PVL基因阳性,均取自重症监护室肺部感染和化脓性感染的患者。结论 MRSA已成为医院感染的重要致病菌,携带致病毒素基因TSST-1、PVL的金葡菌也占有一定的比例,在医院的分布范围较广,致病毒素基因TSST-1、PVL可增强MRSA的致病力,这增加了临床治疗难度。     

糖尿病足湿性坏疽多重耐药菌耐药特点分析

目的探讨糖尿病足湿性坏疽多重耐药菌的分布特点及耐药情况。方法选择2010年1月至2014年12月该院脉管炎科及糖尿病科送检的糖尿病足湿性坏疽患者分泌物1 845份,分离多重耐药菌,分析其分布情况及革兰阴性菌、革兰阳性菌、厌氧菌、真菌的多重耐药特点。结果分离出465株多重耐药菌株中,革兰阴性杆菌274株(58.92%),革兰阳性球菌191株(41.08%)。革兰阴性多重耐药菌株中以大肠埃希菌株为主占37.60%,革兰阳性多重耐药菌株中以凝固酶阴性葡萄球菌为主占51.30%;多重耐药阴性杆菌中,产超广谱β-内酰胺酶(ESBLs)的大肠埃希菌及肺炎克雷伯菌、鲍曼溶血不动杆菌、铜绿假单胞菌表现高耐药性,耐药率达到50.00%以上;多重耐药阳性球菌中,凝固酶阴性葡萄球菌及金黄色葡萄球菌均为耐甲氧西林阳性菌株,这些菌株表现高耐药性,耐药率达85.00%以上。结论糖尿病足湿性坏疽多重耐药菌中大肠埃希菌、肺炎克雷伯菌、凝固酶阴性葡萄球菌及金黄色葡萄球菌表现高耐药性,临床应结合耐药鉴定分析选择用药。     

下呼吸道感染病原菌分布及耐药性分析

目的了解下呼吸道感染疾病常见致病菌的分布情况及抗菌药耐药情况,为临床合理使用抗菌药物提供依据。方法收集2009年5月至2011年5月下呼吸道感染患者合格痰标本进行细菌学培养、鉴定及药敏试验分析。结果下呼吸道感染住院患者痰液培养所分离409株非重复的致病菌株,革兰阴性杆菌是主要致病菌,革兰阴性杆菌216株(52.8%),其中铜绿假单胞菌78株(19.1%)位居第一,其次是肺炎克雷伯杆菌42株(10.3%),大肠埃希菌17株(4.2%)。肺炎克雷伯菌、大肠埃希菌超广谱β-内酰胺酶(ESBLs)阳性率分别为38.1%、64.7%。非发酵阴性杆菌106株(25.9%),铜绿假单胞菌78株,不动杆菌13株。革兰阳性球菌22株(5.4%),葡萄球菌属21株,金黄色葡萄球菌7株(1.7%),耐甲氧西林金黄色葡萄球菌(MRSA)4株,检出率为57.1%,凝固酶阴性的葡萄球菌14株,耐甲氧西林凝固酶阴性的葡萄球菌(MRCNS)10株,检出率为71.4%。真菌171株(41.8%),多数革兰阴性杆菌对阿米卡星、左氧氟沙星有很高敏感率;革兰阳性球菌尤其是耐甲氧西林金葡菌对青霉素、左氧氟沙星等耐药,但对万古霉素敏感。结论医院下呼吸道感染以革兰阴性杆菌为主,不同的细菌对同一抗菌药物的敏感差异较大,铜绿假单胞菌耐药情况呈上升趋势,应根据药敏结果选择抗菌药物。     

Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and Staphylococcus epidermidis

Clinical isolates of Staphylococcus aureus (a total of 206) and S. epidermidis (a total of 188) from various countries were tested with multiplex PCR assays to detect clinically relevant antibiotic resistance genes associated with staphylococci. The targeted genes are implicated in resistance to oxacillin (mecA), gentamicin ?aac(6')-aph(2"), and erythromycin (ermA, ermB, ermC, and msrA). We found a nearly perfect correlation between genotypic and phenotypic analysis for most of these 394 strains, showing the following correlations: 98% for oxacillin resistance, 100% for gentamicin resistance, and 98.5% for erythromycin resistance. The discrepant results were (i) eight strains found to be positive by PCR for mecA or ermC but susceptible to the corresponding antibiotic based on disk diffusion and (ii) six strains of S. aureus found to be negative by PCR for mecA or for the four erythromycin resistance genes targeted but resistant to the corresponding antibiotic. In order to demonstrate in vitro that the eight susceptible strains harboring the resistance gene may become resistant, we subcultured the susceptible strains on media with increasing gradients of the antibiotic. We were able to select cells demonstrating a resistant phenotype for all of these eight strains carrying the resistance gene based on disk diffusion and MIC determinations. The four oxacillin-resistant strains negative for mecA were PCR positive for blaZ and had the phenotype of beta-lactamase hyperproducers, which could explain their borderline oxacillin resistance phenotype. The erythromycin resistance for the two strains found to be negative by PCR is probably associated with a novel mechanism. This study reiterates the usefulness of DNA-based assays for the detection of antibiotic resistance genes associated with staphylococcal infections.     

fbl gene as a species‐specific target for Staphylococcus lugdunensis identification

Staphylococcus lugdunensis is an unusually virulent coagulase‐negative species, associated with severe infections. The present report describes the development of a single‐step, species‐specific PCR protocol for S. lugdunensis identification. fbl gene, encoding a fibrinogen‐binding adhesin, was exploited and assessed as a suitable nucleic acid target. The gene was detected in all 17 S. lugdunensis isolates examined, while no amplification product was obtained from 98 isolates representing 11 staphylococcal and 17 nonstaphylococcal species. Forty‐seven percent of the S. lugdunensis strains produced a positive slide coagulase reaction, which is consistent with varying levels of Fbl protein expression within the species. J. Clin. Lab. Anal. 24:119–122, 2010. © 2010 Wiley‐Liss, Inc.     

Foreign body infections due to Staphylococcus epidermidis

Staphylococcal infections are one of the main causes of complications in patients with implanted foreign prosthetic material. Implants are associated with a significant reduction of the threshold at which contaminating Gram-positive bacteria, particularly Staphylococcus epidermidis, become infectious and develop a biofilm with phenotypic resistance to almost all antibiotics. A 1000-fold increase in minimal bactericidal levels against most antibiotics except rifampin has been repeatedly observed. Since only removal of the foreign material reverses these phenomena, the clinical challenge consists in finding approaches to cure the infection without removal of the implanted device. Rifampin combinations with other antibiotics, administration of exceedingly high antibiotic concentrations in situ, and early therapy before biofilm development are efficacious. Although these strategies have dramatically improved the outcome of foreign body infections, an improved understanding of biofilm-grown S. epidermidis is necessary to develop new antibacterial agents. Here, we review the pathogenesis, prevention, and treatment of implant infections due to S. epidermidis and highlight some new compounds with already promising in vitro results.     

Assessment of biofilm-forming ability of coagulase-negative staphylococci isolated from contaminated platelet preparations in Canada

BACKGROUND: Coagulase-negative staphylococci (CoNS) are the most prevalent bacterial contaminants of platelet (PLT) preparations and have been implicated in adverse transfusion reactions worldwide. The most frequently identified contaminant is Staphylococcus epidermidis, which is noted for its ability to maintain chronic hospital-acquired infections by forming biofilms as a chief virulence mechanism. STUDY DESIGN AND METHODS: Strains of S. epidermidis isolated from contaminated PLT preparations in Canada were distinguished via gene-specific polymerase chain reaction (PCR) with divIVA as a marker. Biofilm-forming ability was assessed by the presence of the gene icaD, slime production on Congo red agar, and biofilm formation on polystyrene surfaces. Production of polysaccharide intercellular adhesin (PIA) was resolved by immunofluorescence. RESULTS: Eight of the 13 (62%) CoNS isolates under study were identified as S. epidermidis. Of these, four strains (50%) were classified as strong biofilm producers. Three of the four biofilm-positive strains (75%) produced slime, harbored the icaD gene, and had positive expression of PIA. CONCLUSIONS: Despite the presumable commensal origin of the CoNS isolates, a large proportion of S. epidermidis strains demonstrated a potential for enhanced virulence. Identification of contaminant staphylococci as biofilm producers is thus relevant and informative with regard to treatment approach in the circumstance of inadvertent infection of a PLT recipient.     

表皮葡萄球菌生物膜形成与细菌耐药性的相关性分析

目的探讨表皮葡萄球菌临床分离株生物膜的形成情况,分析其生物膜形成与细菌耐药性的相关性。方法收集2014年1月至2015年2月住院患者血标本分离出的表皮葡萄球菌62株,采用生物膜形成试验和聚合酶链式反应(PCR)扩增试验检测细菌生物膜,并采用纸片扩散法(K-B法)进行细菌药物敏感性试验。结果利用生物膜形成试验检出生物膜阳性菌株23株,检出率为37.1%;PCR扩增试验检出icaA基因27株,检出率为43.5%,差异无统计学意义(P0.05);两种方法同时阳性的菌株为14株。生物膜阳性菌株对所测抗菌药物的耐药率普遍高于生物膜阴性菌株,其中对庆大霉素、青霉素G、苯唑西林、左旋氧氟沙星、头孢西丁的耐药率比较差异有统计学意义(P0.05),所有菌株对万古霉素、利奈唑胺、奎奴普丁/达福普汀均敏感。结论两种方法对表皮葡萄球菌生物膜的检出率无明显差异,生物膜阳性菌株耐药率普遍高于生物膜阴性菌株。     

多中心耐利奈唑胺凝固酶阴性葡萄球菌耐药机制及同源性分析

目的:了解江苏地区利奈唑胺耐药凝固酶阴性葡萄球菌耐药机制及菌株流行性。方法:收集2017—2018年江苏省3家三级医院临床和环境分离利奈唑胺耐药凝固酶阴性葡萄球菌共38株(头状葡萄球菌32株、人葡萄球菌4株、表皮葡萄球菌1株和缓慢葡萄球菌1株),采用微量肉汤稀释法检测常规药物的敏感性,并采用E-test试验检测菌株对利奈唑胺的最低抑菌浓度(MIC);采用PCR扩增和测序技术检测cfr、optrA、23S rRNA第V功能区基因;采用脉冲场凝胶电泳(PFGE)对分离菌株进行同源性分析。结果:38株凝固酶阴性葡萄球菌对利奈唑胺、青霉素和苯唑西林均耐药,对万古霉素均敏感;对克林霉素、左氧氟沙星和环丙沙星的耐药率大于95%;38株菌株中检出cfr基因31株;23S rRNAⅤ功能区检出G2576T突变35株,其中2株人葡萄球菌同时检出C2319T突变,另检出C2319T突变人葡萄球菌和表皮葡萄球菌各1株;所有菌株均未检出optrA基因;32株头状葡萄球菌PFGE具有高度的相似性,均为同一个克隆株;4株人葡萄球菌为同一克隆株。结论:江苏地区流行的耐利奈唑胺凝固酶阴性葡萄球菌主要是由cfr基因和23S rRNAⅤ功能区基因突变造成,并存在头状葡萄球菌和人葡萄球菌的克隆播散。     

Agar diffusion,agar dilution,Etest, and agar screening test in the detection of methicillin resistance in staphylococci

Methicillin resistant Staphylococcus is an important worldwide problem. Resistance is verified in strains harboring the mecA gene and laboratory methods used to detect resistance are object of constant investigation. In the present study, 99 clinical isolates of staphylococci (41 S. aureus, 33 S. epidermidis, 12 S. saprophyticus and 13 members of other species) were submitted to different phenotypic methods and conditions. Detection of the mecA gene by PCR was used as the reference method and detected 14/41, 10/33, and 10/25 isolates of S. aureus, S. epidermidis and other species, respectively. Results showed that, for S. aureus and S. epidermidis, agar diffusion, agar dilution, and the E test incubated during 24h at 35 degrees C correctly discriminated mecA positive from mecA negative isolates. For other species, all methods and conditions presented low specificity (ranging from 20% to 66.7%) and, particularly S. saprophyticus, may need molecular methods to correctly assess methicillin resistance.     

自溶素基因与表皮葡萄球菌生物膜形成相关性研究

目的研究自溶素(atlE)基因与表皮葡萄球菌生物膜形成的相关性。方法收集2015年6月至2016年6月该院临床分离的表皮葡萄球菌64株进行研究,用生物膜形成试验检测细菌生物膜,采用聚合酶链反应扩增atlE基因,分析atlE基因与表皮葡萄球菌生物膜形成的相关性。结果检出生物膜阳性菌株24株,检出率为37.5%;检出atlE基因菌株31株,检出率为48.4%;atlE基因与表皮葡萄球菌生物膜形成有明显的相关性(P0.05)。结论表皮葡萄球菌有生物膜形成能力,atlE基因与表皮葡萄球菌生物膜形成相关。     

Elucidating the role of Staphylococcus epidermidis serine–aspartate repeat protein G in platelet activation

Summary.  Background: Staphylococcus epidermidis is a commensal of the human skin that has been implicated in infective endocarditis and infections involving implanted medical devices. S. epidermidis induces platelet aggregation by an unknown mechanism. The fibrinogen-binding protein serine–aspartate repeat protein G (SdrG) is present in 67–91% of clinical strains. Objectives:  To determine whether SdrG plays a role in platelet activation, and if so to investigate the role of fibrinogen in this mechanism. Methods : SdrG was expressed in a surrogate host, Lactococcus lactis , in order to investigate its role in the absence of other staphylococcal components. Platelet adhesion and platelet aggregation assays were employed. Results:    L. lactis expressing SdrG stimulated platelet aggregation (lag time: 2.9 ± 0.5 min), whereas the L. lactis control did not. L. lactis SdrG-induced aggregation was inhibited by α_IIbβ₃ antagonists and aspirin. Aggregation was dependent on both fibrinogen and IgG, and the platelet IgG receptor FcγRIIa. Preincubation of the bacteria with Bβ-chain fibrinopeptide inhibited aggregation (delaying the lag time six-fold), suggesting that fibrinogen acts as a bridging molecule. Platelets adhered to L. lactis SdrG in the absence of fibrinogen. Adhesion was inhibited by α_IIbβ₃ antagonists, suggesting that this direct interaction involves α_IIbβ₃. Investigation using purified fragments of SdrG revealed a direct interaction with the B-domains. Adhesion to the A-domain involved both a fibrinogen and an IgG bridge. Conclusion:  SdrG alone is sufficient to support platelet adhesion and aggregation through both direct and indirect mechanisms.