金黄色葡萄球菌凝固酶coa基因的克隆、表达及其生物活性分析

目的 体外表达金黄色葡萄球菌(Staphylococcus aureus, S. aureus)凝固酶Coa,评价其生物学活性。方法 根据GenBank中金黄色葡萄球菌凝固酶coa基因序列设计特异性引物,采用PCR方法从S. aureus USA300 CA-MRSA 923 株基因组中扩增coa基因片段,将其克隆至pET-24b(+)表达载体,构建重组表达质粒pET-24b-coa;将经双酶切及测序鉴定正确的重组质粒转化感受态细胞E.coli BL21(DE3) 中进行IPTG诱导表达;通过SDS-PAGE和Western blot对表达的重组Coa(rCoa)进行鉴定,测定rCoa对人血液和兔血浆的凝固活性;最后用纯化的重组蛋白rCoa免疫BALB/c小鼠,经间接ELISA和Western blot分析其免疫原性。结果 构建的重组质粒pET-24b-coa在E.coli BL21(DE3) 中以包涵体形式表达rCoa,分子量约74.8 ku,与预期蛋白大小相符;rCoa在体外具有凝固人全血和兔血浆活性,能刺激小鼠产生高效价的抗Coa特异性抗体。结论 成功地获得了具有良好凝固酶活性和抗原性的金黄色葡萄球菌凝固酶重组蛋白rCoa,为进一步深入研究S. aureus凝固酶Coa的功能及以Coa为靶点的抗金黄色葡萄球菌抑制剂和疫苗的研制奠定基础。     

耐甲氧西林金黄色葡萄球菌杀白细胞素基因检测

目的 调查临床分离的耐甲氧西林金黄色葡萄球菌(MRSA)杀白细胞毒素基因携带率。方法 收集非重复MRSA菌株83株,按照美国疾病预防控制中心的CA-MRSA 定义分为HA-MRSA和CA-MRSA两组。采用多重PCR法进行SCCmec分型,普通PCR+测序法进行spa分型,普通PCR检测杀白细胞素(PVL)基因。结果 83株MRSA中HA-MRSA、CA-MRSA分别占47.0%、53.0%,SCCmec分型中SCCmecⅠ、Ⅱ、Ⅲ、Ⅳa型和未分型各占1.2%、3.6%、65.1%、28.9%、1.2%,spa分型中83株MRSA共检出15个型,主要分型为t437,t062,t015分别占39.8%, 21.7%,10.8%;PVL阳性的MRSA中HA-MRSA和CA-MRSA分别10.3%、36.4%,两者差异有显著性(P=0.006);33株spa t437中有18株携带PVL基因,阳性率54.5%,50株其他spa分型中仅2株携带PVL基因,阳性率4.0%,两者差异有显著性(P=0.000)。PVL基因阳性的MRSA特征CA-MRSA-Ⅳa-t437 9株,HA-MRSA-Ⅲ-t437 4株,HA-MRSA-Ⅳa-t437 3株;20株PVL基因阳性的MRSA中10株分离自皮肤软组织感染病例,6株分离于耳鼻喉科感染病例,3株分离于呼吸道感染病例,1株分离于败血症病例。结论 CA-MRSA菌株较HA-MRSA菌株的 PVL基因阳性率更高,同时也发现携带有更高毒力的CA-MRSA的克隆已经播散到医院的环境中,引起医院获得性相关感染。     

结核患者合并金黄色葡萄球菌感染的耐药性分析

目的了解结核患者合并金黄色葡萄球菌感染对常用抗菌药物的耐药情况。方法对来自结核病人标本分离的99株金黄色葡萄球菌,使用纸片扩散法对药敏结果进行分析。结果共计99株金黄色葡萄球菌检出62株MRSA,MRSA检出率为62.6%。MRSA对万古霉素、利奈唑胺、复方新诺明的敏感率较高,依次为100%、100%和75.8%,但对其他药物呈多重耐药。MRSA耐药率显著高于MSSA。结论金黄色葡萄球菌对常用抗菌药物呈现多重耐药性,特别对于MRSA应有针对性加强监测,根据分离株耐药特点选用不同的治疗方案。     

多重交叉置换扩增联合羟基萘酚蓝检测脓肿分枝杆菌方法的建立

目的 建立一种可视化的恒温扩增方法用于检测脓肿分枝杆菌(MAB)。方法 采用特异性全长erm(41)基因建立基于多交叉置换扩增(MCDA)结合羟基萘酚蓝(HNB)的方法,通过比色法直接检测扩增产物,采用23株脓肿分枝杆菌复合群和8种细菌标准株对该方法进行初步评价。结果 建立的MAB-MCDA-HNB检测限为100 fg的DNA模板。31株细菌菌株中,只有脓肿分枝杆菌检测结果为阳性,其它细菌包括结核分枝杆菌,卡介苗,马赛分枝杆菌、鸟分枝杆菌等非结核分枝杆菌和金黄色葡萄球菌等均为阴性。脓肿分枝杆菌模拟痰标本检测灵敏度为1.7×10³ CFU/mL(每次反应17 CFU)。结论 MAB-MCDA-HNB方法能够快速准确诊断脓肿分枝杆菌的感染。     

人结膜吸吮线虫病2例报告及Cox1基因分析

目的 检测辽宁和山东两地人体结膜吸吮线虫(Thelaziacallipaeda)分离株的形态学及基因型,探讨两地分离株虫体基因差异及其系统发生亲缘关系。方法 采集2例病人感染的虫体进行形态学鉴定,对其线粒体Cox1基因进行PCR扩增测序,将测序结果与基因库中文献比对分析,结合GenBank上已发表的同源序列,对不同地域来源的11个个体进行系统发育分析。结果与结论 经Cox1基因鉴定,2例病人均可确诊为人结膜吸吮线虫病且其感染的虫体均与欧洲种同源(99.1%及99.6%)。两条虫体扩增的Cox1基因碱基存在差异(8/689),但不同地理虫株之间未见明显遗传变异现象(98%~100%)。     

金黄色葡萄球菌ATCC25923人工感染BALB/c 雌鼠肾脏组织PRNP和PRND的表达变化研究

目的 为了研究细胞型朊蛋白和Doppel蛋白在金黄色葡萄球菌感染中的作用。方法 本实验用金黄色葡萄球菌ATCC25923感染BALB/c雌鼠后,分别在1~7 d内解剖小鼠,进行组织抹片革兰氏染色和石蜡切片制作,最后采用荧光定量PCR检测PRNP和PRND在1~7 d中的相对表达量。结果 金黄色葡萄球菌最先出现在肾脏,且对肾脏组织有损伤作用;PRNP和PRND基因在每个时段的表达量都高于空白对照组,其中PRNP在第1 d的表达量最高,为空白对照组的6.1倍,PRND在第2 d的表达量最高,为空白对照组的13.1倍。结论 小鼠肾脏的PRNP和PRND的mRNA表达水平随着金黄色葡萄球菌感染的时间发生变化。     

广东省肺结核临床分离菌株DNA指纹分析

目的　构建结核分支杆菌IS6 110DNA指纹图谱 ,从分子水平探讨广东省结核分支杆菌的特征。方法 参照VanEmbden推荐的结核分支杆菌DNA指纹标准方法 ,构建标准菌株Mt14 32 3和广东省的 74株临床分离株的以RFLP为基础的结核分支杆菌的IS6 110DNA指纹图谱 ;经互联网与世界结核菌DNA指纹库进行相似性比较 ;应用Gelcompar 4 .1(AppliedMaths ,Kortrijk ,Belgium )软件对上述菌株的指纹图谱进行聚类分析。结果 Mt14 32 3标准菌株和 74例临床分离菌株IS6 110DNA指纹图谱结果与国外同类报道一致 ;其中 2 4 .3% (18/74 )的结核菌株的IS6 110DNA指纹相似值在 1～ 0 .6 5之间 ,鉴定结果为它们均是北京家族结核分支杆菌 ;IS6 110 0 1个拷贝和 2个拷贝菌株较多 (2 1/74 )。结论 目前“北京家族”结核分支杆菌菌株一定程度上在广东地区流行。     

延边州结核分枝杆菌DNA指纹图谱特征分析

目的　建立延边州结核分枝杆菌IS6110 RFLP指纹图谱 ,并分析朝鲜、汉两族的图谱特征。方法 97株结核分枝杆菌分离株 ,提取DNA ,经酶切 ,电泳 ,Southem转印 ,探针杂交 ,发光自显影技术得到IS6110 RFLP图谱;按其指纹图谱特征的同源性高低进行分析。结果 延边州结核分枝杆菌IS6110拷贝数目平均为 13个;57.7%菌株可成簇 ,具有“北京基因型”特征;IS6110 RFLP图谱特征分布无地域差异 ,无民族差异。结论 延边州结核分枝杆菌分离株同北京分离株有较近的亲缘关系 ,但其多态性较高。     

山东地区屠宰场猪肉污染沙门氏菌菌株毒力基因筛查与ERIC-PCR分型

目的 了解生猪屠宰加工过程中沙门氏菌的污染状况、分离菌株的毒力基因携带情况及其溯源性追踪。方法 应用液相芯片法对分离沙门氏菌进行血清分型,并对分离菌株进行毒力基因筛查,通过基因间重复序列为引物的聚合酶链式反应(ERIC—PCR)分型技术对代表性沙门氏菌分离株进行基因分型,并用NTSYS-pc2.10软件进行聚类分析。结果 1 480份样品共得到298株沙门氏菌菌株,分离率为20.14%;98.32%的分离菌株携带肠毒素stn基因;96%以上的分离菌株携带毒力岛SPI1、SPI5核心蛋白基因mogA、araB以及菌毛基因;选择实验的沙门氏菌遗传相似性在70%~100%之间,共分为 9个基因型。结论 山东地区屠宰环节猪肉中分离沙门氏菌携带多种耐药基因和毒力基因,德尔卑、鼠伤寒沙门氏菌占试验菌株的45.97%。     

金黄色葡萄球菌医院感染及耐药分析

目的了解金黄色葡萄球菌感染的特点及其对抗菌药物的耐药性,为临床治疗金黄色葡萄球菌感染提供选药依据。方法对2012-01~2014-06该院临床分离出的196株金黄色葡萄球菌进行常规培养分离鉴定,药敏试验应用K-B纸片扩散法,按国家临床实验室标准委员会/美国临床实验室准化委员会(CLSI/NCCLS)规定的标准进行细菌鉴定和药敏检测。结果共分离出耐甲氧西林金黄色葡萄球菌(MRSA)91株(46.4%),甲氧西林敏感金黄色葡萄球菌(MSSA)105株(53.6%)。其中MRSA主要来源于呼吸内科,以老年患者为主。MRSA及MSSA对万古霉素、利奈唑胺、替考拉宁均敏感。MRSA对氨苄西林、苯唑西林、青霉素G、头孢唑林、头孢西丁的耐药率为100%,MSSA耐药率由高至低依次为青霉素G、氨苄西林、红霉素。MRSA对各种抗菌药物的耐药率均高于MSSA,差异有统计学意义(P0.01)。结论 MSSA和MRSA对抗菌药物耐药率不同,须重视金黄色葡萄球菌的耐药监测,控制耐药菌株,更好地指导临床合理选用抗生素。     

医院内气载耐甲氧西林金黄葡萄球菌（MRSA）耐药性及耐药基因分析

目的监测医院中气载耐甲氧西林金黄葡萄球菌（MRsA）对临床常用抗生素的耐药性及相关耐药基因。方法用FA-1型六级筛孔撞击式空气微生物采样器采集5所医院的大厅、ICU及病房等场所的空气,分离金黄葡萄球菌,采用头孢两丁纸片法检测MRSA,应用K-B法测定MRSA药敏情况,应用多重PCR扩增MRSA甲氧西林耐药基因rrlecA,氨基糖苷修饰酶基因aacA-aphD,大环内酯类23srRNA甲基化酶基因ermA、errnC,四环素类核糖体保护蛋白基因tetM、tetK以及金黄葡萄球菌属特异性基因16srDNA。结果共采集空气样品250份,分离气载金黄葡萄球菌219株,其中MRSA88株。所有气载MRSA均对青霉素及头孢曲松耐药,对庆大霉素、红霉素及四环素耐药率〉90％,但均对万古霉及素替考拉宁敏感。所有MRSA菌株iTIeCA基因和16srDNA基因均阳性,vat（A）、vat（B）、vat（C）基因均阴性。96％以上MRSA菌株可同时检出氨基糖苷类、红霉素类和四环素类耐药基因,呈现多重耐药;携带aacA—aphD、erm（A）或（和）erm（C）、tetM或（和）tetK耐药基因的MRSA菌株总检出率分别为96．59％、100％和96．59％。结论5所医院空气中均存在MRSA分布且呈多重耐药性,相关耐药基因检出率高。因此应加强对医院空气中致病菌的监测与消毒,预防和控制医院气源病原微生物感染的发生。     

"Gesundheit!" sneezing, common colds, allergies, and Staphylococcus aureus dispersion

BACKGROUND: Staphylococcus aureus is among the most important pathogens in today's hospital setting. METHODS: The effects of sneezing on the airborne dispersal of S. aureus and other bacteria were assessed in 11 healthy nasal S. aureus carriers with experimentally induced rhinovirus colds. Airborne dispersal was studied by volumetric air sampling in 2 chamber sessions with and without histamine-induced sneezing. After 2 days of preexposure measurements, volunteers were inoculated with a rhinovirus and monitored for 14 days. Daily quantitative nasal- and skin-culture samples for bacteria and nasal-culture samples for rhinovirus were obtained, cold symptoms were assessed, and volunteer activities were recorded during sessions. RESULTS: All participants developed a cold. Sneezing caused a 4.7-fold increase in the airborne dispersal of S. aureus, a 1.4-fold increase in coagulase-negative staphylococci (CoNS), and a 3.9-fold increase in other bacteria (P < .001). An additional 2.83 colony forming units (cfu) of S. aureus/m3/min, 3.24 cfu of CoNS/m3/min, and 474.61 cfu of other bacteria/m3/min were released per sneeze. Rhinovirus exposure did not change the frequency of sneezing or airborne dispersal. Having respiratory allergies increased the spread of S. aureus by 3.8-fold during sneezing sessions (P < .001). CONCLUSION: Nasal S. aureus carriers disperse a significant amount of S. aureus into the air by sneezing. Experimental colds do not alter bacterial dispersal, but respiratory allergies multiply the effect of dispersing S. aureus.     

Fibronectin, fibrinogen, and laminin act as mediators of adherence of clinical staphylococcal isolates to foreign material

Bacterial adherence to polymer surfaces is a required early step in intravenous (iv) device infection. We collected eight strains of Staphylococcus aureus and 19 of coagulase-negative staphylococci from patients with proven iv device bacteremia and studied the role of plasma or connective-tissue proteins in promoting bacterial adherence to polymethylmethacrylate (PMMA) coverslips. Although only a negligible percentage of organisms adhered to albumin-coated PMMA, surface-bound fibronectin significantly promoted adherence of all isolates. Fibrinogen markedly promoted adherence of all S. aureus strains but of only four coagulase-negative strains. Thus, coagulase-negative staphylococci revealed a marked heterogeneity in adherence to fibrinogen-coated surfaces, a result suggesting the existence of heretofore unknown receptors for fibrinogen. Laminin promoted adherence of staphylococci to a much lower extent. Although strain specific, adherence of clinical staphylococcal isolates to foreign surfaces is significantly increased by fibronectin, fibrinogen, and laminin, an observation suggesting the possible contribution of these proteins to the pathogenesis of iv device infection.     

2009年中国12家教学医院革兰阳性球菌耐药性研究

目的 调查2009年我国革兰阳性球菌临床分离株的耐药性.方法 收集2009年6-12月9个城市12家教学医院临床分离的1169株非重复革兰阳性球菌.采用琼脂稀释法测定抗菌药物的最低抑菌浓度(MIC)值.结果 金黄色葡萄球菌和凝固酶阴性葡萄球菌中耐苯唑西林菌株分别占45.3%(211/466)和89.5%(214/239);不同标本苯唑西林耐药金黄色葡萄球菌(MRSA)分离率为33.3%～68.1%.未发现对替考拉宁、万古霉素和利奈唑胺耐药的葡萄球菌.5.5%(7/128)的屎肠球菌对万古霉素耐药,未发现万古霉素耐药的粪肠球菌;粪肠球菌和屎肠球菌对利奈唑胺的敏感率约为99.1%(108/109).肺炎链球菌中青霉素中介株(P1SP)分离率为21.6%(48/222),仅发现1株青霉素耐药株(PRSP),占0.5%(1/222);未发现对替考拉宁、万古霉素和利奈唑胺耐药的肺炎链球菌.结论 葡萄球菌中苯唑西林耐药菌株仍有较高的分离率,不同标本类型MRSA的分离率有所不同.替考拉宁、万古霉素和利奈唑胺对葡萄球菌、粪肠球菌、屎肠球菌和肺炎链球菌具有很好的抗菌活性.     

Inducible clindamycin resistance in staphylococci isolated from clinical samples

This study aimed to determine the levels of the macrolides-lincosamides-streptogramins B (MLS(B)) resistance phenotype of Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolates from clinical samples. A total of 521 strains of staphylococci, comprising 230 S. aureus and 291 CNS isolates from various clinical samples, were identified by conventional methods. The double-disc test was applied by placing erythromycin and clindamycin discs on these isolates to investigate the inducible and constitutive MLS(B) resistance phenotypes and MS phenotype. Among the S. aureus strains, 24.3% showed the constitutive and 7.8% the inducible phenotype, while there was no MS phenotype. In the CNS strains, 40.2% showed the constitutive and 14.7% the inducible MLS(B) resistance phenotype, and 18.2% had the MS phenotype. In both S. aureus and CNS strains, the constitutive MLS(B) resistance rate was found to be higher than the rate of inducible resistance. By applying double-disc tests on a routine basis to detect inducible MLS(B) resistance, clindamycin can be effectively used on staphylococcal infections. Additionally, it can be used to survey the MLS(B) resistance of staphylococci strains from specific geographical regions or hospitals.     

Bacteremia and candidemia in hematological malignancies: microbiological findings and antibiotic susceptibilities

The microorganisms isolated in 1981-1985 from 171 cases of septicemia in patients with hematological malignancies were on the whole the same as those found in 1970-1972. The distribution between species was also quite similar for the two periods except within staphylococci, where the isolation rate of coagulase-negative staphylococci was higher in the latter period while that of Staphylococcus aureus was lower. Of 67 strains of Enterobacteriaceae tested for an aminoglycoside, 6% were found to be resistant, whereas 8% of 48 Enterobacteriaceae strains were found to be cefotaxime resistant. Methicillin- or aminoglycoside resistant S. aureus did not occur.     

猪源耐甲氧西林金黄色葡萄球菌的耐药表型及其SCCmec基因分型研究

目的 了解上海猪场及屠宰场耐甲氧西林金黄色葡萄球菌(MRSA)的流行状况、耐药谱特征及其SCCmec基因分型特点。方法 我们于2011年8月—2012年5月分别在上海市5个规模化猪场和1个屠宰场,采集猪鼻腔拭子样品共计232份。通过7.5%氯化钠肉汤预增菌,显色培养基显色培养分离金黄色葡萄球菌(SA),采用双重PCR方法扩增其中是否含有nuc基因和mecA基因进行MRSA鉴定;以肉汤稀释法进行药物敏感性试验;应用多重PCR方法进行SCCmec基因分型;同时检测杀白细胞素(PVL)基因。结果 共计分离得到139株SA,其中70株MRSA,MRSA的检出率为30.2%(70/232)。基因分型显示均为SCCmecⅣb型,未检测到PVL基因,药敏结果显示MRSA除对利奈唑胺,万古霉素,阿米卡星全部敏感外,对头孢噻呋,庆大霉素,诺氟沙星耐药率分别为74.3%,62.9%和42.9%,对阿奇霉素,红霉素,氟苯尼考,氯霉素,苯唑西林的耐药率均为100%,值得注意的,本研究中分离的全部MRSA都对截短侧耳类药物泰妙菌素、沃尼妙林以及人医新药瑞他帕林表现高水平耐药。结论 结果显示了上海地区猪源MRSA主要流行SCCmecⅣb型,并且具有多重耐药的特点,尤其对截断侧耳类人医新药全部耐药,这也提示我们要防控动物源耐药菌或耐药基因通过食物链或者环境向人类的传播的潜在风险。     

Nosocomial bloodstream infections in United States hospitals: a three-year analysis.

Nosocomial bloodstream infections are important causes of morbidity and mortality. In this study, concurrent surveillance for nosocomial bloodstream infections at 49 hospitals over a 3-year period detected >10,000 infections. Gram-positive organisms accounted for 64% of cases, gram-negative organisms accounted for 27%, and 8% were caused by fungi. The most common organisms were coagulase-negative staphylococci (32%), Staphylococcus aureus (16%), and enterococci (11%). Enterobacter, Serratia, coagulase-negative staphylococci, and Candida were more likely to cause infections in patients in critical care units. In patients with neutropenia, viridans streptococci were significantly more common. Coagulase-negative staphylococci were the most common pathogens on all clinical services except obstetrics, where Escherichia coli was most common. Methicillin resistance was detected in 29% of S. aureus isolates and 80% of coagulase-negative staphylococci. Vancomycin resistance in enterococci was species-dependent--3% of Enterococcus faecalis strains and 50% of Enterococcus faecium isolates displayed resistance. These data may allow clinicians to better target empirical therapy for hospital-acquired cases of bacteremia.     

甘肃武威地区2006～2007年常见葡萄球菌感染及耐药状况分析

目的了解甘肃武威地区常见葡萄球菌感染及耐药状况,以更好地指导临床用药。方法将2006～2007年甘肃省武威市医院采集的320株葡萄球菌采用常规方法分离,用Vitek-32全自动微生物分析仪进行菌种鉴定及药敏试验。结果分离到金黄色葡萄球菌192株,表皮葡萄球菌86株,溶血性葡萄球菌42株。其中耐甲氧西林葡萄球菌213株,包括耐甲氧西林金黄色葡萄球菌118株（36．9％）,耐甲氧西林表皮葡萄球菌60株（18．8％）,耐甲氧西林溶血性葡萄球菌35株（10．9％）。耐甲氧西林金黄色葡萄球菌、表皮葡萄球菌和溶血性葡萄球菌均成多重耐药特征,未发现耐万古霉素、替考拉丁的葡萄球菌。结论耐甲氧西林葡萄球菌检出率呈明显上升趋势,合理使用抗生素,尤其万古霉素的使用,已成为当务之急。     

Characterization of macrolide resistance in Gram-positive cocci from Colombian hospitals: a countrywide surveillance.

OBJECTIVE: The characterization of macrolide resistance in Gram-positive cocci recovered from Colombian hospitals. METHODS: The resistance profiles and mechanism of macrolide resistance were investigated in isolates of Streptococcus pneumoniae (1679), Staphylococcus aureus (348), coagulase-negative staphylococci (CoNS) (175), and Enterococcus spp (123). Minimum inhibitory concentrations (MICs) for erythromycin (ERY) and clindamycin (CLI), detection of macrolide resistance genes, phenotypic characterization, and pulsed field gel electrophoresis (PFGE) of macrolide-resistant pneumococci were performed. RESULTS: Resistance to ERY and CLI was 3.3% and 2.3% for S. pneumoniae, 58% and 57% for S. aureus (94% for both compounds in methicillin-resistant Staphylococcus aureus (MRSA)), and 78.6% and 60.7% in methicillin-resistant Staphylococcus epidermidis, respectively. ERY resistance was 62% in Enterococcus faecalis and 82% in Enterococcus faecium. The MLS(B)-type accounted for 71% of S. pneumoniae and 100% of MRSA. The erm(A) gene was prevalent in MRSA, erm(B) in S. pneumoniae and enterococci, and erm(C) in CoNS isolates. Efflux pump genes (mef(A) genes) were mostly identified in S. pneumoniae (24%). The most common genotype amongst ERY-resistant pneumococci was the Spain(6B)-2 clone. CONCLUSIONS: The prevalence of macrolide resistance is low in Colombian pneumococci and high in MRSA (cMLS(B)-type).