2017—2018年上海某医院耐甲氧西林金黄色葡萄球菌分子生物学特征研究

目的分析上海市某医院中不同来源耐甲氧西林金黄色葡萄球菌(MRSA)的分子生物学特征,明确上海某医院MRSA的流行特点。方法收集上海市某二甲医院2017—2018年期间72株MRSA,综合应用多位点序列分型(MLST)、脉冲场凝胶电泳(PFGE)分型方法对MRSA菌株进行分子生物学特征分析。结果 72株MRSA中,46株分离自临床病例,26株分离自医院环境。46株临床分离菌株包括33株社区获得性MRSA(CA-MRSA),以及13株医院获得性MRSA(HA-MRSA),且来自医院的不同的病区。感染MRSA的住院患者所在科室以老年科(34.8%,16株)、内科(26.1%,12株)和外科(26.1%,12株)为主。MLST分型结果显示,不同来源MRSA菌株的ST型差别较大,但ST764是不同来源的MRSA菌株的优势克隆群(40.3%,29株),且PFGE聚类结果显示12株不同来源的ST764型MRSA菌株具有较高的同源性(>90%)。结论 ST764型MRSA可能在社区、医院及环境中存在播散,应持续监测MRSA及其变异情况。     

47株金黄色葡萄球菌临床血流感染分离株的基因分型研究

目的 研究金黄色葡萄球菌临床血流感染分离株的分子流行病学特征.方法 收集2006年1月至2008年12月解放军总医院分离的临床血流感染金黄色葡萄球菌菌株(共47株),标本来源均为患者静脉血.采用琼脂稀释法检测所有菌株对多种抗生素的耐药性,PCR方法 检测pvl毒素基因,DiversiLab~(TM)Rep-PCR分型系统分析菌株的同源性;对耐甲氧西林金黄色葡萄球菌(MRSA)进行葡萄球菌染色体/mec(SCCmec)分型以及ST239型别的快速筛查;综合分型和药敏试验结果,挑选部分代表菌株进行多位点序列分型(MLST).结果 47株血流感染金黄色葡萄球菌中,pvl基因检出率为4.3%.MRSA占51.1%.MRSA均为SCCmec Ⅲ型菌株;Rep-PCR分为A～L共12个型,其中A型为最主要的型,共22株(46.8%),所有的MRSA均属于A、B两型.结论 47株临床血流感染金黄色葡萄球菌中的MRSA绝大部分为多重耐药克隆ST239-MRSA-SCCmecⅢ型.     

MLST和PFGE在耐碳青霉烯类肺炎克雷伯菌医院感染监测中的应用

 目的 探讨某院重症监护病房(ICU)患者、医院环境中耐碳青霉烯类肺炎克雷伯菌(CRKP)菌株的同源性,为医院感染防控提供理论依据。方法 收集2017年9—12月某三甲医院ICU患者、环境中连续分离的CRKP菌株,进行耐药表型、碳青霉烯酶基因检测,采用多位点序列分型(MLST)和脉冲场凝胶电泳(PFGE)进行分子同源性分析。结果 共收集10株CRKP菌株,其中9株分离自5例患者的临床感染标本,1株分离自气压治疗仪面板。10株菌均携带产KPC酶基因;MLST分型均为ST11型;共有5种PFGE带型,其中5株带型完全一致,为流行菌株,1株菌株与流行菌株带型仅相差1条条带,其余4株菌带型相近,但与流行株带型差异较大。气压治疗仪面板分离的1株CRKP菌株与患者来源的4株CRKP菌株耐药表型、PFGE型别及ST型别完全一致,考虑为仪器共用造成的医院感染。分离自同一重症胰腺炎患者腹腔引流液、血标本的CRKP菌株PFGE带型完全一致,推测CRKP可能通过腹腔感染入血。结论 PFGE和MLST对明确医院感染细菌传播路径,个体细菌感染路径以及遗传变异有重要意义,有助于从切断感染途径方面入手指导控制多重耐药菌的传播。     

MRSA分子流行病学研究

目的采用脉冲场凝胶电泳(PFGE)分型及多重聚合酶链反应(PCR)SCCmec分型技术对耐甲氧西林金黄色葡萄球菌(MRSA)进行基因分型,以了解医院感染MRSA的流行情况。方法收集天津市南开医院2007年1月-2008年12月住院患者送检标本分离的MRSA 40株,进行耐药谱分析、PFGE分型和SCCmec分型。结果 40株菌耐药谱高度相似,可被分为A-E5个PFGE型,其中A型21株,B型8株,C型4株,D型6株,E型1株;SCCmec分型:Ⅰ型1株,Ⅲ型33株,Ⅳ型1株,Ⅴ型5株。通过对医院内菌株发生的时间和空间分析,明确了感染的发生和传播情况,即在2007年1月-2008年12月存在MRSA散发感染。结论 PFGE可有效鉴定菌株间的亲缘关系,SCCmec分型对是否为医院感染菌株有提示作用,两种分型方法相结合可更好地鉴定流行菌株的特征,为医院感染的监控提供依据。     

MRSA呼吸系统医院感染的分子流行病学调查

目的对广州呼吸疾病研究所2001年7月～2003年3月间,金黄色葡萄球菌引起的呼吸系统医院感染进行病原学监测. 方法收集临床分离菌株,应用脉冲场凝胶电泳的方法(PFGE)进行分型,确定菌株亲缘关系,结合临床资料分析. 结果在各种呼吸道采集标本中共收集到76株金黄色葡萄球菌,耐甲氧西林金黄色葡萄球菌(MRSA)68株,占金黄色葡萄球菌检出率89.47%,MRSA在我所病区中引起59例医院感染,属于暴发形式发病的有23例,属于聚集性发病的有19例,散发病例17例,PFGE分型表明,绝大多数MRSA属于同一型(A型)共有60株,其中A1亚型28株,A2亚型14株,A3亚型17株,A4亚型1株. 结论MRSA引起的呼吸系统医院感染十分严重,并主要由少数菌株的传播引起,及时采取有效措施控制MRSA的流行和播散非常必要.     

云南省迪庆藏族自治州19株耐甲氧西林的金黄色葡萄球菌分子流行病学分析

[目的]对云南省迪庆藏族自治州2012-2013年两家医院分离的金黄色葡萄球菌(MRSA)菌株进行了分子流行病学分析,以此了解该地区MRSA耐药情况。[方法]采用脉冲场凝胶电泳(Pulsed-Feld Gel Electrophoresis,PFGE)、多位点测序分析(multilocus sequence typing,MLST),药物敏感试验和毒力基因检测等方法对19株MRSA进行分析研究。[结果]所有菌株对β-内酰胺类抗生素耐药,部分菌株对喹诺酮类、大环内酯类、四环素以及磺胺类药物耐药;毒力基因检测结果表明,所有菌株均携带有溶血素基因以及不同组合的肠毒素基因;PFGE结果显示19个菌株共产生11个PFGE带型,具有非常高的分辨率水平;MLST分型结果显示,19株MRSA共产生3个ST型别,其中以ST59和ST121型菌株为主。[结论]云南省迪庆藏族自治州MRSA菌株耐药水平不容忽视,菌株的毒力基因携带情况增加了病原菌的致病能力;MRSA的流行株在迪庆藏族自治州地区均有不同程度的分布,同时PFGE对MRSA的分型水平高于MLST,同时又与MLST分型方法具有良好的相关性。     

MRSA分子流行病学研究 FREE

目的 采用脉冲场凝胶电泳（PFGE）分型及多重聚合酶链反应（PCR）SCCmec分型技术对耐甲氧西林金黄色葡萄球菌（MRSA）进行基因分型,以了解医院感染MRSA的流行情况。方法 收集天津市南开医院2007年1月-2008年12月住院患者送检标本分离的MRSA 40株,进行耐药谱分析、PFGE分型和SCCmec分型。结果40株菌耐药谱高度相似,可被分为A-E 5个PFGE型,其中A型21株,B型8株,C型4株,D型6株,E型1株;SCCmec分型：Ⅰ型1株,Ⅲ型33株,Ⅳ型1株,V型5株。通过对医院内菌株发生的时间和空间分析,明确了感染的发生和传播情况,即在2007年1月-2008年12月存在MRSA散发感染。结论 PFGE可有效鉴定菌株间的亲缘关系,SCCmec分型对是否为医院感染菌株有提示作用,两种分型方法相结合可更好地鉴定流行菌株的特征,为医院感染的监控提供依据。     

耐甲氧西林金黄色葡萄球菌多位点序列分析

目的 对2000年和2005年耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)进行多位点序列分析(multilocus sequencing typing,MLST),初步了解MRSA的分子分型特征.方法 采用基因测序的方法 分别对29株MRSA和2株甲氧西林敏感金黄色葡萄球菌(methicillin-sensitive Staphylococcus aureus,MSSA)的7个等位基因进行序列分析,同时进行网上数据库比对,得到每个菌株相应的等位基因号.结果 2000年选取的12株MRSA均为序列型(sequence type,ST)239,2005年的17株MRSA中10株仍为ST239(58.82%,10/17),同时也出现了新的型别ST5(41.18%,7/17).在选取菌株范围内ST239为主要多位点序列分析型别(75.86%,22/29),其次为ST5(24.14%,7/29).2株MSSA分型结果分别为ST6和ST630.结论 ST239和ST5为本次研究的优势型别,MRSA与MSSA在多位点序列分析上存在极大的差异.     

ICU患者分离金黄色葡萄球菌的耐药性及分子流行病学特征

目的了解某院重症监护病房（ICU）金黄色葡萄球菌的耐药特点及分子流行病学特征。方法收集2014年1—12月该院ICU分离的金黄色葡萄球菌,进行细菌鉴定及药物敏感性试验,采用金黄色葡萄球菌A蛋白(spa)分型及多位点序列分型(MLST)方法进行分型。结果160株金黄色葡萄球菌中耐甲氧西林金黄色葡萄球菌（MRSA）120株（占75.00%）。MRSA对红霉素、克林霉素、左氧氟沙星的耐药率均＞80%;MSSA对头孢唑林敏感,对红霉素、克林霉素、左氧氟沙星的耐药率分别为62.50%、35.00%、10.00%。spa分型和MLST结果显示,120株MRSA主要为ST239 t030、ST239 t037、ST5 t2460 3种型别,其中ST239 t030（105株,87.50%）为主要流行菌株,8个ICU均有检出;MSSA存在较多型别,ST59 t437仅在神经内科(8株)和消化科（2株）检出,ST6 t701、ST398 t3625、ST398 t1793和ST121 t2092分别仅在神经内科（7株）、麻醉科（5株）、神经外科（4株）和心外科（4株）检出。结论该院ICU MRSA分离率较高,以ST239 t030克隆株为主,存在医院内流行;不同型别MSSA在各科室内存在流行趋势。     

北京市丰台区腹泻患者肠毒素性大肠埃希菌的分布与分子分型特征

目的了解北京市丰台区腹泻患者肠毒素性大肠埃希菌(ETEC)的分布与分子分型特征,分析2种分型方法的应用价值,为ETEC的分子流行病学研究提供参考依据。方法对腹泻患者便标本分离培养,采用脉冲场凝胶电泳技术(PFGE)和多位点序列分型(MLST) 2种方法进行分型比较。结果 55株ETEC分离株PFGE分型得到40种型别,呈多态性; MLST分型得到19个型别,ST-6010与ST-69是最主要的ST型别,部分试验菌株形成2个ST克隆群,分别为STC-6007、STC-3103。多数菌株的遗传关系较远,可能与菌株来源多样有关。结论 ETEC感染在腹泻患者中长期存在,遗传关系呈多元化。2种分子分型方法的结合,从基因水平上解释了ETEC的流行特征及变化趋势。     

International spread of major clones of methicillin resistant staphylococcus aureus: nosocomial endemicity of multi locus sequence type 239 in Saudi Arabia and Romania

Phenotypically identified methicillin resistant Staphylococcus aureus (MRSA) strains from several hospitals in Romania and Saudi Arabia (n = 103 and 68, respectively) were confirmed to be MRSA by mecA PCR and PBP-2' based latex agglutination. Subsequently, strains were differentiated at the sub-species level using pulsed field gel electrophoresis (PFGE) of SmaI DNA macro-restriction fragments. Comparison of the PFGE fingerprints identified major clusters of strains, persistently present in the various hospitals. Endemicity of certain strains was identified, amongst others one due to a particularly methicillin resistant type in the burn wound sector of the Romanian hospital. No PFGE-based overlap was found between the Saudi and Romanian strains. However, multi locus sequence typing (MLST), performed for 20% of all strains, revealed that genuine genetic similarity was obscured by the PFGE analysis. In both the Romanian and Saudi hospitals the renowned sequence type (ST) 239 was very over-represented. This was especially apparent in Saudi Arabia, where all strains except two shared the ST 239 genotype. This clonal type has previously been identified in a variety of other countries. Despite the MLST concordance, PFGE data indicate that ST 239 diversifies while maintaining its core genome intact. ST 80, another previously but less frequently identified clone, was introduced in 2000 in the Romanian institutes and persisted over the past 3 years as a frequent cause of infections in a surgical department. The successful MRSA types can acquire prominent positions in hospitals of previously low-endemicity MRSA status.     

Clinical and molecular characteristics of nosocomial meticillin-resistant Staphylococcus aureus skin and soft tissue isolates from three Indian hospitals

We analysed risk factors for nosocomial meticillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infections (SSTIs) in three Indian hospitals. We also determined antimicrobial resistance patterns and genotypic characteristics of MRSA isolates using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and staphylococcal cassette chromosome (SCCmec) typing. Medical records of 709 patients admitted to three tertiary hospitals with nosocomial S. aureus SSTIs were clinically evaluated. Antimicrobial susceptibility testing of patient isolates was performed in accordance with Clinical and Laboratory Standards Institute guidelines, with meticillin and mupirocin resistance confirmed by multiplex polymerase chain reaction. PFGE analysis of 220 MRSA isolates was performed, followed by MLST and SCCmec typing of a selected number of isolates. MRSA was associated with 41%, 31% and 7.5% of infections at the three hospitals, respectively. Multiple logistic regression analysis identified longer duration of hospitalisation [odds ratio (OR): 1.78; OR: 2.83 for ≥20 days], intra-hospital transfer (OR: 1.91), non-infectious skin conditions (3.64), osteomyelitis (2.9), neurological disorders (2.22), aminoglycoside therapy (1.74) and clindamycin therapy (4.73) as independent predictors for MRSA SSTIs. MRSA isolates from all three hospitals were multidrug resistant, with fifteen clones (I–XV) recognised. A majority of the strains possessed type III cassette. The common sequence type (ST) 239 was considered the signature MLST sequence for PFGE clone III. This major MRSA clone III was closely related to the UK EMRSA-1 and was significantly more resistant to antibiotics. Dissemination of multidrug-resistant MRSA clones warrants continuous tracking of resistant genotypes in the Indian subcontinent.     

A major methicillin-resistant Staphylococcus aureus clone predominates in Malaysian hospitals

This study was conducted to determine the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in Malaysian hospitals. A total of 264 MRSA isolates from eight hospitals were subjected to typing by pulsed-field gel electrophoresis (PFGE) of SmaI restricted DNA. Antibiotic disk susceptibility testing was also carried out to determine their resistance patterns. Thirty-one PFGE pattern types were identified. Three major pattern types A, ZC and K were found with type A the predominant profile in c. 80% of strains and present in all hospitals. Unlike type A, other DNA pattern types were unique to the hospitals in which they were isolated. PFGE type A also consisted of strains that were multiply antibiotic resistant. The presence of a single predominant PFGE type in Malaysian hospitals is an important finding which suggests that inter-hospital spread of MRSA had occurred frequently and regularly.     

2011-2012年杭州市肠道沙门菌临床分离株的分子型别分析

目的研究2011-2012年杭州市肠道沙门菌临床分离株的型别,了解本地菌株分子流行病学特征。方法对66株肠道沙门菌临床分离株进行血清分型和多位点序列分型(MLST)。对其中主要血清型:鼠伤寒、甲型副伤寒、萨雷甲尼和肠炎沙门菌菌株进行脉冲场凝胶电泳(PFGE)分型。结果分布于21个血清型的66株沙门菌分成26个ST型别。发现一株纽波特沙门菌为新型ST1690。菌株血清型与MLST型别数据库中所对应的血清型符合率为100.00%。9株甲型副伤寒沙门菌的PFGE带型完全一致(P7型),与先前杭州流行菌株有差异(P1-P6型)。6株肠炎沙门菌分成4个PFGE型,型间最小相似性为92.70%。13株鼠伤寒沙门菌分为11个PFGE型,型间最小相似性为71.70%。7株萨雷甲尼沙门菌分成4个PFGE型别,型间最小相似性为91.00%。结论近年杭州腹泻病人中流行的肠道沙门菌菌株主要血清型为鼠伤寒、甲型副伤寒、萨雷甲尼和肠炎等。甲型副伤寒沙门菌菌株在杭州出现了新PFGE型别。MLST数据可以对沙门菌血清学鉴定提供一定的帮助。     

2017-2018年江西地区耐甲氧西林金黄色葡萄球菌的分子流行特征

目的 了解江西各地区耐甲氧西林金黄色葡萄球菌（MRSA）的分子特征及主要流行克隆型别,探讨流行克隆可能的竞争优势,为后续的机制研究提供资料。方法 收集2017年1月-2018年12月江西省11个地市11所三甲医院临床分离的MRSA,同时应用mecA+femB双重PCR法鉴定为MRSA菌株后纳入研究,应用spa分型、SCCmec分型、PFGE和MLST进行分子分型,采用PCR方法检测杀白细胞素（PVL）毒力基因。结果 spa分型主要为t437（74株,占31.22%）、t030（32株,占13.50%）;不同地区的spa型存在很大差异,南昌地区以t030为主,其余地区以t437为主。SCCmecIVa为优势型别（占66.67%）,SCCmecⅢ占15.19%;除南昌地区以SCCmecⅢ为主要型别外（占59.38%）,其余各地区均以SCCmecIVa为主要优势型别。经PFGE分型和MLST,优势克隆型别为ST239、ST59、ST1和ST338;优势克隆群为CC239、CC59和CC1。共检出32株（13.19%）PVL阳性菌,其中鹰潭地区PVL检出率最高（29.63%,8/27）,其次为景德镇地区（26.32%,5/19）。结论 ST59-MRSA-IVa-t437为江西地区的主要优势流行克隆型,且各地区之间存在一定程度的克隆播散。ST239-MRSA-Ⅲ-t30为次要优势流行克隆型,主要在南昌地区多见,其他地区散发。江西地区PVL阳性菌株的主要型别为ST59-MRSA-t437-IVa型。     

2008 - 2014年北京市山夫登堡沙门菌耐药性及分子流行病学特征分析

目的 分析北京市2008 - 2014年山夫登堡沙门菌临床分离株的分子流行病学特征。方法 对从腹泻病例分离到的91株山夫登堡沙门菌进行药敏试验、脉冲场凝胶电泳（PFGE）分子分型及多位点序列分型（MLST）。结果 2011 -2012年山夫登堡沙门菌在北京地区高水平流行,占所有分离株的87.9%,2008 - 2010年,2013 - 2104年各年份高度散发,病例主要集中在20～59岁成人组,5 - 6月为发病高峰;所有菌株对萘啶酸有较高耐药性（76.9%）,对磺胺异噁唑（53.9%）和链霉素（49.5%）中度耐药,对其他抗菌药物均较敏感;PFGE分型将91株腹泻株分成23种型别,形成三大克隆群,引起2011 - 2012年暴发流行的64株菌形成绝对优势克隆株;42株菌的MLST分型结果显示包括暴发流行在内的绝大多数菌株属于常见型ST14,5株散发菌株为新型ST1923。结论 北京市山夫登堡沙门菌具有明显的克隆株优势带型,引起2011 - 2012年的本地流行,对抗生素敏感性较高。     

食品及环境阪崎肠杆菌分离株的MLST和PFGE分型方法比较

目的:对PFGE和MLST方法在阪崎肠杆菌分型研究中的分辨力和潜在价值进行了比较研究和论述。方法:采用PFGE和MLST方法对本实验室分离的19株阪崎肠杆菌和一株标准菌株ATCC51329进行分型研究。结果:PFGE方法可将20株阪崎肠杆菌分为6大类群,共16个PFGE型,分辨力为0.9474。而MLST方法可将20株菌株分为12个ST型,分辨力为0.9263。结论:PFGE法较MLST法具有较高的分辨力,可用于阪崎肠杆菌的溯源研究,而MLST分型方法能通过全球比对数据库得到更多的关于进化和亲缘关系的分析资料,在致病研究、流行病学调查、进化研究方面优于PFGE法。     

Coagulase gemne variants associated with distinct populations of Staphylococcus aureus

An identifying characteristic of Staphylococcus aureus is the production of staphylocoagulase (coagulase). The aim of this study was to determine the clonal distribution of coagulase gene (coa) variants within populations of S. aureus defined by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and protein A variation. The N-terminal region of the coa gene from 43 methicillin-susceptible (MSSA) and 252 methicillin-resistant (MRSA) S. aureus human isolates and 9 animal S. aureus isolates was amplified and digested with HinfI. Twelve types were identified amongst the MSSA isolates and the majority (93%) of MRSA isolates were assigned to 5 of the 12 types. MLST and PFGE analysis identified epidemic populations of MRSA and each epidemic population was characterized by a different coagulase type. Nine of the 12 MLST-defined clonal complex ancestral genotypes recently described each carried a different coagulase type suggesting that coagulase evolution and the evolution of the clonal complexes are intimately related.     

Bovine mastitis Staphylococcus aureus: Antibiotic susceptibility profile,resistance genes and molecular typing of methicillin-resistant and methicillin-sensitive strains in China

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) infection in dairy animals is of great concern for livestock and public health. The aim of present study was to detect new trends of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) towards antibiotic susceptibility, resistance genes and molecular typing by methods of disc diffusion, multiplex PCR assay and multilocus sequence typing (MLST). A total of 219 S. aureus strains were isolated from bovine mastitis cases from six provinces of China, including 34 MRSA strains. The results revealed that more than 70% isolated strains showed resistance to various antibiotics, and multiple-drugs resistance to more than five categories of antibiotics was found more common. The ermC was the most prevalent resistance gene, followed by other genes; however, ermA was the least frequently detected gene. Twenty-eight mecA-negative MRSA and six mecA-positive MRSA strains were detected, and in which three strains were ST97-MRSA-IV, others were ST965-MRSA-IV, ST6-MRSA-IV and ST9-MRSA-SCCmec-NT. The mecA-negative MRSA strains were found resistant to most of the antibiotics, and harbored aac(6′)/aph(2′′), aph(3′)-III and tetM genes higher than MSSA strains. The resistance to most of the antibiotics was significantly higher in MRSA than in MSSA strains. The MLST profiles showed that these strains mainly belonged to CC5, CC398, CC121 and CC50 lineage, especially within ST97 and ST398, while some novel sequence types (ST2154, ST2165 and ST2166) were identified and deposited in the MLST database. This indicates that the resistance of S. aureus is becoming more complicated by changes in multi-drug resistance mechanism and appearance of mecA-negative MRSA isolates, and importantly, MRSA-IV strains in different MLST types are emerging.