深圳市金黄色葡萄球菌耐药性分析及分子分型

目的了解深圳市金黄色葡萄球菌的耐药性特点及分子分型特征。方法收集2012年来自深圳市7所医院的428株金黄色葡萄球菌,以琼脂稀释法测定其对12种抗菌药物的最低抑菌浓度(MIC),采用聚合酶链式反应(PCR)检测杀白细胞毒素(PVL),并对携带PVL基因的菌株进行多位点基因序列分型(MLST)。结果428株金黄色葡萄球菌中耐甲氧西林金黄色葡萄球菌(MRSA)共116株(26.2%),甲氧西林敏感金黄色葡萄球菌(MSSA)共312株(73.8%)。在12种抗菌药物中,该菌对青霉素和红霉素的耐药率最高,分别为88.8%和44.2%;未发现替考拉宁、利奈唑胺和万古霉素的耐药株。MRSA对青霉素和环丙沙星的耐药率显著高于MSSA。428株金黄色葡萄球菌中,有60株(14.02%)携带PVL基因。MLST分型结果显示共有14种已知序列型和4种新的序列型,其中ST59和ST338最多,分别为16株和12株。结论深圳地区金黄色葡萄球菌MRSA检出率以及对多种抗菌药物的耐药率均低于全国平均水平,PVL基因阳性率处于中等水平;存在多种ST分型,以ST59和ST338多见,具有遗传多样性和独特的遗传背景。     

金黄色葡萄球菌儿童临床分离株携带PVL基因状况的分析

目的了解金黄色葡萄球菌儿童分离株携带Panton-Valentine杀白细胞素（PVL）基因的状况及感染类型。方法采用多重PCR同时检测金黄色葡萄球菌16SrRNA基因、PVL基因和mecA基因;多重PCR检测MR—SA的SCCmec基因型及亚型。结果66株金黄色葡萄球菌JL童临床分离株经多重PCR检测,其中MRSA有7株（10．6％）,MSSA有59株（89．4％）;携带PVL基因金黄色葡萄球菌有31株,总阳性率为47．O％（31／66）,其中2株为MRSA,29株为MSSA,阳性率分别为28．6％（2／7）和49。2％（29／59）。2株MRSA都属于SCCmecIV型;31株PVL基因阳性分离株有21株分离自脓液,7株分离自血液,仅1株分离自痰液。结论儿童MSSA是携带PVL基因的主要菌株,携带PVL基因的金黄色葡萄球菌主要引起化脓性感染和血流感染。     

社区获得性和医院获得性耐甲氧西林金黄色葡萄球菌PVL基因检测

目的 调查本地区患者各种标本中分离耐甲氧西林金黄色葡萄球菌(MRSA)的杀白细胞素(PVL)基因携带情况,为临床MRSA的治疗及流行病学调查提供合理的依据.方法 对所分离的MRSA菌株进行药敏试验分析,同时采用PCR法检测mecA基因和PVL基因,比较社区获得性MRSA (CA-MRSA)和医院获得性MRSA (HA-MRSA)之间耐药性的比较及PVL基因携带率的比较.结果 对不同来源的9l株MRSA分离株耐药性分析,CA-MRSA对环丙沙星、利福平、庆大霉素和左旋氧氟沙星的敏感性明显高于HA-MRSA.经PCR检测发现,所有菌株均携带有mecA基因,21株携带有PVL基因,其中65株HA-MRSA有仅8株携带有PVL基因,而26株CA-MRSA中有13株携带有PVL基因,携带率差异有统计学意义.结论 本地区CA-MRSA是携带PVL基因的主要菌株,HA-MRSA对抗菌药物的耐药性明显高于CA-MRSA,尚未发现对万古霉素和替考拉宁的耐药菌株.     

耐甲氧西林金黄色葡萄球菌的分子流行病学研究

了解我院患者耐甲氧西林金黄色葡萄球菌（MRSA）的分子流行病学特点,为临床抗感染治疗提供依据。收集2007年1月~2008年9月我院分离的耐甲氧西林金黄色葡萄球菌共54株,采用PCR进行SCCmec基因分型、葡萄球菌A蛋白（SPA）分型,并检测杀白细胞毒素（PVL）基因,同时应用脉冲场凝胶电泳（PFGE）进行同源性分析。54株MRSA菌株SCCmec基因分型为SCCmecⅡ型17株,SCCmecⅢ型33株,SCCmecⅣ型2株,SCCmecⅤ型2株;SPA基因分型将28株归属为t030,9株为t002,8株为t037,5株为t570,2株为t437,t163和t796各1株;PVL毒素检测只有2株SCCmecⅣ型菌株阳性;PFGE证实院内MRSA感染主要为2种克隆株传播,同时还有其他型别出现。本院MRSA流行传播的SCCmec基因型主要以Ⅲ型占优势,同时发现有携带PVL毒素的CA-MRSA分离株流行,应引起密切关注。     

携带TSST-1和PVL基因的金黄色葡萄球菌的耐药特点、分布特征及与致病性的关系

目的:探讨携带中毒休克综合征毒素-1(TSST-1)和杀白细胞毒素(PVL)基因的金黄色葡萄球菌的耐药特点、分布特征及其与致病性的关系。方法:收集金黄色葡萄球菌临床分离菌株93株,采用聚合酶链反应(PCR)检测TSST-1基因和PVL基因,采用琼脂扩散法检测金黄色葡萄球菌菌株对青霉素(PEN)、苯唑西林(OXA)、头孢噻吩(CEF)、氨苄西林(AMP)、头孢噻肟(CTX)、阿莫西林/克拉维酸(AMC)、亚胺培南(IPM)、克拉霉素(CLR)、万古霉素(VAN)、环丙沙星(CIP)、庆大霉素(GM)、左氧氟沙星(LVX)和利福平(RA)13种抗菌药物的耐药性。结果:耐甲氧西林金黄色葡萄球菌(MRSA)占总数的88.2%,甲氧西林敏感金黄色葡萄球菌(MSSA)占总数的11.8%。TSST-1+菌株在MRSA、MSSA中分别占12.2%、0,差异无统计学意义(P0.05);PVL+菌株在MRSA、MSSA中分别占40.2%、9.1%,差异有统计学意义(P0.05)。MRSA存在明显的耐药性,且表现出多药耐药性,而携带TSST-1与PVL基因的MRSA耐药性更严重。结论:MRSA在金黄色葡萄球菌中的分离率高,耐药性严重,携带TSST-1与PVL基因的MRSA耐药性与致病力增加。     

金黄色葡萄球菌的耐药性分析及基因分型研究

目的通过分析上海地区院内分离金黄色葡萄球菌的药敏谱型及对耐甲氧西林的金黄色葡萄球菌（MRSA）进行基因谱型的研究,了解金黄色葡萄球菌的院内流行状况。方法对临床分离出的43株金黄色葡萄球菌进行药敏试验和SCCmec基因盒的多重PCR检测,并将结果整合后用MEGA3．1软件分析其进化相关关系。结果药敏结果显示43株金葡菌对青霉素和甲氧西林的耐药率最高。甲氧西林的耐药率达到62．8％。MecA阳性菌株SCCmec的分型显示均为Ⅱ型或Ⅲ型,且所占比例相近,未见Ⅰ型和Ⅳ型。进化树分析发现了在同一医院中亲缘关系相近的菌株,为院内感染流行株。结论MecA基因介导的MRSA在分离菌株中所占比例高,存在院内感染爆发性流行。     

性病患者金黄色葡萄球菌感染耐药性调查

目的：分析和探讨性病后泌尿生殖系统金黄色葡萄球菌及耐甲氧西林金黄色葡萄球菌(MRSA)感染及其对各类抗生素的耐药性,以协助指导临床合理用药。方法：采用常规培养鉴定方法和1999年美国NCCLS药敏试验纸片扩散法检测泌尿生殖系标本中金黄色葡萄球菌、MRSA分离率及其对抗生素的敏感性。结果：427例标本共分离出金黄色葡萄球菌236株,其中MRSA占43．6％。金黄色葡萄球菌对常用16种抗生素的耐药率小于20％有万古霉素、呋喃坦啶、阿米卡星、利福平。MRSA对上述外的抗生素均有不同程度的耐药,且耐药性均高于MRSS．呈多重耐药。结论：金黄色葡萄球菌在性病泌尿系统感染占首位,这些菌株对各类抗生素有较高的耐药性,且呈多重耐药．应密切关注MRSA流行和播散。     

2016-2017年宁夏地区牛源金黄色葡萄球菌的全基因组分析

[目的] 了解宁夏地区奶牛乳腺炎金黄色葡萄球菌（Staphylococcus aureus,SA）代表菌株的基因组序列基本特征,进一步探究其耐药基因型、毒力及进化关系,为兽医临床防治提供理论依据。[方法] 采用纸片法对97株金黄色葡萄球菌临床分离株进行抗菌药物敏感性试验,同时进行葡萄球菌蛋白A（Staphylococcus aureus protein A,spa）分型、多位点序列分型（multilocus sequence typing,MLST）,根据分型结果选取16株代表菌株进行全基因组测序,并对获得的测序序列进行处理分析。[结果] 药敏试验结果显示97株分离株对18种抗菌药物存在不同程度的耐药,其中9株耐甲氧西林金黄色葡萄球菌（methicillin-resistantStaphylococcus aureus,MRSA）对青霉素、氨苄西林、苯唑西林、头孢噻呋、磺胺异噁唑、红霉素、庆大霉素和克林霉素等8种抗菌药物完全耐药,甲氧西林敏感金黄色葡萄球菌（methicillin-sensitiveStaphylococcus aureus,MSSA）菌株对青霉素、氨苄西林、磺胺异噁唑耐药率较高。耐药基因数据库（antibiotic resistance genes database,ARDB）注释分析显示16株代表菌株共携带21种耐药基因,其中norA、tet38、bacA、mepA的携带率较高,与药敏试验结果具有一定的相关性。毒力基因数据库（virulence factors of pathogenic bacteria,VFDB）注释分析显示所有菌株携带多种与粘附、宿主免疫逃逸、分泌、胞外酶编码、铁摄取等疾病相关的毒力基因,MRSA菌株均携带较多毒力因子,MSSA菌株携带毒力因子数目不等。基因岛预测结果显示16株代表菌株存在不同数量的基因岛且MRSA菌株携带基因岛数目及毒力基因岛较多,但耐药基因岛数目与MSSA差异不明显。SNP分析结果显示部分分离株同源性较高,同源性较高的两株MRSA的全基因组基本序列特征差异较小,携带的耐药、毒力基因情况相似。[结论] 宁夏地区牛源SA分离株耐药性情况严重且具有较高的毒力水平,本研究为家畜相关MRSA（livestock-associated MRSA,LA-MRSA）与MSSA基因组序列信息的比较分析及宁夏地区SA感染的临床防控提供参考依据。     

MRSA中mecA及femB基因的检测与耐药相关性

研究femB、mecA基因在耐甲氧西林金黄色葡萄球菌(MRSA)中的表达与耐药的关系.运用PCR对MRSA的femB、mecA基因进行检测,MRSA耐药检测采用头孢西丁纸片法.40 株金黄色葡萄球菌(下简称金葡菌)通过头孢西丁纸片法,检出 30 株耐头孢西丁的菌株,通过PCR检测这 40 株金葡菌mecA基因,30 株MRSA全部为阳性, femB基因在 30 株MRSA中全部表达,而甲氧西林敏感的金黄色葡萄球菌(MSSA)的未表达.结果可见,PCR能快速准确地鉴定MRSA, mecA基因是MRSA的耐药基因,femB基因是MRSA的耐药相关基因.     

深圳住院患儿MRSA的分子分型和耐药性

目的了解深圳住院患儿耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)分子型别及其抗生素的耐药情况,为深圳地区和我国其他地区儿童MRSA感染的监控提供实验依据,为研究该地区儿童MRSA菌株的耐药机制提供依据。方法收集2014年1月至2015年10月深圳市儿童医院临床分离的社区获得性MRSA(community-acquired,CA-MRSA)、院内获得性MRSA(hospital-acquired,HA-MRSA)共129株,应用SCCmec、spa、MLST等方法进行分子分型及追溯同源性。结果 129株MRSA SCCmec分型中只有SCCmecⅤ型、SCCmecⅣ和未分出型别(non-typeableNT型),spa分型中t437是最主要型别,MLST分型ST59为最主要型别。两类MRSA对非β-内酰胺酶抗生素如红霉素、克林霉素耐药高达70%以上。结论深圳住院患儿MRSA中的ST59-SCCmecⅣ-t437为主要流行克隆。CA-MRSA与HA-MRSA在分子分型上无差异性。MRSA对部分非β-内酰胺酶抗生素高度耐药,暂无耐万古霉素MRSA菌株。     

Molecular identification and genotyping of MRSA isolates

The aim of this study was to identify and characterize 97 methicillin-resistant Staphylococcus aureus (MRSA) isolates. Two conventional multiplex PCR assays, a real-time PCR assay and two PCR-based genotyping techniques including the spa - and hypervariable region (HVR)-typing methods were used to identify and characterize 97 MRSA strains isolated between April 2006 to September 2007 from the Steve Biko Academic Hospital. All MRSA isolates were positive for 16S rRNA gene, 99% were positive for the mec A gene and 4% positive for the Panton–Valentine leukocidin (PVL) gene. Staphylococcal cassette chromosome mec (SCC mec ) typing showed 67% of isolates were SCC mec II [health-care-associated MRSA (HA-MRSA)], 14% were SCC mec III (HA-MRSA) and 4% were SCC mec IVd [community-associated MRSA (CA-MRSA)]. These CA-MRSA isolates showed a prevalence of 100% for the PVL gene. Using spa typing, three distinct clusters could be identified while HVR typing revealed six different clusters. CA-MRSA isolates were clustered together using spa and HVR typing. This study showed the prevalence of the CA-MRSA strains, PVL genes, the SCC mec types and the clonality of the MRSA strains. The high prevalence of the PVL gene in CA-MRSA isolates already residing in intensive care units was alarming and indicated the emergence of new MRSA lineages with a particular fitness for community and hospital transmission.     

Molecular nature of methicillin-resistant Staphylococcus aureus derived from explosive nosocomial outbreaks of the 1980s in Japan

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) with Panton-Valentine leukocidin (PVL) genes is increasing worldwide. Nosocomial outbreak-derived (hospital-acquired) MRSA (HA-MRSA) in Japan in the 1980s was also largely PVL(+). PVL(+) HA-MRSA and CA-MRSA shared the same multi-locus sequence type (ST30) and methicillin resistance cassette (SCCmecIV), but were divergent in oxacillin resistance, spa typing, PFGE analysis or clfA gene analysis. PVL(+) HA-MRSA, which probably originated in PVL(+)S. aureus ST30, was highly adhesive (carrying cna and bbp genes), highly-toxic (carrying luk(PV) and sea genes) and highly drug-resistant. PVL(+) HA-MRSA was once replaced by other PVL(-) HA-MRSA (e.g., ST5), and is re-emerging as CA-MRSA.     

Gene sequences and specific detection for Panton-Valentine leukocidin

A new category of methicillin-resistant Staphylococcus aureus (MRSA), called community-acquired MRSA (CA-MRSA), has emerged worldwide. In contrast to previous MRSA, most CA-MRSA carries the Panton-Valentine leukocidin (PVL) genes (lukPVSF) as a virulence genetic trait. Sequence analysis of the lukPVSF gene of a Japanese isolate demonstrated that the gene has more similarity to methicillin-susceptible S. aureus from France than MRSA from the United States. Based on the sequences, we developed a real-time PCR assay for the three key genes of CA-MRSA; that is, lukPVSF, mecA (for methicillin resistance), and spa (for S. aureus). Dual or triple assay for lukPVSF, mecA, and spa in one test tube became possible. The detection limit of the assay with probe and SYBR Green methods was between 2.7 and 2.7 x 10(1) CFU/ml. The assay detected PVL-positive MRSA in clinical (blood) isolates.     

Comparison of methods for the detection of methicillin resistance in Staphylococcus aureus isolates from food products

AIMS: To compare several methods for detection of methicillin resistance in Staphylococcus aureus isolates from food. METHODS AND RESULTS: Two hundred S. aureus isolates from food of animal origin were screened for methicillin resistance by a PCR assay specific for the mecA gene, an oxacillin agar screen test and a cefoxitin disk diffusion test. Six out of 200 strains (3%) were found to be methicillin-resistant Staphylococcus aureus (MRSA) by PCR. The oxacillin agar screen test detected only one of the MRSA isolates (sensitivity of 16.7%) and mischaracterized three additional strains as MRSA (specificity of 98.45%). None of the MRSA strains was detected by the cefoxitin test (sensitivity of 0%), while 15 methicillin-susceptible S. aureus (MSSA) strains were misclassified as resistant (specificity of 92.3%). Fifteen MSSA strains displayed a beta-lactamase hyperproducer-like phenotype. The six MRSA (mecA-positive) strains resembled the characteristics of heteroresistant strains. CONCLUSIONS: As MRSA of animal origin may display atypical phenotypes, PCR appears to be more reliable for detection of methicillin resistance in animal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The study stresses the need for implementing the methods of screening S. aureus from food of animal origin for methicillin resistance.     

烧伤病房患者创面金葡菌分布及耐药性分析

目的分析烧伤病房患者不同创面金葡菌的分布及耐药性,为临床合理选用抗菌药提供依据。方法对2006年1月至2013年12月间中国人民解放军第八五医院烧伤病房患者创面分离出金葡菌,采用K—B纸片扩散法进行药物敏感试验。分析金葡菌的耐药性,并对难愈性创面、非难愈性创面的耐甲氧西林金葡菌（MRSA）与甲氧西林敏感金葡菌（MSSA）的耐药性进行对比分析。结果分离出金葡菌112株,其中难愈性创面有70株MRSA和17株MSSA来自难愈性创面,16株MRSA和9株MSSA来自非难愈性创面。金葡菌对青霉素、红霉素、克林霉素的耐药率较高（分别为94．64％、81．25％和74．11％）,对复方新诺明、呋喃妥因的耐药率较低（分别为16．07％和1．79％）,对万古霉素、利奈唑烷的耐药率为0。MRSA的耐药率高于MSSA。来源于难愈性创面与非难愈性创面的MRSA仅在对利福平的耐药率上有明显差异,而来源于两创面的MSSA的耐药率无明显差异。结论创面金葡菌中MRSA的构成比高,难愈性创面MRSA耐药严重,应积极防控创面MRSA感染和扩散。     

Prospective surveillance of community-onset and healthcare-associated methicillin-resistant Staphylococcus aureus isolated from a university-affiliated hospital in Japan

We conducted a prospective comparative study of community-onset (CO) and healthcare-associated (HA) methicillin-resistant Staphylococcus aureus(MRSA) strains between 2000 and 2001 at Tokyo Women's Medical University Hospital (1,500 beds) in Japan. Of the 172 consecutive MRSA isolates analyzed, 13 (8%) were categorized as CO-MRSA. The mean age of patients with CO-MRSA was significantly younger than that of patients with HA-MRSA. Most CO-MRSA strains were isolated from skin and more likely to be susceptible to erythromycin, clindamycin, tetracycline, levofloxacin, and spectinomycin compared to HA-MRSA isolates. Pulsed-field gel electrophoresis (PFGE) analysis, staphylococcal cassette chromosome mec(SCCmec) typing, and multi-locus sequence typing (MLST) revealed that CO-MRSA strains were divided into the following multi-clones: 3 clone A: II: ST5 (PFGE type: SCCmec type: MLST sequence type); 1 L: II: ST5; 1 H: IV: ST1; 1 I: IV: ST81; 2 D: IV: ST8; 1 B: IV: ST89; 1 B: IV: ST379; and 3 B: IV: ST91. Of the 159 HAMRSA strains, 124 (78%) belonged to a single clone (PFGE clone A: SCCmec type II: tst and sec positive: coagulase type II: multi-drug resistance). Four CO-MRSA strains belonging to PFGE clone B: SCCmec type IV: MLST clonal complex 509 (ST89, 91, 379) had the exfoliative toxin B (etb) genes, but all CO-MRSA and HA-MRSA strains did not possess the Panton-Valentine leukocidin (pvl) genes. These results demonstrate that multiple lineages of CO-MRSA have the potential for dissemination in the community in Japan.     

Identification of the third type of PVL phage in ST59 methicillin-resistant Staphylococcus aureus (MRSA) strains

The genes lukS-PV and lukF-PV for Panton-Valentine leukocidin (PVL) that confers high virulence to Staphylococcus aureus are located on the prophages (PVL phages) which have been classified into group 1 and 2 sfi21-like Siphoviridae. We report novel PVL phages lysogenized in ST59 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in Japan (JCSC7247) and Taiwan (JCSC5967). The genomes of φ7247PVL and φ5967PVL showed more than 99% identity, and the regions containing the five genes located at both ends of the prophages, int (integrase), hol (holin), ami (amidase), lukS-PV, and lukF-PV, are highly homologous to extant PVL phages. The genes for the structural module are less homologous to these phages, but are highly homologous to non-PVL phages belonging to group 3 Sfi21-like Siphoviridae, for example φN315. Subsequent PCR identification and nucleotide sequencing of an additional 11 Taiwanese ST59 MRSA isolates suggested they all carry the same phage as φ5967PVL, which differed from φ7247PVL by a single base. This study adds evidence to the notion that novel PVL phages would be generated through illegitimate recombination events by acquiring the region at which hol, ami, luk, and int genes would line up upon lytic growth, and suggests that the PVL-positive MRSA clones that have emerged worldwide may carry distinct phages.