MRSA的7种新SCCmec型别及其抗药特性

为探明海口地区耐甲氧西林金黄色葡萄球菌(MRSA)的耐药性和携带的葡萄球菌染色体mec盒(SCCmec)型别,对收集的1174株金黄色葡萄球菌用PBP2a检测法确证为MRSA有686株,用多重PCR对58株进行SCCmec分型测定,并用K-B琼脂扩散法和E-test法测定其对临床常用7类抗生素的代表性药物耐药性。结果在17株中又发现了7种新的SCCmec型别,其结构特点为:New3含A、F、H、M4个位点,New4型含F、H、M3个位点,New5含D、B、M3个位点,New6型含A、B、M3个位点,New7型含H、E、C、M4个位点,New8型含A、M两个位点,New9型含A、C、M3个位点;它们均与报道型别的结构特点存在明显差异;且携带新型的MRSA菌株,其分布特点及抗药性也与已报道的菌株存在差异:多分自门诊病人,且耐药性高,抗药谱较广,值得引起高度重视和关注。     

苯唑西林敏感、mecA基因阳性的猪源金黄色葡萄球菌的流行、分子分型及耐药性研究

[目的] 为了解我国猪源苯唑西林敏感-mecA阳性金黄色葡萄球菌（Oxacillin-susceptible,mecA-positive Staphylococcus aureus,OS-MRSA）的流行情况、菌株分子特征及耐药性,本研究对我国中西部4个省份（甘肃、陕西、河南和广西）的9个规模化养猪场进行鼻腔拭子样本采集。[方法] 运用PCR扩增nuc和mecA基因及苯唑西林耐药性检测对OS-MRSA菌株进行分离鉴定。然后对分离所得的OS-MRSA菌株进行26种毒素编码基因、16种抗生素耐药性以及spa、MLST和SCCmec分型检测。[结果] 结果表明,采集的884份样本中,67份样本7.6%（67/884）分离到金黄色葡萄球菌,包括50株甲氧西林敏感菌株（Methicillin-sensitive Staphylococcus aureus,MSSA）、8株苯唑西林耐受-mecA阳性金黄色葡萄球菌（Oxacillin-resistant mecA-positive,OR-MRSA）和9株OS-MRSA菌株。26种被检毒素编码基因中有9种毒素编码基因被检出,其中hla基因检出率最高,其次为hld、hlb、hlg、sei、sem、seg、sen和seo。此外,67株分离株中仅有16株携带肠毒素编码基因,其中OR-MRSA和OS-MRSA菌株分别占37.5%（6/16）和50.0%（8/16）,且携带毒素编码基因的菌株克隆型均为ST9-t899。16种所测试抗生素中,菌株对12种抗生素表现为耐药,其中MSSA、OR-MRSA和OS-MRSA分离株分别主要对1-8、10-12和7-11种抗生素耐药。所有分离株共有4种克隆型ST398-t571、ST9-t899、ST398-t034和t11241,其中ST9-t899为MRSA菌株唯一克隆型和ST398-t571为MSSA优势克隆型。除4株分离株未检测到SCCmec分型外,IV_b（76.5%,13/17）是MRSA分离株的唯一分型。[结论] 结果表明,我国猪源MRSA分离株对苯唑西林药物敏感性发生了改变,出现了较多的苯唑西林敏感菌株。此外,MSSA和MRSA分离株优势克隆型分别为ST398-t571和ST9-IVb-t899。研究还发现,克隆型与毒素编码基因有显著相关性,携带毒素编码基因的菌株克隆型均为ST9-t899。通过了解我国猪源MSSA、OR-MRSA和OS-MRSA的流行、分子特征和耐药性,可以为我国猪源金黄色葡萄球菌的防控提供数据支持。     

耐甲氧西林金黄色葡萄球菌的两种新SCCmec型别及其抗药性研究

为探明本地区耐甲氧西林金黄色葡萄球菌(Staphylococcusaureus)的耐药性、流行病学分布状况及携带的葡萄球菌染色体mec盒(SCCmec)型别,用K-B琼脂扩散法、E-test和多位点PCR,对临床分离的金黄色葡萄球菌菌株进行了SCCmec分型及耐药性测定。结果发现了两种新的SCCmec型别,新1型含Ⅱ型的mecA上游特异性位点B和位于mecA内的M位点以及Ⅲ型的下游位点F,缺乏Ⅱ型上游位点C和下游位点D、G;新2型含Ⅰ、Ⅱ型的上游特异性位点A、B和两个Ⅲ型的下游位点F、H,同样缺乏位点C、D、G,可能分别为原有Ⅱ型和Ⅰ、Ⅱ型与Ⅲ型的基因重组株;且携带有新SCCmec型别的MRSA菌株,其流行病学分布特点及抗药性也与国外已报导的菌株不同,多分自门诊病人,且耐药性高,抗药谱广,值得引起高度重视和关注。     

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。     

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。     

以agr基因为靶点快速检测金黄色葡萄球菌方法的建立

旨在建立一种以agr基因为靶点,快速检测金黄色葡萄球菌的环介导等温扩增（LAMP)方法。针对金黄色葡萄球菌附属基因调控基因agr序列,设计了4条特异性引物;优化了LAMP体系中甜菜碱、dNTP浓度、长短引物比等因素;通过对14株不同金黄色葡萄球菌菌株和4株其他常见食品致病菌株进行检测,评估引物特异性。结果表明,在Mg²⁺浓度为2.4 mmol/L,dNTP浓度为0.8 mmol/L,甜菜碱浓度为0.1 mol/L,长短引物比为1:8,65 ℃反应50 min条件下扩增可达最佳效果。优化后的LAMP对14株金黄色葡萄球菌均表现为阳性,对4株非金黄色葡萄球菌菌株表现为阴性,证明引物具有特异性。本文首次利用agr基因作为靶基因片段,建立了一个简单、快速、特异性强的检测金黄色葡萄球菌的方法,在食品安全检测方面极具意义。     

金黄色葡萄球菌tst-1基因敲除菌株的构建

抽提金黄色葡萄球菌834菌株的基因组DNA,PCR克隆扩增tst-1及tst-1的上、下游基因,通过将tst-1上、下游基因分别重组到载体质粒pAULA中,形成同源重组质粒pAULA Δtst-1,将pAULA-Δtst-1电转入细菌内,进行同源重组,以PCR、Western blot鉴定tst-1基因敲除菌株无tst-1基因片段,且无TSST-1蛋白表达,表明已成功构建金黄色葡萄球菌tst-1基因的敲除菌株。     

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。     

金黄色葡萄球菌所致肺部感染的耐药特点及其Panton—Valentine杀白细胞素基因的携带状况分析

目的分析金黄色葡萄球菌所致肺部感染的耐药性特点及其Panton—Valentine杀白细胞素基因的携带状况。方法回顾性调查了温州医学院第一附属医院2005年1月至2006年1月医院感染的金黄色葡萄球菌所致肺部感染患者132例,对其体外药敏试验进行分析;并利用多重PCR检测其PVL基因,应用多位点基因序列分型（multilocus sequence typing,MLST）技术对PVL基因阳性的菌株进行序列分型。耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus,MRSA）的SCCmec基因分型采用多重聚合酶链反应。结果致肺部感染的132株金黄色葡萄球菌的耐药现象较为严重,仅对万古霉素、呋喃妥因及复方新诺明等药物的敏感率较高;其中经多重PCR筛选出10株携带PVL基因的金葡菌,全部为MRSA菌株,3株为ST239-SCCⅢ,2株为ST398-SCCmecⅢ,2株为ST398-SCCmecⅣ,ST25-SCCmecⅢ、ST59-SCCmecⅠ和ST88-SCCmecⅢ各1株。结论肺部感染的金黄色葡萄球菌对多种抗生素耐药,呈多重耐药性;其携带PVL基因占一定比例。     

生鲜猪肉中金黄色葡萄球菌的耐药特征、毒力基因及agr分型

为研究市售生鲜猪肉中金黄色葡萄球菌的污染状况,分两个季节从雅安市雨城区采集351份生鲜猪肉样本,采用K-B法检测分离株的耐药性,PCR技术对分离株的mecA基因、传统肠毒素基因、中毒休克综合征毒素基因、杀白细胞毒素基因、溶血素基因和脱皮毒素基因进行分析和agr分型。结果表明,夏、冬季总体检出率分别为15.28%和28.69%;分离株对青霉素、四环素、红霉素的耐受性最强;5种传统肠毒素的检出率为80%,sea、sed的检出率均为4.11%,seb、sec的检出率均为12.33%;中毒休克综合征毒素基因的检出率为15.07%,溶血毒素基因检出率最高,杀白细胞毒素基因和脱皮毒素基因检出率最低。分离株以agrⅠ型为主（50.68%）,未检测出agrⅣ 。综上表明,该地区生鲜猪肉中金黄色葡萄球菌的污染较为严重,分离株呈多重耐药谱,毒力基因分布多样,部分菌株致病性较强,具有的潜在危险应引起重视。     

The emergence and evolution of methicillin-resistant Staphylococcus aureus

Significant advances have been made in recent years in our understanding of how methicillin resistance is acquired by Staphylococcus aureus. Integration of a staphylococcal cassette chromosome mec (SCCmec) element into the chromosome converts drug-sensitive S. aureus into the notorious hospital pathogen methicilin-resistant S. aureus (MRSA), which is resistant to practically all beta-lactam antibiotics. SCCmec is a novel class of mobile genetic element that is composed of the mec gene complex encoding methicillin resistance and the ccr gene complex that encodes recombinases responsible for its mobility. These elements also carry various resistance genes for non-beta-lactam antibiotics. After acquiring an SCCmec element, MRSA undergoes several mutational events and evolves into the most difficult-to-treat pathogen in hospitals, against which all extant antibiotics including vancomycin are ineffective. Recent epidemiological data imply that MRSA has embarked on another evolutionary path as a community pathogen, as at least one novel SCCmec element seems to have been successful in converting S. aureus strains from the normal human flora into MRSA.     

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.     

Characterisation of virulence genes in methicillin susceptible and resistant Staphylococcus aureus isolates from a paediatric population in a university hospital of Medellín, Colombia

Virulence and antibiotic resistance are significant determinants of the types of infections caused by Staphylococcus aureus and paediatric groups remain among the most commonly affected populations. The goal of this study was to characterise virulence genes of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains isolated from a paediatric population of a Colombian University Hospital during 2009. Sixty MSSA and MRSA isolates were obtained from paediatric patients between zero-14 years. We identified the genes encoding virulence factors, which included Panton-Valentine leucocidine (PVL), staphylococcal enterotoxins A-E, exfoliative toxins A and B and toxic shock syndrome toxin 1. Typing of the staphylococcal chromosome cassette mec (SCCmec) was performed in MRSA strains. The virulence genes were more diverse and frequent in MSSA than in MRSA isolates (83% vs. 73%). MRSA strains harboured SCCmec types IVc (60%), I (30%), IVa (7%) and V (3%). SCCmec type IVc isolates frequently carried the PVL encoding genes and harboured virulence determinants resembling susceptible strains while SCCmec type I isolates were often negative. PVL was not exclusive to skin and soft tissue infections. As previously suggested, these differences in the distribution of virulence factor genes may be due to the fitness cost associated with methicillin resistance.     

Bridges from hospitals to the laboratory: genetic portraits of methicillin-resistant Staphylococcus aureus clones

Methicillin-resistant Staphylococcus aureus (MRSA) emerged in the early 1960's after the acquisition of the methicillin resistance gene mecA, which is carried by the staphylococcal cassette chromosome mec (SCCmec). MRSA seemed to have arisen by multiple introductions of SCCmec into successful methicillin-susceptible S. aureus (MSSA) lineages. MRSA is one of the most common agents of nosocomial infections worldwide increasing the cost and mortality compared to MSSA infections. Little by little, MRSA has acquired resistance to all antibiotics available in clinical practice, which complicates treatment. This situation was further aggravated by the recent reports of vanA-mediated vancomycin-resistant S. aureus. As a reaction to the emergence and spread of multidrug-resistant MRSA worldwide, international surveillance systems such as the CEM/NET initiative have been created. The characterization of over 3000 MRSA isolates from different regions of the world evidenced the existence of only a few epidemic clones spread worldwide, namely the Iberian, Brazilian, Hungarian, New York/Japan, Pediatric and EMRSA-16 clones. It was found that in surveillance or evolutionary studies strains should be characterized by a combination of different typing methods, namely pulsed-field gel electrophoresis, multi-locus sequence typing and SCCmec typing. In recent years, community-acquired MRSA (CA-MRSA) has become a growing public health concern. However, although many authors reported the emergence of CA-MRSA isolates, a standard definition has not been created and the prevalence of MRSA among persons without risk factors seems to remain very low. CA-MRSA has distinct properties compared to epidemic nosocomial clones and its origin is still unclear. Certain authors suggest there is MRSA transmission from the hospital setting to the community, namely transfer of nosocomial MRSA minor clones or sporadic isolates showing a high degree of similarity with CA-MRSA; others believe CA-MRSA strains represent new acquisitions of SCCmec DNA in susceptible backgrounds. Many questions concerning this extraordinarily versatile and threatening pathogen remain unanswered, needing future investigation     

SCCmec遗传元件及其在耐甲氧西林金黄色葡萄球菌分子分型中的应用

携带mec基因簇的葡萄球菌盒式染色体(Staphylococcal chromosome cassette mec, SCCmec)遗传元件的获得是耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus, MRSA)耐药的主要原因。SCCmec由一个mec基因簇、一个染色体重组酶(ccr)基因簇及3个J区组成。mec基因簇含有mecA及其调控基因,mecA基因编码的耐药决定簇使MRSA对β-内酰胺类抗生素耐药;ccr基因簇编码的重组酶负责SCCmec元件的整合与切离;J区差异大,导致不同来源MRSA菌株携带SCCmec的大小不一,在组成上也具有多样性。这些特征为利用SCCmec元件进行MRSA分型创造了条件。文章介绍了SCCmec元件的结构和功能,综述了基于SCCmec的MRSA分型研究。     

Molecular characterization of methicillin-resistant Staphylococcus aureus in hospitals in Niigata, Japan: divergence and transmission

The major methicillin-resistant Staphylococcus aureus(MRSA) distributed among hospitals in Japan is New York/Japan clone [multilocus sequence type 5 (ST5), agr type 2 and methicillin resistance locus type (SCC mec) II] which possesses both the toxic shock syndrome toxin 1 gene (tst) and staphylococcal enterotoxin C gene (sec). In this study, we collected 245 MRSA strains from four hospitals during 2001 to 2005 in Niigata, Japan, and analyzed tst and sec genes and SCC mec type among them. A total of 13 strains were further examined for their genotypes, virulence gene patterns and drug resistance. Among the 245 strains four tst sec genes patterns were observed; tst(+) sec(+) strains represented a majority of 86.5% and 9.4% were tst(-) sec(-). SCCmec typing revealed that 91.4% had type II, 4.1% type IV and 4.1% type I. Multilocus sequence typing (MLST) revealed that 10 of the 13 typed strains belonged to clonal complex 5 (7 had ST5 while 3 were single locus variants of ST5) with similar characteristics to the New York/Japan clone and possessed multi-drug resistance with high virulence gene content. The remaining 3 strains were ST8 (n=2) and ST91 (n=1). The ST91 strain had SCC mec IV and seemed to originate in the community, while ST8 strains exhibited SCC mec type I, which is distinct from community type IV. The data suggest that MRSA in hospitals in Niigata now mainly includes the New York/Japan clone (undergoing genomic divergence and clonal expansion) and other minor types (e.g. ST8) as well as the community type.