耐甲氧西林金黄色葡萄球菌染色体盒的研究进展

耐甲氧西林金黄色葡萄球菌（MRSA）的产生是由甲氧西林敏感的金黄色葡萄球菌（MSSA）获得外源性的SCCmec所致。MRSA菌株可以产生一种新的青霉素结合蛋白PBP2a,PBP2a降低了与β-内酰胺类抗生素的亲合力,从而对β-内酰胺类抗生素产生耐药性。PBP2a由mecA基因编码,mecA基因存在于葡萄球菌盒式染色体（Staphylococcal cassette chromosome mec,SCCmec）中,SCCmec是一种可移动的遗传元件,该元件还携带除mecA基因外的其他抗菌药物的耐药基因,造成多重耐药（Multidrug-resistance,MDR）。SCCmec目前主要分为8型,其中又分为若干亚型。SCCmec的基因型与MRSA的流行背景有关,不同地区的SCCmec基因分型分布可能不同。     

Roles of CcrA and CcrB in Excision and Integration of Staphylococcal Cassette Chromosome mec,a Staphylococcus aureus Genomic Island

The gene encoding resistance to methicillin and other β-lactam antibiotics in staphylococci, mecA, is carried on a genomic island, SCCmec (for staphylococcal cassette chromosome mec). The chromosomal excision and integration of types I to IV SCCmec are catalyzed by the site-specific recombinases CcrA and CcrB, the genes for which are encoded on each element. We sought to identify the relative contributions of CcrA and CcrB in the excision and integration of SCCmec. Purified CcrB but not CcrA was shown to mediate the gel shift of chromosomal target integration sequences (attB) in electrophoretic mobility shift assays. However, preincubation of CcrB-DNA complexes with increasing concentrations of CcrA blocked gel shift. The interaction of CcrB and CcrA was confirmed by Escherichia coli two-hybrid analysis. SCCmec excision mediated by plasmid-encoded and inducible ccrA, ccrB, or both genes was assessed by PCR in Staphylococcus aureus. CcrB alone could mediate excision but excision was at an alternate att site (attR2) within the right extremity of SCCmec. In contrast, both CcrB and CcrA were required to mediate excision at the chromosomal attB site (called attR when SCCmec is integrated). Insertion of a plasmid containing the SCCmec att site (attS) into the chromosome required both CcrA and CcrB, but CcrA overexpression lowered integration frequency. Thus, while CcrB binds DNA, interaction between CcrA and CcrB, in a precise ratio, is required for attB site-specific excision and SCCmec chromosomal insertion.Staphylococcus aureus is one of the most common causes of serious human bacterial infections, both in the hospital and the community (33). Therapy of these infections is made more difficult by the development of resistance to drugs with antistaphylococcal activity such as the beta-lactam antibiotics. Resistance to beta-lactam antibiotics in staphylococci is mediated by a beta-lactamase and by a beta-lactam-resistant target transpeptidase, penicillin-binding protein 2a (PBP2a) (4, 5, 8). However, while the beta-lactamase has a narrow substrate specificity, limited to penicillins, PBP2a resists inactivation by all beta-lactam antibiotics and can cross-link peptidoglycan when all other target PBPs are rendered nonfunctional by beta-lactams. The latter is called methicillin resistance and is the most important clinical resistance phenotype among staphylococci (8) The gene for PBP2a, mecA, is located on a genomic island called SCCmec (for staphylococcal cassette chromosome mec) that is integrated into the staphylococcal chromosome at a specific site. In addition to mecA, all SCCmec elements carry intact or mutant mecA regulators (mecR1/mecI) and genes that mediate the site-specific integration and excision of SCCmec (ccr genes) (14). SCCmec elements have been typed according to the sequences of the ccr and mec complexes with five cores (types I to V) being prevalent but with considerable variation in the genetic organization within each element (14-17, 22).SCCmec is presumed to be a mobile genetic element, which can integrate into and excise from the chromosome by site-specific recombination between a site on SCCmec (attS) and one on the chromosome (attB). attB comprises the last 15 bp of a highly conserved gene called orfX that is located near the S. aureus origin of replication (15, 19). When SCCmec is inserted, the attB sequence is duplicated at the other end of the element with the site in orfX now called attR and the one abutting the non-orfX end of SCCmec designated attL. When SCCmec excises, the attB site is reconstituted in the chromosome and the two ends of the element come together to form attS within a nonreplicating circular version of SCCmec.The site-specific recombination of SCCmec is catalyzed by its encoded ccr recombinases, CcrA and CcrB for types I to IV and CcrC for type V. CcrA and CcrB belong to a family of large serine invertase and resolvases which consist of resolvases, invertases, phage integrases, and transposases (6, 10, 29, 31). All of them contain a conserved catalytic motif and some contain DNA-binding domains at either the N or the C terminus. The catalytic domains can either function as both integrases and excisases or as only integrases that require additional proteins to mediate excision (6, 29, 30, 31).The ccrA and ccrB genes are part of two-gene operons of 1,350 and 1646 bp in S. aureus strain N315 encoding proteins of 52.6 and 62.7 kDa, respectively. Although there is considerable variation at the amino acid level among the CcrA and CcrB proteins found in types I to IV SCCmec, plasmid-encoded CcrA and CcrB recombinases from each type can excise SCCmec from any of the others (23). However, CcrC can only excise type V SCCmec (16). There has been little examination of the role of each of these proteins in recombination or in DNA binding. In the present study we sought to define the precise roles of CcrA and CcrB in DNA binding and in the excision and integration of SCCmec in S. aureus. This is the first step in understanding the host range of SCCmec and how it may move among staphylococcal isolates in nature.     

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec)typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.     

Characterization of the Staphylococcus aureus rRNA Methyltransferase Encoded by orfX,the Gene Containing the Staphylococcal Chromosome Cassette mec (SCCmec) Insertion Site

The gene orfX is conserved among all staphylococci, and its complete sequence is maintained upon insertion of the staphylococcal chromosome cassette mec (SCCmec) genomic island, containing the gene encoding resistance to β-lactam antibiotics (mecA), into its C terminus. The function of OrfX has not been determined. We show that OrfX was constitutively produced during growth, that orfX could be inactivated without altering bacterial growth, and that insertion of SCCmec did not alter gene expression. We solved the crystal structure of OrfX at 1.7 Å and found that it belongs to the S-adenosyl-l-methionine (AdoMet)-dependent α/β-knot superfamily of SPOUT methyltransferases (MTases), with a high structural homology to YbeA, the gene product of the Escherichia coli 70 S ribosomal MTase RlmH. MTase activity was confirmed by demonstrating the OrfX-dependent methylation of the Staphylococcus aureus 70 S ribosome. When OrfX was crystallized in the presence of its AdoMet substrate, we found that each monomer of the homodimeric structure bound AdoMet in its active site. Solution studies using isothermal titration calorimetry confirmed that each monomer bound AdoMet but with different binding affinities (K_d = 52 ± 0.4 and 606 ± 2 μm). In addition, the structure shows that the AdoMet-binding pocket, formed by a deep trefoil knot, contains a bound phosphate molecule, which is the likely nucleotide methylation site. This study represents the first characterization of a staphylococcal ribosomal MTase and provides the first crystal structure of a member of the α/β-knot superfamily of SPOUT MTases in the RlmH or COG1576 family with bound AdoMet.     

Comparative Genotypes,Staphylococcal Cassette Chromosome mec (SCCmec) Genes and Antimicrobial Resistance amongst Staphylococcus epidermidis and Staphylococcus haemolyticus Isolates from Infections in Humans and Companion Animals

This study compares the characteristics of Staphylococcus epidermidis (SE) and Staphylococcus haemolyticus (SH) isolates from epidemiologically unrelated infections in humans (Hu) (28 SE-Hu; 8 SH-Hu) and companion animals (CpA) (12 SE-CpA; 13 SH-CpA). All isolates underwent antimicrobial susceptibility testing, multilocus sequence typing and DNA microarray profiling to detect antimicrobial resistance and SCCmec-associated genes. All methicillin-resistant (MR) isolates (33/40 SE, 20/21 SH) underwent dru and mecA allele typing. Isolates were predominantly assigned to sequence types (STs) within a single clonal complex (CC2, SE, 84.8%; CC1, SH, 95.2%). SCCmec IV predominated among MRSE with ST2-MRSE-IVc common to both Hu (40.9%) and CpA (54.5%). Identical mecA alleles and nontypeable dru types (dts) were identified in one ST2-MRSE-IVc Hu and CpA isolate, however, all mecA alleles and 2/4 dts detected among 18 ST2-MRSE-IVc isolates were closely related, sharing >96.5% DNA sequence homology. Although only one ST-SCCmec type combination (ST1 with a non-typeable [NT] SCCmec NT9 [class C mec and ccrB4]) was common to four MRSH-Hu and one MRSH-CpA, all MRSH isolates were closely related based on similar STs, SCCmec genes (V/V_T or components thereof), mecA alleles and dts. Overall, 39.6% of MR isolates harbored NT SCCmec elements, and ACME was more common amongst MRSE and CpA isolates. Multidrug resistance (MDR) was detected among 96.7% of isolates but they differed in the prevalence of specific macrolide, aminoglycoside and trimethoprim resistance genes amongst SE and SH isolates. Ciprofloxacin, rifampicin, chloramphenicol [fexA, cat-pC221], tetracycline [tet(K)], aminoglycosides [aadD, aphA3] and fusidic acid [fusB] resistance was significantly more common amongst CpA isolates. SE and SH isolates causing infections in Hu and CpA hosts belong predominantly to STs within a single lineage, harboring similar but variable SCCmec genes, mecA alleles and dts. Host and staphylococcal species-specific characteristics were identified in relation to antimicrobial resistance genes and phenotypes, SCCmec and ACME.     

Novel Rearrangements in the Staphylococcal Cassette Chromosome Mec Type V Elements of Indian ST772 and ST672 Methicillin Resistant Staphylococcus aureus Strains

Staphylococcus aureus is a commensal gram positive bacteria which causes severe and non severe infections in humans and livestock. In India, ST772 is a dominant and ST672 is an emerging clone of Staphylococcus aureus. Both cause serious human diseases, and carry type V SCCmec elements. The objective of this study was to characterize SCCmec type V elements of ST772 and ST672 because the usual PCR methods did not amplify all primers specific to the type. Whole genome sequencing analysis of seven ST772 and one ST672 S. aureus isolates revealed that the SCCmec elements of six of the ST772 isolates were the smallest of the extant type V elements and in addition have several other novel features. Only one ST772 isolate and the ST672 isolate carried bigger SCCmec cassettes which were composites carrying multiple ccrC genes. These cassettes had some similarities to type V SCCmec element from M013 isolate (ST59) from Taiwan in certain aspects. SCCmec elements of all Indian isolates had an inversion of the mec complex, similar to the bovine SCCmec type X. This study reveals that six out of seven ST772 S. aureus isolates have a novel type V (5C2) SCCmec element while one each of ST772 and ST672 isolates have a composite SCCmec type V element (5C2&5) formed by the integration of type V SCCmec into a MSSA carrying a SCC element, in addition to the mec gene complex inversions and extensive recombinations.     

MRSA感染的血清学分型及质粒研究

探明MRSA感染的血清学特点及质粒分布 ,为其预防和治疗提供科学依据。做血浆凝固抑制试验—中和法和质粒DNA提取用碱裂解法。结果血浆凝固酶Ⅱ型的检出阳性率为 5 4.0 % ( 47/ 87) ,Ⅳ型为 2 7.5 % ( 2 4/ 87) ,其余为Ⅶ和Ⅲ型。按菌株来源分析 :病房工作人员和住院患者的MRSA以Ⅱ型居多 ,Ⅳ型次之 ,有明显的偏重集中趋势 ,而门诊患者MRSA血浆凝固酶型别分布散乱。多数菌株有质粒 ,分布复杂。表明沈阳地区流行的主要血浆凝固酶型别为Ⅱ型和Ⅳ型。     

858例血感染患者病原菌分布及耐药情况

目的:通过对某地区中心医院收集的临床血感染患者感染病原菌的分析,了解该地区血感染患者病原菌构成、分布及耐药特点,为临床治疗提供参考和指导。方法:收集2012年6月至2013年8月期间在某院就诊的858例血感染患者血液标本,采用BACTEC9050全自动血培养仪培养,采用VITEK 2 Compack系统和K-B琼脂纸片扩散法对阳性标本进行菌种鉴定和药敏检测。结果:血培养结果显示,在858份血培养标本中共检出阳性标本109份,每份标本都只检出一种病菌,总检出率为12.7%,革兰阳性菌占64.22%(70/109),革兰阴性菌占33.03%(36/109),真菌占0.35%%(3/109);药物敏感试验结果显示:葡萄球菌对青霉素、红霉素和复方新诺明耐药率40%;肠杆菌科细菌对氨苄西林和氯霉素耐药率40%;非发酵菌科细菌对氨苄西林,头孢他啶,头孢噻肟和氯霉素耐药率40%。结论:目前本地区临床血感染患者革兰阳性菌感染率高,以金黄色葡萄球菌和凝固酶阴性葡萄球菌为主,治疗可以首选糖肽类抗菌药物;革兰阴性菌以大肠杆菌和绿脓杆菌为主,对氨苄西林、氯霉素耐药率高,大肠杆菌对头孢类抗生素的耐受较绿脓杆菌低,两种细菌感染治疗可以考虑选择单环-内酰胺类抗生素。及时准确的血培养结果及药敏试验可为临床合理选择抗菌药物提供重要依据。     

缺血性脑卒中患者颅内外动脉狭窄的发生规律及分布特征

目的:观察脑卒中患者颅内外动脉狭窄的发生规律及分布特征.方法:收集2003年2月～2006年10月在我科住院治疗并入选的1025例脑卒中患者,572患者均行头颅CT/MRI,全脑血管数字减影血管造影术(DSA)检查.狭窄程度的计算遵照NASCET测量标准,计算颅内外动脉狭窄的发生率,并对惠者发病的危险因素与颅内、外动脉狭窄或闭塞情况的相关性进行对比分析.结果:颅内外动脉狭窄的发生规律研究结果显示,颅内外动脉狭窄患者在高血压、脂代谢紊乱、缺血性心脏病、短暂性脑缺血发作史、吸烟史方面与非狭窄患者比较差异均有统计学意义(P＜0.05).结论:通过筛查颅内外动脉狭窄的危险因素,及早发现并对高危人群进行积极干预,对预防缺血性脑卒中的发生具有重要意义.     

巨细胞病毒感染与SARI肺炎病例的相关性研究

探讨巨细胞病毒感染与住院严重急性呼吸道感染(SARI)肺炎的相关性.以67例符合严重急性呼吸系统感染临床诊断标准的住院病例为研究对象,同时以81例流感样门诊病例作为对照,使用荧光定量PCR方法检测所有研究对象的巨细胞病毒感染情况.采用二分类logistic回归模型分析巨细胞感染与住院严重急性呼吸道感染肺炎病例的关联.巨细胞病毒在呼吸道感染病例中有较高的阳性率,阳性率随年龄呈下降趋势.80％以上巨细胞病毒阳性病例存在与其他常见呼吸道病原共感染情况.Logistic回归分析表明:年龄和多病原共感染是SARI肺炎发生的危险因素,单独CMV阳性与SARI肺炎的发生没有显著相关性.SARI肺炎中CMV与其他呼吸道病原共感染概率高,临床上应加强对呼吸道感染病例的巨细胞病毒检测.     

240例咽部细菌感染患者病原菌分布及耐药分析

目的:探讨咽部细菌感染患者病原菌分布及其耐药情况.方法:收集我院240例咽部细菌感染患者病原菌,采用常规方法进行鉴定,药敏试验采用K-B法.结果:占咽部感染病原菌分布前两位的为链球菌属(30.0％)和奈瑟菌属(18.3％),其次为肺炎克雷伯菌(12.5％)、铜绿假单胞菌(11.3％)和真菌(5.0％).药敏结果显示,致病菌肺炎克雷伯菌对环丙沙星、阿米卡星、头孢吡肟、哌拉西林/他唑巴坦、头孢哌酮/舒巴坦、亚胺培南和美罗培南有较低的耐药率(小于20.0％);铜绿假单胞菌对环丙沙星、阿米卡星和头孢吡肟有较低的耐药率(小于20.0％).结论:咽部细菌感染常见致病菌为肺炎克雷伯菌和铜绿假单胞菌,对喹诺酮类药物、氨基糖苷类药物和第四代头孢菌素都有较低的耐药率,可以做为治疗咽部主要致病菌感染的可选药物.     

肿瘤患者深部真菌感染的菌株分布及耐药性分析

探讨肿瘤患者深部真菌医院感染的病原菌分布特点及耐药现状。回顾性分析2008年1月至2009年12月哈尔滨医科大学第一临床医学院住院肿瘤患者送检标本分离出的173株真菌的分布及耐药情况。肿瘤患者深部真菌医院感染以下呼吸道感染为主,占76.3%,真菌种类主要是白假丝酵母菌(74.6%);真菌药敏试验结果表明,深部真菌对两性霉素B和5-氟胞嘧啶耐药率均5%;对伊曲康唑及伏立康唑的耐药率为0~6.5%;对氟康唑耐药率有上升趋势,为2.5%~25.0%。临床分离的真菌主要集中在呼吸道标本,以白假丝酵母菌为主,对抗真菌药物普遍敏感,应积极治疗,合理利用抗真菌药物,改善患者预后,减少耐药菌株的产生。     

心血管病患者并发肺部感染的病原菌分布及危险因素分析

目的:探讨心血管病患者并发肺部感染的病原菌分布特点及相关危险因素,以降低感染率。方法:选择2015年8月~2016年3月因心血管病入院治疗的154例患者为研究对象,收集其相关临床数据,进行回顾性分析,采集医院感染患者痰标本进行细菌培养,分析病原菌分布及危险因素。结果:154例心血管病患者并发肺部感染者25例,感染率为16.23%。共培养出病原菌27株,革兰阴性菌16株(占59.26%)、革兰阳性菌7株(占25.93%)及真菌4株(占14.81%)。单因素分析显示年龄、基础疾病、侵入性操作、抗生素使用、住院时间等与医院感染率有关,差异有统计学意义(P0.05);logistic回归分析发现,年龄(≥60岁)、有基础疾病、有侵入性操作、使用抗生素、住院时间(14天)是心血管病患者并发肺部感染的危险因素。结论:心血管病患者肺部感染的主要病原菌为大肠埃希菌、肺炎克雷伯菌以及金黄色葡萄球菌等,应针对相关危险因素进行干预,以降低医院感染率。     

幽门螺杆菌感染与急性心肌梗死患者血清炎症反应的关系

目的:探讨幽门螺杆菌(Hp)感染与急性心肌梗死(AMI)患者血清炎症反应的关系,为临床防治AMI提供参考。方法:选取2013年4月-2014年11月我院收治的74例AMI患者作为研究组,另选取同期在我院进行体检的74例健康人作为对照组。采用酶联免疫法检测和比较两组患者Hp免疫球蛋白G(Immunoglobulin G,IgG)浓度及血清IL-6、IL-8、IL-18、TNF-α和hs-CRP水平,分析Hp IgG阳性与血清炎症因子水平的相关性。结果:研究组患者的Hp IgG浓度为(60.92±45.15)KU/L,相比于对照组的(32.36±24.08)KU/L明显偏高(P0.05),且其阳性率为72.97%,明显高于对照组的51.35%(P0.05);Hp阳性患者IL-6、IL-8、IL-18、TNF-α、hs-CRP明显高于Hp阴性患者的(P0.05)。Pearson相关分析显示Hp IgG浓度与血清IL-6、IL-8、IL-18、TNF-α、hs-CRP水平均呈显著正相关,相关系数分别为0.735、0.644、0.798、0.674、0.616(P0.05)。结论:Hp感染与AMI患者血清炎症反应之间存在着密切的关系。     

Possible Correlation between Borna Disease Virus Infection and Japanese Patients with Chronic Fatigue Syndrome

Borna disease virus (BDV) is a neurotropic, as yet unclassified, non-segmented, negative-sense, single-strand RNA virus. Natural infection with this virus has been reported to occur in horses and sheep. In addition, antibodies to BDV in plasma or BDV RNA in peripheral blood mononuclear cells (PBMCs) were also found in patients with neuropsychiatric diseases. We describe here the possible link between the patients with chronic fatigue syndrome (CFS) and infection with BDV.     

尿毒症维持性血液透析患者血清CRP与营养状态及感染的相关性

目的:探讨尿毒症维持性血液透析患者的血清C-反应蛋白(CRP)与营养状态及感染的相关关系。方法:按照是否发生感染将2013年2月-2015年8月我院收治的238例尿毒症维持性血液透析患者分为感染组(118例)和非感染组(120例),对两组患者的血清CRP、营养状态相关指标进行比较,并分析CRP水平与营养状态及感染的相关性。结果:感染组的体质量指数(BMI)、白蛋白、前白蛋白、转铁蛋白、血红蛋白水平低于非感染组,重组人促红细胞生成素、血清CRP水平高于非感染组,肱三头肌皮褶厚度低于非感染组,差异均有统计学意义(P0.05)。经Pearson积矩相关分析,血清CRP与BMI、白蛋白、前白蛋白、转铁蛋白、血红蛋白呈负相关关系(r=-0.81、-0.93、-0.85、-0.79、-0.91),差异有统计学意义(P0.05),血清CRP与肱三头肌皮褶厚度(TSF)呈负相关关系(r=-0.76,P0.05),与重组人促红细胞生成素呈正相关关系(r=0.89,P0.05)。结论:尿毒症维持性血液透析患者的血清CRP水平可作为评估患者营养状态及感染的重要指标,具有重要的参考价值。     

Histopathological Features and Viral Antigen Distribution in the Lung of Fatal Patients with Enterovirus 71 Infection

正Dear Editor,Enterovirus 71(EV71)infection causes hand-foot-andmouth disease(HFMD)in infants and children.Patients with HFMD usually have good prognosis;however,in some extreme cases the infection can be accompanied by central nervous system diseases,eventually leading to cardiorespiratory failure,and even death.Currently,EV71     

靖江地区宫颈病变患者高危型人乳头瘤病毒感染及其基因型分析

目的:研究靖江地区各组宫颈病变中高危型人乳头瘤病毒(HPV)感染率及其基因型分布。方法:采用实时荧光PCR检测方法检测334例宫颈脱落细胞标本中高危型HPV及其基因型。结果:高危型HPV在宫颈炎组、湿疣组、低度鳞状上皮细胞内病变(LSIL)组、高度鳞状上皮细胞内病变(HSIL)组中的感染率分别为24.2%、58.2%、49.3%、69.5%;HPV 16、18、33、58型在宫颈病变中是最常见的HPV型别,其中,HPV16型在宫颈炎组、湿疣组、LSIL组、HSIL组中所占比例逐渐增高,分别为17.9%、18.9%、30.8%、41.9%。结论:高危型HPV感染率随着宫颈病变程度的加重而升高,HPV16型是高危型HPV感染中的主要亚型,HPV18、33、52、58也较常见,其余型别很少。