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。     

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.     

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.     

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。     

Increased Neopterin Levels in Brains of Patients with Human Immunodeficiency Virus Type 1 Infection

Postmortem levels of native neopterin (D-erythro-neopterin) were measured in cerebral cortical samples from 44 human immunodeficiency virus type 1-infected and eight uninfected, nonneurological control patients. Cerebral cortical gray and white matter neopterin levels for the controls ranged from 0.5 to 7.2 pmol/mg of protein in contrast to neopterin levels in brains of the virus-infected patients, which frequently were more than threefold and occasionally more than 30-fold higher than mean control levels. Cortical neopterin levels did not correlate with severity of the acquired immunodeficiency syndrome dementia complex, but subcortical levels correlated with the presence of active human immunodeficiency virus type 1 infection, as reflected by pathological evidence of multinucleated giant cell encephalitis. Evidence of opportunistic cytomegalovirus infections in approximately 25% of the human immunodeficiency virus type 1-infected patients was associated with enhanced levels of neopterin in frontal cortex.     

HPV和HCMV在慢性宫颈炎病人中的分布及相关性研究

本文采用DNA-DNA分子杂交技术对病理组织学确诊的87例慢性宫颈炎患者和25例健康宫颈的宫颈活检组织DNA进行了HPV 6、11、16、18型DNA及HCMV HindⅢE片段检测。结果表明,对照宫颈检出率全部为0,慢性宫颈炎检出率HPV 6为16％,HPVll为12.6％,HPV16为11.5％,HPVl8为5％,总的HPV DNA相关序列为29.0％,HCMV为12.64％。经显著性检验,HPV DNA相关序列检出率在慢性宫颈炎与对照组间具显著性差异,HPV DNA相关序列阳性者患慢性宫颈炎的危险性为HPV DNA相关序列阴性者20.81的倍。本实验结果还表明HCMV阳性患者中72.7％的病人同时有HPV感染,提示HCMV感染与HPV感染有关。     

胃大部切除后不同吻合术式对胃癌合并2型糖尿病患者血糖的影响

目的:探讨胃大部切除后不同吻合术式对胃癌合并2型糖尿病患者血糖的影响.方法:选择66例在本院行手术治疗的胃癌合并2型糖尿病的患者为研究对象,随机分为毕Ⅰ式组26例,毕Ⅱ式组24例,Roux-en-y组16例.检测和比较三组患者术前和术后随访3月时患者的OGTT空腹及负荷后血糖值、糖化血红蛋白等水平.结果:术前三组OGTT空腹血糖、2h血糖、糖化血红蛋白水平、空腹胰岛素水平、稳态模型评估胰岛素抵抗(HOMA-IR)的比较均无统计学差异(P均＞0.05).术后3月,与毕Ⅰ式组比较,毕Ⅱ式组与Roux-en-y组未达2型糖尿病诊断标准的患者百分率显著升高(X2=7.866,P=0.020),OGTT空腹血糖(F=6.472,P=0.005)水平、OGTT 2 h血糖水平(F=3.431,P=0.041)、糖化血红蛋白水平(F=5.456,P=0.009)、空腹胰岛素水平(F=6.120,P=0.008)、HOMA-IR(F=8.091,P＜0.001)均显著降低.毕Ⅱ式组与Roux-en-y组之间以上指标比较均无统计学差异(P＞0.05).结论:胃癌合并2型糖尿病的患者在消化道重建术后部分可得缓解,采用毕Ⅱ式或Roux-en-y吻合术可能更适宜胃癌合并2型糖尿病的患者.     

老年糖尿病患者尿路感染的病原菌分布及耐药性监测

目的：了解我院老年糖尿病患者尿路感染的病原茵分布及耐药情况,指导临床合理用药。方法：选择我院2010-2012年收治的477例老年糖尿病合并尿路感染患者为研究对象,对其中段尿进行细菌培养及药敏分析。结果：本院老年糖尿病患者尿路感染的细菌以革兰氏阴性菌为主,占67．5％,常见致病菌为大肠埃希菌、肠球菌、肺炎克雷伯茵和表皮葡萄球菌,并发现有2种细菌混合感染及真菌并存感染。革兰阴性菌对亚胺培南、阿米卡星和头孢哌酮／舒巴坦的耐药率较低,而对其他抗菌药物的耐药率较高;革兰阳性茵对万古霉索、阿米卡星和呋喃妥因的耐药率较低,表皮葡萄球菌对万古霉素敏感。结论：本院老年糖尿病患者尿路感染的主要病原菌是大肠埃希菌,其对亚胺培南、阿米卡星和头孢哌酮／舒巴坦的耐药率较低,临床可参考应用。     

Eggshell Types and Their Evolutionary Correlation with Life-History Strategies in Squamates

The eggshell is an important physiological structure for the embryo. It enables gas exchange, physical protection and is a calcium reserve. Most squamates (lizards, snakes, worm lizards) lay parchment-shelled eggs, whereas only some gekkotan species, a subgroup of lizards, have strongly calcified eggshells. In viviparous (live-bearing) squamates the eggshell is reduced or completely missing (hereafter “shell-less”). Recent studies showed that life-history strategies of gekkotan species differ between species with parchment- and rigid-shelled eggshells. Here we test if the three different eggshell types found in the squamates are also associated with different life-history strategies. We first investigated the influence of the phylogeny on the trait “eggshell type” and on six life-history traits of 32 squamate species. Phylogenetic principal component analysis (pPCA) was then conducted to identify an association between life-history strategies and eggshell types. Finally, we also considered adult weight in the pPCA to examine its potential effect on this association. Eggshell types in squamates show a strong phylogenetic signal at a low taxonomical level. Four out of the six life-history traits showed also a phylogenetic signal (birth size, clutch size, clutches per year and age at female maturity), while two had none (incubation time, maximum longevity). The pPCA suggested an association of life-history strategies and eggshell types, which disappeared when adult weight was included in the analysis. We conclude that the variability seen in eggshell types of squamates is weakly influenced by phylogeny. Eggshell types correlate with different life-history strategies, and mainly reflect differences in adult weights of species.