一种鉴别临床常见致病性葡萄球菌方法的建立与初步评价

目的建立一种鉴别临床常见致病性葡萄球菌的聚合酶链反应-限制性片段长度多态性（PCRRFLP）方法。方法采用经全自动微生物鉴定系统和分子生物学方法准确鉴定的金黄色葡萄球菌、表皮葡萄球菌、溶血葡萄球菌各3株,提取细菌DNA,PCR扩增tuf基因,扩增产物Alu I、Hinf I双酶切后进行琼脂糖凝胶电泳,分析不同葡萄球菌酶切后带型的差异。收集临床分离葡萄球菌142株,采用建立的PCRRFLP对其进行分类鉴别,随机选择分类鉴别的葡萄球菌各20株,PCR扩增16S r DNA,扩增产物测序,将结果与Gen Bank数据库进行比对,初步评价该方法的准确性。结果金黄色葡萄球菌,表皮葡萄球菌,溶血葡萄球菌均能扩增出长668 bp DNA片段。扩增产物经Alu I、Hinf I双酶切后电泳条带不同,金黄色葡萄球菌出现三条带（108 bp/192 bp/217 bp）,表皮葡萄球菌出现两条带（192 bp/304 bp）,溶血葡萄球菌出现两条带（192 bp/217 bp）。PCR-RFLP结果显示,142株葡萄球菌中金黄色葡葡球菌、表皮葡葡球菌和溶血葡葡球菌分别为67、29和46株。随机挑选的20株不同种葡萄球菌16S r DNA测序结果与Gen Bank数据库对应序列的相似性均〉99%,说明建立的PCR-RFLP方法能准确区分三种常见葡萄球菌。结论 PCRRFLP能准确鉴别临床常见的致病性葡萄球菌,为葡萄球菌病的分子诊断奠定了基础。     

临床常见G^＋致病菌的种类及耐药性的研究

目的：调查我院常见G^＋致病菌的种类及其对目前常用抗生素的耐药现状,为临床医生合理选用抗生素提供数据资料。方法：从3286个样本中分离到206株G^＋致病菌,用全自动微生物分析仪-VITEK32进行菌种鉴定及药敏实验。结果：206株G^＋致病菌经鉴定属于6大类,其中金黄色葡萄球菌,74株,占35,9％;溶血葡萄球菌,47株,占22．8％;表皮葡萄球菌,47株,占22．8％;肠球菌属,33株,占16．0％;肺炎链球菌,3株,占1．5％;β-溶血链球菌,2株,占1．0％。肺炎链球菌和β-溶血链球菌仅对检测的14种抗生素中的少数敏感。在对14种抗生素的耐药性实验中,药效普遍较差的抗生素为：青霉素、庆大霉素、苯唑西林。它们三者的平均药敏感率依次为：16．52％、18．42％和21．07％：结论：临床G^＋致病菌的耐药性问题已非常严峻,合理的选择和使用抗生素已十分重要．     

细菌性前列腺炎患者前列腺液菌群调查及耐药谱分析

目的了解细菌性前列腺炎患者菌群分布及其耐药现状,更有效地对细菌性前列腺炎进行预防控制。方法对我院2005年10月至2007年9月拟诊为细菌性前列腺炎的患者266例,取前列腺液做细菌需氧及5%CO2环境培养,以Phoenix 100型全自动细菌分析系统进行菌种鉴定及药物敏感试验,综合分析结果。结果266例患者中前列腺液培养阳性者204例,阳性率为76.69%。其中葡萄球菌152株,占检出菌株数的74.51%。检出主要细菌为：溶血葡萄球菌82株（40.2%）,表皮葡萄球菌34株（16.67%）,粪肠球菌25株（12.25%）,头状葡萄球菌10株（4.9%）,金黄色葡萄球菌5株（2.45%）。葡萄球菌对氨苄西林、青霉素耐药率达到100%,对万古霉素、呋喃妥因、吗啉噁酮敏感率分别为100%、95.9%、95.1%。结论细菌性前列腺炎患者多由条件致病菌感染所致,尤其以凝固酶阴性葡萄球菌感染多见,患者年龄呈逐渐年轻化趋势;万古霉素、呋喃妥因、吗啉噁酮对葡萄球菌高度敏感。     

新生儿耐甲氧西林凝固酶阴性葡萄球菌败血症40例分析

目的：探讨耐甲氧西林凝固酶阴性葡萄球菌（MRCNS）在新生儿败血症中的分布,临床特征及耐药性特点。方法：应用全自动血培养系统（BACTE/9050）,ATB细菌鉴定仪,从80例新生儿血培养阳性瓶中分离出40株MRCNS。应用ATB药敏系统,测定了青霉素等17种抗生素的耐药性。结果：80株细菌中,分离出40株MRCNS（占50%）,其中溶血葡萄球菌17株（42.5%)居首位,表皮葡萄球菌15株（37.5%),其他葡萄球菌8株（20%）。药敏结果显示多重耐药。结论：凝固酶阴性葡萄球菌（CNS）已成为小儿血培养的第一位检出菌,主要为溶血葡萄球菌和表皮葡萄球菌,MRCNS占主导地位（达50%左右）,成为新生儿病房院内感染的主要致病菌。药敏结果显示MRCNS呈多重耐药,万古霉素及其他糖肽类抗生素是治疗MRCNS感染的首选药物,MRCNS在新生儿病房造成院内感染的危害性以及耐药性应引起广泛关注。     

丹东市熟肉制品中食源性致病菌污染状况的调查研究

为了解丹东市熟肉制品中沙门菌、金黄色葡萄球菌和单核细胞增生性李斯特菌的污染及菌相分布状况,为丹东市熟肉制品中致病菌污染提供本地资料,以及熟肉制品卫生监督提供科学依据。依据国标方法以及采用全自动酶联免疫荧光快速筛选仪进行筛选和BBL Crystal细菌鉴定仪鉴定,对130份样品分别进行上述致病菌分离、血清学和生化鉴定。结果共检出致病菌15株,总检出率为11．5％,酱卤类检出率为14．0％、烧烤类检出率为7．5％、白切类检出率为12．5％,其中沙门菌4株、单核细胞增生性李斯特菌4株和金黄色葡萄球菌7株。说明丹东市熟肉制品中存在食源性致病菌污染,其中金黄色葡萄球菌污染最严重。     

基于单碱基延伸标签反应的常见食源性致病菌基因芯片检测方法的建立

针对8种常见的食源性致病菌(金黄色葡萄球菌、副溶血弧菌、单核细胞增生李斯特菌、沙门氏菌、阪崎肠杆菌、志贺氏菌、肠出血性大肠杆菌O157:H7和空肠弯曲杆菌),建立了基于单碱基延伸标签反应原理的基因芯片检测方法。筛选和整合8种食源性致病菌基因组中的特异性序列和相应PCR引物,致病菌靶DNA片段被扩增和纯化作为单碱基延伸标签反应的模板,反应产物在DNA芯片上与探针进行杂交反应,然后通过扫描基片的荧光强度进行判断。实验结果表明,可采用基于单碱基延伸标签反应的基因芯片方法同时特异性检测8种食源性致病菌,基因组DNA多重检测灵敏度可达到0.1pg,以鼠伤寒沙门氏菌为单一检测对象的细菌纯培养物灵敏度可达到5×102CFU/mL。本方法可以快速灵敏地检测食源性致病菌,为食源性疾病的诊断和防治提供了一个有效的方法。     

372株溶血葡萄球菌的临床分布及耐药性

目的了解医院4年来溶血葡萄球菌的临床分布特征及耐药性变化,为临床合理用药提供参考依据。方法回顾性分析2010年1月至2013年12月住院患者标本分离的372株溶血葡萄球菌,采用美国BD Phoenix 100全自动细菌鉴定药敏分析仪及其配套GP检测卡进行药敏分析。结果分离的372株溶血葡萄球菌主要分布于ICU、呼吸内科和外科,主要来源于痰液、尿液和伤口分泌物;未检测到万古霉素耐药菌株,对阿莫西林/克拉维酸、氨苄西林、苯唑西林、红霉素、青霉素、头孢西丁均保持90%以上的高耐药性,对其它抗生素均存在不同程度耐药性。结论溶血葡萄球菌耐药现象日益严重,且多重耐药现象呈上升趋势,临床在治疗此类细菌感染时,应根据药敏试验结果制定用药方案,减少经验用药。     

286株溶血葡萄球菌的临床分布及药敏分析

【摘 要】 目的 了解宁波市妇女儿童医院溶血葡萄球菌的临床分布和耐药情况,为临床合理用药提供依据。方法 对该院2010年1月1日至2012年12月31日期间分离的286株溶血葡萄球菌进行分析,采用法国生物梅里埃公司的VITEK-60型全自动微生物分析仪进行菌株鉴定和药敏试验,用参考菌株做质量控制。结果 分离的溶血葡萄球菌主要来自儿科(59.8%);标本来源主要是阴道/尿道分泌物(29.7%),其次是创口分泌物(22.7%)。286株溶血葡萄球菌中耐甲氧西林溶血葡萄球菌(MRSH)261株,占91.3%;抗生素耐药分析显示,溶血葡萄球菌对青霉素G、苯唑西林、氨苄西林/舒巴坦表现出高度耐药性,耐药率分别为94.1%、91.3%、90.6%。对利福平、莫西沙星、呋喃妥因的耐药率较低,分别为2.8%、2.4%、1.0%;未发现耐万古霉素溶血葡萄球菌。结论 本院溶血葡萄球菌对某些药物的耐药率很高,有必要对其进行耐药率监测,指导临床合理选择抗菌药物。     

儿童血液凝固酶阴性葡萄球菌的感染情况及耐药性分析

目的了解本院儿童血培养凝固酶阴性葡萄球菌（CNS）的感染率及其药物敏感情况,为儿科合理使用抗生素提供依据。方法对本院2006年1月至2007年12月间住院及门诊儿童血液培养的结果进行回顾性统计分析。结果在1265例儿童血培养中共检出CNS117株,其中表皮葡萄球菌41株（占35．0％）,人葡萄球菌27株（占23．1％）,溶血葡萄球菌21株（占18．0％）,其他凝固酶阴性葡萄球菌28株（占23．9％）;耐甲氧西林凝固酶阴性葡萄球菌（MRCNS）分离率为79．5％;MRCNS药敏结果显示多重耐药。结论CNS已成为儿童血液感染的重要致病菌,MRCNS检出率高且多重耐药,万古霉素、喹宁始霉素-达福普汀、呋西地酸是治疗MRCNS感染的首选药物。     

抗生素对临床常见G+致病菌的效价分析

目的:调查我院常用抗G~ 致病菌的抗生素的种类效价情况,为抗G~ 致病菌的合理用药提供依据。方法:从2978个样本中分离到953株G~ 致病菌,用全自动微生物分析仪-VITEK32进行菌种鉴定及16种抗生素的药敏实验。结果:953株G~ 致病菌经鉴定属于11大类,其中金黄色葡萄球菌、表皮葡萄球菌、溶血葡萄球菌、草绿色链球菌和肠球菌属是五类主要的G~ 致病菌,依次分别占总分离量的30.3%、17.3%、15.1%、9.8%和6.8%;在检测的16种抗生素对排在前五位的G~ 致病菌的平均效价最低的五类抗生素是:青霉素(24.22%)、红霉素(26.22%)、头孢唑林(28.06%)、与链霉素(30.52%)和庆大霉素(31.06%)。而抗G~ 致病菌整体效果最好的是:螯合新泰林(93.72%)、万古霉素(82.62%)和四环素(78.68%)。结论:研究结果表明越来越多抗生素抗临床常见G~ 致病菌的效价呈现逐年递减的趋势。     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.

The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals.     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent.faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium -like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum , shared characteristics with Ent. columbae.
The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol.
Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.     

Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission

The molecular characterisation of species and genotypes of Cryptosporidium and Giardia is essential for accurately identifying organisms and assessing zoonotic transmission. Results of recent molecular epidemiological studies strongly suggest that zoonotic transmission plays an important role in cryptosporidiosis epidemiology. In such cases the most prevalent zoonotic species is Cryptosporidium parvum. Genotyping and subtyping data suggest that zoonotic transmission is not as prevalent in the epidemiology of giardiasis. Molecular characterisation of Cryptosporidium and Giardia is a relatively recent application that is evolving as new genes are found that increase the accuracy of identification while discovering a greater diversity of species and yet unnamed taxa within these two important genera. As molecular data accumulate, our understanding of the role of zoonotic transmission in epidemiology and clinical manifestations is becoming clearer.     

Comparison of BMI and percentage of body fat of Indian and German children and adolescents

Today, serious health problems as overweight and obesity are not just constricted to the developed world, but also increase in the developing countries (Prentice 2006, Ramachandram et al. 2002). Focusing on this issue, BMI and percentage of body fat were compared in 2094 schoolchildren from two cross-sectional studies from India and Germany investigated in 2008 and 2009. The German children are in all age groups significantly taller, whereas the Indian children show higher values in BMI (e.g. 12 years: Indian: around 22 kg/m2; German: around 19 kg/m2) and in the percentage of body fat (e.g. 12 years: Indian: around 27%; German: around 18-20%) in most of the investigated age groups. The Indian children have significantly higher BMI between 10 and 13 (boys) respectively 14 years (girls). Indian children showed significant higher percentage of body fat between 10 and 15 years (boys) and between 8 and 16 years (girls). The difference in overweight between Indian and German children was strongest at 11 (boys) and 12 (girls) years: 70% of the Indian but 20% of the German children were classified as overweight. In countries such as India that undergo nutritional transition, a rapid increase in obesity and overweight is observed. In contrast to the industrialized countries, the risk of overweight in developing countries is associated with high socioeconomic status. Other reasons of the rapid increase of overweight in the developing countries caused by different environmental or genetic factors are discussed.     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000