金黄色葡萄球菌核酸酶基因缺失突变株RN4220△nuc1的构建

金黄色葡萄球菌存在两个核酸酶编码基因,一个是葡萄球菌核酸酶(Staphylococcal nuclease,SNase),命名为nuc1,另一个是耐热核酸酶(Thermonuclease,TNase),命名为nuc2,nuc2是一个新的候选基因,以往认为金黄色葡萄球菌中的核酸酶只源于一个编码基因nuc1,为了进一步研究nuc2基因的功能,首先要将金黄色葡萄球菌nuc1基因缺失.研究目的就是通过构建同源重组质粒pBT2莫玭uc1,将其电转入金黄色葡萄球菌菌株RN4220中,获得nuc1基因缺失突变株.经过了7轮培养和筛选,同源重组几率为2%(7/345),筛选出的nuc1突变株用PCR方法和RT-PCR进行了验证,从而获得了nuc1基因缺失突变株RN4220△nuc1.     

金黄色葡萄球菌hmp基因缺失突变株的构建及抗一氧化氮能力分析

摘要：【目的】:构建金黄色葡萄球菌RN6390黄素血红蛋白(flavohaemoglobin, HMP)基因缺失突变株,研究其抗一氧化氮(Nitric Oxide, NO) 能力及其在细菌生物被膜形成中的作用。【方法】：根据同源重组技术的原理,利用PCR扩增RN6390的hmp基因上下游同源臂,经过抗生素和温度交替培养筛选hmp基因缺失突变株,利用基因组PCR、定量PCR对突变菌株进行鉴定。以硝普钠（SNP）为NO供体,检测了hmp基因缺失菌株的抗NO能力,并初步研究了hmp基因在生物被膜形成中的作用。【结果】：成功构建了RN6390的hmp基因缺失突变株,外源NO能够诱导菌株hmp基因的表达,hmp基因缺失菌株抗NO能力明显下降,但其生物被膜形成能力有明显提高。【结论】：获得了RN6390的hmp基因缺失突变株,该突变株的获得为进一步研究hmp基因的生物功能,以及细菌内源性NO的作用奠定了良好的技术平台。     

小菌落突变株：一种利于持续复发性感染的细菌致病形式

小菌落突变株是一种具有独特表型及致病特征且生长缓慢的细菌亚群。在表型上,小菌落突变株表现为生长率低、菌落形态不规则及生化特性异常,这使得临床微生物学家在鉴定时遇到了挑战。在临床上,小菌落突变株比相应的野生株更能持续存在于哺乳动物细胞中,且对抗生素更不敏感。当宿主细胞形成应急的保护性环境时,小菌落突变株会引发隐性或复发性感染。这篇综述涵盖了小菌落突变株相关表型、遗传及临床上的特征,重点描述目前研究最多的葡萄球菌。     

一株金黄色葡萄球菌噬菌体的分离鉴定与生物学特性

[背景]金黄色葡萄球菌作为一种条件致病菌,在临床感染中扮演着重要的角色,需要研究更多更有效的防治手段.[目的]分离金黄色葡萄球菌噬菌体,研究其生物学特性,从而为金黄色葡萄球菌的替代防治提供理论借鉴.[方法]以金黄色葡萄球菌D085为宿主从污水中分离得到一株噬菌体,命名为vB_SauS_SAP3,用PEG8000浓缩、氯...     

金黄色葡萄球菌的分离及其抗药性的研究

从土壤中分离出金黄色葡萄球菌后,以其为出发菌株,采用梯度培养皿法,利用青霉素、四环素、红霉素、氯霉素和链霉素5种抗生素,对自然界中和经过NaNO2诱变的菌株进行了耐药性菌株的分离及抗性水平的确定。在自然界中分离的金黄色葡萄球菌只对青霉素(80μg/mL)和四环素(60μg/mL)有抗性,而对红霉素、氯霉素和链霉素则没有抗性。经过NaNO2诱变后,金黄色葡萄球菌对四环素(40μg/mL)的抗性降低,但对青霉素(120μg/mL)和其他3种抗生素的抗性均有所增加。     

猪源致病性金黄色葡萄球菌的分离鉴定及其耐药性分析

目的鉴定引起猪渗出性皮炎的病原,并分析猪源致病性金黄色葡萄球菌的耐药性,为临床用药提供依据。方法采集患渗出性皮炎的仔猪标本进行细菌分离培养,联合应用形态学检查、生理生化试验和PCR方法鉴定分离菌株,并进行致病性和药物敏感性试验。结果先后从病猪标本中分离鉴定获得PSA1、PSA2、PSA3和PSA4四株金黄色葡萄球菌,其中PSA1和PSA3分离株的致病性较强。药敏试验结果显示PSA1、PSA2和PSA3分离株为MRSA菌株,PSA4分离株为MSSA菌株。MRSA菌株对14种抗菌药物均呈现不同程度的耐药,尤其是对青霉素、链霉素、四环素、强力霉素、环丙沙星和氧氟沙星等6种抗菌药物的耐药率达100%。所有分离株对万古霉素与替考拉宁均敏感。结论合肥地区猪渗出性表皮炎的病原为金黄色葡萄球菌。猪源致病性金黄色葡萄球菌合肥分离株具有多重耐药性,治疗猪渗出性皮炎应建立在体外药敏试验的基础上,有针对性选择抗菌药物。     

雪貂葡萄球菌的分离与鉴定

目的对雪貂分离菌株进行鉴定。方法通过菌落、菌体形态、生化特征等细菌学检测方法和药敏实验对所分离菌株进行鉴定。结果根据生物学特性和药敏实验确定所分离菌株为金黄色葡萄球菌和中间葡萄球菌。结论分离菌株为金黄色葡萄球菌和中间葡萄球菌。     

鹦鹉嗜衣原体禽鸟株的分离鉴定及小鼠呼吸道感染模型的建立

摘要：【目的】优化鹦鹉热嗜衣原体（Chlamydophila psittaci,C. psittaci,Cps）禽鸟株分离培养技术,建立Cps呼吸道感染的小鼠模型。【方法】从禽鸟肝组织中抽提总DNA,PCR法体外扩增Cps ompA基因,初步鉴定Cps阳性标本;同时将阳性标本的肝组织匀浆液接种到HeLa和Vero细胞中培养,Giemsa和免疫荧光染色法鉴定衣原体包涵体。将临床株扩大培养后,用2×104 、2×105 、2×106 IFU三个剂量鼻内接种小鼠,分别于感染后5d和10d处死小鼠,显微镜观察受染小鼠各脏器病理变化。【结果】采用PCR扩增Cps ompA基因,从100只禽鸟标本中检测到阳性Cps标本6例（6%）,并成功地在HeLa及Vero细胞中培养出3例,且Vero细胞内的衣原体包涵体体积大,结构致密,对衣原体感染引起的宿主细胞溶解耐受性较HeLa细胞强,更适合用于Cps的分离培养及体外研究。成功建立Cps的鼠呼吸道感染模型,105IFU 是建立Cps呼吸道感染小鼠模型的适宜菌量。【结论】优化了Cps临床株的分离培养技术,成功建立了Cps呼吸道感染小鼠模型,为Cps流行病学调查及研究衣原体的致病性和致病机制奠定基础。     

Small colony variants of Staphylococcus aureus reveal distinct protein profiles

Small-colony variants (SCVs) of Staphylococcus aureus represent a slow-growing subpopulation causing chronic and relapsing infections due to their physiological adaptation on an intracellular lifestyle. In this first proteomic study on physiological changes associated with a natural, clinically derived SCV, its proteomic profile was investigated in comparison to corresponding isogenic strains displaying normal (clinical wild-type strain, complemented hemB mutant and spontaneous revertant of the clinical SCV) and SCV phenotypes (hemB mutant and gentamicin-induced SCV). Applying an ultra-high resolution chromatography and high mass accuracy MS(E) -based label-free relative and absolute protein quantification approach, the whole cytoplasmic proteome of this strain sextet was investigated in a growth phase-controlled manner covering early-exponential, late-exponential and stationary phases. Of 1019 cytoplasmic proteins identified, 154 were found to be differently regulated between strains. All SCV phenotypes showed down-regulation of the tricarboxylic acid (TCA) cycle-related proteins and of a protein cluster involved in purine/pyrimidine and folate metabolism. In contrast to hemB mutant and gentamicin-induced SCVs, the clinically derived SCVs showed no prominent up-regulation of glycolytic proteins. The spontaneous switch into the normal phenotype resulted in up-regulation of TCA cycle-related parts, while oxidative stress-related proteins were down-regulated. However, the natural revertant from the clinical SCV retained also dominant protein features of the clinical SCV phenotype. In conclusion, physiological changes between normal and SCV S. aureus phenotypes are more complex than reflected by defined electron transport chain-interrupting mutants and their complemented counterparts.     

Characteristics of Staphylococcus aureus, isolated from airways of cystic fibrosis patients, and their small colony variants

The colonization of respiratory tract by Staphylococcus aureus is a frequent feature of cystic fibrosis (CF), especially in pediatric patients. The formation of small colony variants (SCVs), which produce reduced amounts of alpha-toxin, is one of the proposed ways of staphylococcal accommodation in an intracellular niche. The aim of the present study was to compare some properties of S. aureus SCVs and their parent strains. A site-directed S. aureus hemB mutant and parent strain 8325-4 were included in the study (control pair). Normal and SCV strain pairs from CF patients as well as control strains were tested for the susceptibility to defensins, killing activity of professional phagocytes and adhesion to A549 cell line. Because S. aureus are exposed to many cationic proteins in the host, we challenged a clinical isolate with minimal subinhibitory concentration (subMIC) of protamine and found that hemin and menadione auxotrophic SCVs emerged. SCVs were more resistant than normal strains to protamine but not to dermaseptin. The susceptibility to the bactericidal activity of magainin was the same for normal and SCV strains. The protamine resistance of normal as well as SCVs was strongly enhanced by high salt concentration. The adhesion of some SCVs to A549 cells was higher than adhesion of parental strains. However, the number of adherent bacteria (SCVs) was diminished in the presence of hemin for hemin auxotrophs. The uptake of SCVs by granulocytes was lower than ingestion of normal strains, but SCVs were killed with equal or greater potency. SCVs are adapted to intracellular survival and persistence in the host under certain circumstances. The ability to form a variant subpopulation affords S. aureus additional survival options.     

Diversity of staphylocoagulase and identification of novel variants of staphylocoagulase gene in Staphylococcus aureus

Staphylocoagulase (SC) is a major phenotypic determinant of Staphylococcus aureus. Serotype of SC (coagulase type) is used as an epidemiological marker and 10 types (I-X) have been discriminated so far. To clarify genetic diversity of SC within a single and among different serotype(s), we determined approximately 1500 bp-nucleotide sequences of SC gene encoding D1, D2, and central regions (N-terminal half and central regions of SC; SC(NC)) for a total of 33 S. aureus strains comprising two to three strains from individual coagulase types (I-VIII, X) and 10 strains which were not determined as previously known SC serotypes (ND-strains). Amino acid sequence identities of SC(NC) among strains with a single coagulase type of II, III, IV, V, VI and X were extremely high (more than 99%), whereas lower identity (56-87%) was observed among different types. In contrast, within a single coagulase type of I, VII, or VIII, sequence divergence was found (lowest identity; 82%). SC(NC) sequences from the ND-strains were discriminated into two genetic groups with an identity of 71% to each other (tentatively assigned to genotypes [XI] and [XII]), and exhibited less than 86% sequence identities to those of most known coagulase types. All the types [XI] and [XII] strains were methicillin susceptible and belonged to different sequence types from those of coagulase types I-X strains reported so far by multilocus sequence typing. These findings indicated genetic heterogeneity of SC in coagulase types I, VII, and VIII strains, and the presence of two novel SC genotypes related to antigenicity of SC serotypes.     

Evaluation of six commercial identification kits for the identification of Staphylococcus aureus isolated from bovine mastitis

AIMS: Comparison of six commercially available in human medicine well-established slide agglutination systems for the identification of Staphylococcus aureus. METHODS AND RESULTS: Slide agglutination tests were compared with the conventional tube coagulase test, biochemical identification and with the molecular identification by polymerase chain reaction (PCR) amplification of species-specific parts of the gene encoding the 23S RNA. Systems evaluated included Masta-Staph (Mast Diagnostics), Staphylase-Test (Oxoid), Staphytect-Plus (Oxoid), Staphyloslide Latex (Becton Dickinson), Slidex Staph Plus (bioMerieux) and Dry Spot Staphytect Plus (Oxoid). A total of 141 staphylococcal strains isolated from cases of bovine mastitis including 90 S. aureus, 14 Staphylococcus epidermidis, 10 Staphylococcus warneri, 13 Staphylococcus xylosus, 11 Staphylococcus haemolyticus and three other coagulase-negative staphylococci were tested with each method. Staphylococcus aureus strains were selected by macrorestriction analysis with pulsed field gel electrophoresis (PFGE). Only genetically unrelated strains were included in the study. The sensitivities and specificities of the test were as follows: Masta-Staph 86.7 and 90.1%, Staphylase-Test 78.4 and 85.1%, Staphytect-Plus 81.1 and 86.5%, Staphyloslide Latex 77.8 and 84.4%, Slidex Staph Plus 77.8 and 84.4%, Dry Spot Staphytect Plus 75.6 and 83.0%. CONCLUSIONS: The results of this evaluation suggest that the six slide agglutination methods tested can provide rapid identification of S. aureus also from bovine mastitis. The sensitivity and specificity seems to be less than those reported from human S. aureus isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first comparative reported investigations about the applicability of different commercially available slide agglutination tests for the detection of S. aureus from bovine mastitis using PFGE selected clinical isolates.     

一株高效异养硝化菌的选育、鉴定及其硝化条件

【目的】针对现阶段异养硝化菌硝化速率较低的问题,选育更高效的异养硝化菌,进而鉴定该菌株的种属,了解其硝化特性和硝化条件。【方法】分别从污水处理厂活性污泥、化肥厂土壤以及农田土壤中取样,以柠檬酸钠为碳源,NH4Cl为氮源,采用污泥驯化、驯化过程中驯化液连续梯度稀释、平板划线分离及颜色指示剂快速硝化效果检测等步骤,筛得一株高效的异养硝化菌。经生理生化和16SrDNA序列的系统发育分析鉴定其种属;将该菌接入人工氨氮废水,定时检测水中含氮化合物的变化,了解其硝化特性;通过改变培养基碳源、溶氧量、C/N比、温度和pH考察其硝化条件。【结果】获得的高效异养硝化菌为革兰氏阴性杆菌,不利用葡萄糖发酵,氧化酶、接触酶阳性,不产吲哚,能由有机酸盐产碱;其与产碱菌属菌株Alcaligenessp.ES-SDK-3的16SrDNA同源性高达99.7%。用该菌株处理初始氨氮浓度为182.30mg/L的废水,30h后氨氮去除率为99.8%,指数期平均氨氮去除速率为9.61mg-N/L/h,其在硝化过程中几乎没有亚硝酸盐氮和硝酸盐氮产生;最佳碳源为柠檬酸钠;高的溶氧量和高的C/N比有利于其降解氨氮,当C/N比为12时即可达到较好的效果;该菌株在温度为30℃-35℃,pH为5.0-9.0范围内均能较彻底地降解氨氮。【结论】该菌株为产碱菌属,命名为Alcaligenessp.HN-S;其在硝化速率与处理的氨氮浓度方面均高于目前国内外筛出的大多数异养硝化菌;通过考察其硝化条件,为其走向实际污水脱氮工艺提供了依据。