嗜水气单胞菌国内分离株的毒力因子分布与致病性相关性分析

为了研究分析国内致病性嗜水气单胞菌的毒力因子的分布及与致病性的相关性及其防治,选取5种主要毒力因子气溶素(aerA)、溶血素(hlyA)、丝氨酸蛋白酶(ahpA)、抗金属蛋白酶(ast)、肠毒素(altA)设计5对特异性引物,用PCR方法进行检测,采用急性毒性实验方法测定菌株对异育银鲫的半数致死量(50%lethal dose,LD50),细菌药物敏感试验纸片扩散(K-B)法检测对抗生素的敏感性,结果显示:除菌株Hong12的LD50约为109 cfu,毒力弱外,其他毒力均较强,LD50均在106 cfu左右。aerA、ahpA、hlyA、altA、ast 5种毒力基因的携带率分别为77.8%、88.9%、100%、100%、77.8%。通过比较嗜水气单胞菌毒力基因的分布情况与其对鲫鱼致病性试验结果可以发现,aerA、ahpA为嗜水气单胞菌致病的主要毒力因子,与致病性存在相关性,ast与致病力存在一定相关性,但不是主要毒力因子,hlyA、altA与致病性不存在相关性。9株嗜水气单胞菌对青霉素、羧苄青霉素、万古霉素不敏感,对复合磺胺、头孢噻肟、利福平中毒敏感,对呋喃妥因、氯霉素、四环素、氧氟沙星高度敏感。     

湖北地区暴发病池塘中嗜水气单胞菌的遗传多样性和毒力特征研究

为了调查引起鱼类运动型气单胞菌败血症(俗称暴发病)的嗜水气单胞菌的遗传多样性和毒力特征, 阐明其流行规律, 研究于2006-2009年度从湖北省内3个不同地区的6个发病鱼塘中分离了30株嗜水气单胞菌, 其中20株为临床株(分离自血液、肝脏、肾脏或腹水), 6株为肠道株, 4株为池水株。基于所有菌株gyrB基因序列, 构建了系统发育树; 通过ERIC (Enterobacterial repetitive intergenic consensus, 肠道细菌基因间重复序列)指纹图谱进行菌株的遗传分型; 用PCR方法检测了7个毒力基因在菌株中的分布模式。这7个基因包括气溶素(aerA)、溶血素(hlyA)、热不稳定性细胞兴奋性肠毒素(alt)、热稳定性细胞兴奋性肠毒素(ast)、弹性蛋白酶(ahpB)、脂酶(lip)和鞭毛基因(fla)。此外, 以斑马鱼为感染对象, 通过腹腔注射测定了15株代表菌株的毒力。结果表明: 不同来源的20株临床株、1株肠道株和3株池水株具有相同的遗传特性, 体现为在系统树上聚为一枝, 序列相似性为100%, 具有相同的ERIC指纹图谱, 毒力基因分布模式为: aerA+hlyA+alt+ast+ahpB+lip+fla+, 且均为强毒株(LD50 9.74104cfu/尾)。与临床株相比, 其余5株肠道株和1株池水株或具有不同的ERIC指纹图谱或具有不同的毒力基因分布模式, 显示出了遗传多样性, 且毒力均弱于临床株(LD501.01106cfu/尾)。这说明在一定时间、一定区域内, 作为暴发病病原的嗜水气单胞菌为同一克隆系在流行, 不存在明显的变异或遗传多样性。此结果有助于阐明嗜水气单胞菌引起的暴发病的流行规律, 制定相应的防御措施。多种毒力基因在致病性菌株中的联合流行为发病机理的解析奠定了基础。此外, 鉴于毒力基因谱与致病性之间的相关性, 表明毒力基因可作为标记基因, 用于致病性菌株的检测。       

三重PCR检测鱼类致病性嗜水气单胞菌

[目的]建立一种能够快速准确地检测致病性嗜水气单胞菌的PCR.方法.[方法]根据嗜水气单胞菌的16S rRNA、气溶素基因(aer)和丝氨酸蛋白酶基因(ahp)的保守序列设计了3对引物,然后进行了PCR反应条件的优化、特异性和敏感性的检测并与普通的细菌分离鉴定进行了临床样本和人工攻毒样本检出率的比较.[结果]该方法特异性好,只对致病性嗜水气单胞菌呈阳性扩增;敏感性高,最低可检测100fg的细菌DNA模版.对临床疑似黄鳝(Monopterus albus)样本的检出率为81.8%,高于细菌分离的40.9%;对人工攻毒鲫鱼(Carassius auratus)样本的检出率为87.5%,高于细菌分离的67.5%.[结论]本方法的成功建立,实现在同一反应管中同时对16SrRNA、aer和ahp的检测,避免了只针对aer或ahp单个毒力基因的PCR检测方法可能存在的漏检和误检,为致病性嗜水气单胞菌的诊断、大规模检疫、流行病学调查等提供了一种快速、准确而有效的检测方法.     

和厚朴酚对嗜水气单胞菌气溶素表达的抑制作用

为了研究不同浓度和厚朴酚对嗜水气单胞菌(Aeromonas hydrophila)致病力的影响, 筛选抗嗜水气单胞菌感染的天然化合物, 通过溶血试验、免疫印记试验、荧光定量PCR试验和动物试验进行了研究。结果发现, 和厚朴酚能在亚抑菌浓度下降低嗜水气单胞菌培养物上清中的溶血活性; 蛋白免疫印迹试验发现和厚朴酚能降低嗜水气单胞菌气溶素的分泌; 荧光定量PCR试验进一步表明和厚朴酚与嗜水气单胞菌共培养后降低了气溶素编码基因aerA的转录而降低气溶素的分泌。此外, 通过动物试验发现和厚朴酚治疗能显著提高斑点叉尾鮰( Ictalurus punctatus )嗜水气单胞菌感染模型的存活率。以上研究表明, 和厚朴酚能通过降低气溶素编码基因aerA的转录而降低嗜水气单胞菌的致病力, 和厚朴酚是一种潜在的新型抗嗜水气单胞菌感染的先导化合物。     

一种检测大鲵致病性嗜水气单胞菌的四重PCR法

建立一种快速诊断致病性嗜水气单胞菌的PCR法。根据嗜水气单胞菌的16S rDNA、外膜蛋白、溶血素及丝氨酸蛋白酶基因设计4对引物,经PCR反应条件优化后进行检测。结果表明,仅嗜水气单胞菌呈阳性,其检测敏感性高,可检测100 fg的DNA模板。20株大鲵源嗜水气单胞菌经四重PCR法检测时,有18株呈阳性。其中,毒力基因种类较多的阳性菌株经人工感染试验和胞外产物活性检测时显示出较高的致病性。利用该PCR法进行的其他检测中,还发现33份人工感染样本全部呈阳性,34份疑似样本有21份呈阳性,说明四重PCR法可应用于临床检测。本研究建立的四重PCR法具有高度敏感性和特异性,可用于嗜水气单胞菌快速诊断。     

嗜水气单胞菌气溶素基因的克隆与序列分析

以嗜水气单胞菌BZ和NK分离株的DNA为模板,采用PCR技术,扩增气溶素基因(aerA)的DNA片段,将其克隆到pMDl8-T载体上.通过序列测定,分析结果表明:所克隆的1393 bp片段为aerA部分序列,编码产生464个氨基酸.BZ与NK之间aerA核苷酸同源性为97.6%.氨基酸同源性为98.3%,与其它分离物核苷酸同源性为71.6%～97.5%,氨基酸同源性为68.0%～98.9%.利用邻接法构建了aerA分子树状图,树状图分析表明:气单胞菌属各分离物聚为三支,其中嗜水气单胞菌各菌株之间关系密切,被聚类为同一支.     

致病性维罗纳气单胞菌检测方法的建立

发掘维罗纳气单胞菌特异性更强的检测靶点和毒力相关基因靶点,建立能够检测致病性维罗纳气单胞菌的PCR检测方法.通过序列比对分析气单胞菌的16S rRNA基因序列,筛选对维罗纳气单胞菌特异的引物,用于检测种特异性,利用气单胞菌气溶素基因保守引物,检测菌株的致病性,并进行反应条件和反应体系的优化,灵敏度试验和特异性试验.发掘并设计的维罗纳气单胞菌16S rRNA特异性引物结合气单胞菌气溶素基因保守引物建立的检测方法,对12株气单胞菌和10株非气单胞菌的检测结果显示,所有致病性维罗纳气单胞菌都能扩增到大小分别为343 bp和232 bp的特异性条带,而非维罗纳气单胞菌的致病性气单胞菌只能扩增到232 bp的气溶素基因特异性条带,其它菌株都不能扩增到目的条带.灵敏度试验表明,该反应体系的检测灵敏度为1.35×10-3 mg/L.我们建立的致病性维罗纳气单胞菌检测方法能特异地检测致病性维罗纳气单胞菌,并具有高度灵敏性.     

嗜水气单胞菌气溶素基因的克隆与序列分析

以嗜水气单胞菌BZ和NK分离株的DNA为模板, 采用PCR技术, 扩增气溶素基因(aerA)的DNA片段, 将其克隆到pMD18-T载体上。通过序列测定, 分析结果表明：所克隆的1393 bp片段为aerA部分序列, 编码产生464个氨基酸。BZ与NK之间aerA核苷酸同源性为97.6%, 氨基酸同源性为98.3%, 与其它分离物核苷酸同源性为71.6%~97.5%, 氨基酸同源性为68.0%~98.9%。利用邻接法构建了aerA分子树状图, 树状图分析表明：气单胞菌属各分离物聚为三支, 其中嗜水气单胞菌各菌株之间关系密切, 被聚类为同一支。     

翘嘴鳜病原嗜水气单胞菌分子特征及LAMP检测方法的建立

嗜水气单胞菌(Aeromonas hydrophila)是一种危害鳜鱼养殖生产的重要病原细菌, 为进一步明确该病原菌的分子特征及建立快速检测技术, 实验对引起翘嘴鳜(Siniperca chuatsi)暴发性死亡的病原嗜水气单胞菌进行了致病性、菌株毒力特征研究, 同时以嗜水气单胞菌气溶素基因aerA为分子靶标设计引物, 利用环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP)建立了病原嗜水气单胞菌的快速检测方法。结果表明, 本次引起翘嘴鳜暴发性死亡的病原嗜水气单胞菌半致死浓度为1.6×106 CFU/mL, 携带aerA等14种毒力基因, 此14种毒力基因可用于其致病性分析及分子检测。以气溶素基因aerA设计引物进行的环介导恒温扩增, 结果显示可扩增出阶梯状条带, 加入SYBR Green I染色后呈现绿色的阳性反应, 而对照组均未出现任何扩增条带且反应体系呈现橙色, 表明LAMP检测方法对于嗜水气单胞菌检测具有很好的特异性; 灵敏度检测的最低检测限为4.6×101 CFU/mL; 10种经人工感染的淡水养殖鱼虾组织匀浆增菌液, 提取DNA后进行LAMP方法检测, 结果均可获得阳性扩增结果, 而对照未染菌组呈阴性, 表明该方法具有较好的应用性, 可应用于嗜水气单胞菌引起的水生动物疾病的检测。     

嗜水气单胞菌毒力基因在传代过程中的稳定性研究

为了解嗜水气单胞菌毒力基因在传代过程中的稳定性,对传代前后嗜水气单胞菌(Aeromonas hydrophila,A.hydrophila)的毒力和毒力基因进行测定和检测.采用急性毒性试验方法测定10株嗜水气单胞菌原代、第10代及第20代的菌株对异育银鲫(Carassais auratus gibebio)的半数致死量,通过聚合酶链式反应(PCR)检测原代、第10代及第20代的菌株中5种毒力基因aerA、hlyA、ahpA、altA及ast的变化情况.试验结果表明,嗜水气单胞菌在传代过程中毒力基因分布相对较稳定.但是ahpA基因极易发生变异,在培养基上连续多次传代后,毒力逐渐减弱甚至消失.可推知,ahpA基因与毒力相关性最大,并易受传代影响.     

High frequency of hemolytic and cytotoxic activity in Aeromonas spp. isolated from clinical, food and environmental in Rio de Janeiro, Brazil

Molecular study of aerolysin and cytotonic enterotoxin genes by PCR and colony blot hybridization was performed in 117 strains of Aeromonas spp. isolated from different sources. Homogeneous distribution of these genes in A. hydrophila complex strains was observed. For A. caviae and A. sobria complex strains, aerolysin genes were more frequent than cytotonic enterotoxins genes. Of 64 A. caviae complex strains, only one (1.5%) amplified the 451 bp product for the aer gene, however, the same primers detected a 400 bp product in 50 (78%) strains. This product was sequenced and had two short regions with homology to several hemolysin genes. The genotype aer ⁺/aerA⁺/hly ⁺/ast ⁺/alt ⁺ was detected in six A. hydrophila strains from food and environmental source. The most common genotype found in A. hydrophila strains was hly ⁺ (85%) and aerA⁺ (78.7%), while in A. caviae complex strains was aerA⁺ (32.8%). All A. veronii complex sobria strains were aer ⁺/aerA⁺. All A. caviae and A. hydrophila were positive when tested with aer probe using the colony blot test. Thirty-seven percent of A. hydrophila and 53% of A. caviae tested were positive for ast probe. Eighty-nine percent of samples were cytotoxic in Vero cells. Our data demonstrated that Aeromonas spp. can harbor and express virulence genes and reinforce the potential of Aeromonas as a human pathogen.     

Detection of toxigenic strains of <Emphasis Type="Italic">Aeromonas</Emphasis> species in foods by a multiplex PCR assay

Aeromonas hydrophila and other aeromonads are ubiquitous organisms found in meat, vegetables, drinking water and various other food items. They cause diarrhea and extra-intestinal infections in normal and immunocompromised patients. The aim of the study was to develop a multiplex PCR assay for the detection of virulence-associated genes of Aeromonas including hemolysin (hlyA), aerolysin (aerA), glycerophospholipid-cholesterol acyl transferase (GCAT) alongwith a 16S rRNA gene. Internal amplification control (IAC), which was coamplified with aerA primers, was also included in this study. The results showed that all cultures of Aeromonas were accurately identified by the assay without showing non-specificity. A. hydrophila could be detected at a range of 10–50 CFU ml⁻¹ from experimentally spiked fish, chicken and milk samples following overnight enrichment in alkaline peptone water supplemented with 10 μg/ml ampicillin (APW-A) by this multiplex PCR (mPCR). When evaluated on a total of 74 naturally occurring food samples, four samples were identified to contain Aeromonas by mPCR. All these results were compared to the conventional culture, isolation and biochemical identification procedures. The high throughput and cost-effective mPCR method developed in this study could provide a powerful tool for detection of pathogenic Aeromonas spp. from food and environmental samples and in addition, the method has advantages in terms of specificity, sensitivity and ease of use compared to other reported PCR methods and DNA hybridization assays.     

Detection of three virulence genes alt, ahp and aerA in Aeromonas hydrophila and their relationship with actual virulence to zebrafish

Aims: To evaluate the frequency of the aerolysin (aerA), cytotoxic enterotoxin (alt) and serine protease (ahp) genes in Aeromonas hydrophila isolates from different sources, and to determine the relationship between the presence of these genes and virulence of A. hydrophila in zebrafish. Methods and Results: Aeromonas hydrophila isolates from clinical cases (n = 40), from healthy fish (n = 22) and from water environment (n = 21) were analysed with respect to the prevalence of aerA, alt and ahp genes by PCR assay. These virulence factors occur among clinical isolates as well as among isolates from healthy fish and water environment. The majority (97·6%) of the strains examined carried one or more virulence genes. The isolates were divided into seven genetic profiles on the basis of PCR result: aerA⁺alt⁺ahp⁺ (62·7%), aerA⁺alt⁺ahp^? (13·3%), aerA⁺alt^?ahp⁺ (10·8%), aerA^?alt⁺ahp⁺ (4·8%), aerA^?alt^?ahp⁺ (3·6%), aerA⁺alt^?ahp^? (2·4%) and aerA^?alt^?ahp^? (2·4%). A higher frequency of genetic group aerA⁺alt⁺ahp⁺ was determined in the isolates from diseased animals compared to those from healthy fish or water environments. Virulence properties of 26 representative strains belonging to the seven genetic profiles were further characterized. Results demonstrated that as the present of virulence genes increased, the proteolytic, haemolytic and cytotoxic activities of extracellular products also increased. And the 50% lethal doses (LD₅₀s) of aerA⁺alt⁺ahp⁺ isolates (<10⁵) in zebrafish were lower when compared with the strains expressing one or combinations of two virulence genes (>10⁶). Conclusions: Virulence properties of A. hydrophila correlated well with the presence of virulence genes tested. aerA⁺alt⁺ahp⁺ was more frequent virulence genotype in A. hydrophila isolates from clinical diseases than from healthy fish and water environment, and the aerA⁺alt⁺ahp⁺ isolates were more virulent to zebrafish compared to the other six genetic profiles. Significant and Impact of the Study: The detection for aerA, alt and ahp can be used for virulence typing of A. hydrophila isolates.     

连翘提取物对嗜水气单胞菌群体感应系统的影响

【背景】传统抑菌剂的大量使用导致细菌产生多重耐药性与抗性,而基于细菌群体感应靶点调控的新型抑菌剂可缓解细菌耐药性与抗性,是未来抑菌剂的发展方向之一。【目的】研究连翘(Forsythiasuspensa)提取物对嗜水气单胞菌(Aeromonashydrophila)群体感应系统的影响及可能的作用机制,为新型抑菌剂的开发提供理论依据。【方法】以紫色杆菌(Chromobacterium violaceum)CV026为报告菌株,以嗜水气单胞菌为供试菌株,采用倍比稀释法测定连翘提取物对2种菌的最小抑菌浓度(minimal inhibitory concentration,MIC),通过微量法测定提取物对嗜水气单胞菌生长、群集运动及蛋白酶活性的影响,利用高效液相色谱串联质谱法分析提取物中的主要成分,采用分子对接模拟探究提取物对嗜水气单胞菌群体感应系统的作用机制。【结果】连翘提取物对紫色杆菌CV026和嗜水气单胞菌的MIC均为16.00mg/mL。在亚抑菌浓度下,连翘提取物处理显著抑制了CV026紫色菌素的产生,最大抑制率高达56.30%。经8.00mg/mL连翘提取物处理后,嗜水气单胞菌的群集运...     

牛源多杀性巴氏杆菌的分离与初步鉴定

【目的】本研究旨在对引起犊牛呼吸道综合征的多杀性巴氏杆菌进行分离鉴定,分析其亲缘关系和毒力基因的分布情况。【方法】收集2017年8月至2018年4月疑似患有犊牛呼吸道综合征的病牛鼻拭子进行细菌分离培养,对菌落形态和染色疑似巴氏杆菌的菌株进行16S rRNA测序和血清型鉴定,选择巴氏杆菌7类23种毒力基因,筛查临床分离株的毒力基因的分布。【结果】从8个省份的237份病料中分离出31株多杀性巴氏杆菌,分离率为13.1%。16S rRNA测序分析表明31株A型多杀性巴氏杆菌属于同一亚群,其序列同源性与中国分离株HB01以及国外分离株USDA-ARS-USMARC-60712、USDA-ARS-USMARC-60214、ATCC 43137以及36950亲缘关系较近。对分离出的31株A型多杀性巴氏杆菌的7类共23种毒力基因鉴定,结果显示31株多杀性巴氏杆菌所携带的毒力因子大多分布在17–19个,且集中度较高。【结论】A型多杀性巴氏杆菌为犊牛呼吸道综合症的主要流行血清型,通过对多杀性巴氏杆菌的临床分离株进化树和毒力基因分析,内蒙古、黑龙江、新疆、山西以及河北的7株分离株进化来源于同一分支,且均缺失毒力基因tadD和hgbA及携带毒力基因hsf-1,提示着其亲缘关系可能与其携带的特定毒力基因存在一定相关性。该研究为犊牛呼吸道综合征的病原学调查和多杀性巴氏杆菌流行病学调查提供了参考数据。     

黄芩苷对嗜水气单胞菌生物膜形成的影响及体内外的抑菌作用

[目的] 探讨中药单体黄芩苷对嗜水气单胞菌在体内外生长及生物膜形成的影响。[方法] 体外实验中,利用牛津杯法检测抑菌圈直径,结晶紫法检测生物膜的形成,通过泳动实验检测黄芩苷对嗜水气单胞菌运动性的影响,紫外吸收法检测细胞膜完整性,用透射电镜技术观察黄芩苷对细菌形态的影响。体内实验利用草鱼为对象检测黄芩苷对嗜水气单胞菌增殖的影响。[结果] 黄芩苷在体外对嗜水气单胞菌有明显的抑菌效果,通过对生物膜的研究发现黄芩苷对生物膜形成具有抑制作用,并同时抑制其运动性。同时黄芩苷可以破坏细胞结构,并增加了细胞膜通透性。体内实验结果显示黄芩苷对嗜水气单胞菌具有清除作用,且具有一定的浓度依赖性。[结论] 黄芩苷在体内外均具有抑制嗜水气单胞菌增殖的作用,有望在水产养殖病害防治工作中得到应用。     

16S rRNA及rpoC1基因用于螺旋藻、节旋藻系统发育的比较北大核心CSCD

【目的】利用16S rRNA和rpoC1基因分子标记研究螺旋藻、节旋藻的系统发育关系,并对其区分能力进行比较。【方法】以84株螺旋藻、节旋藻为研究对象,对其进行16S rRNA、rpoC1基因序列的扩增、测序及分析,并对构建的系统发育树进行对比。【结果】rpoC1基因序列保守位点所占比例49.7%、平均G+C百分含量47.7%和序列相似度76%–100%明显低于16S rRNA基因序列的79.4%、55.6%和91%–100%,其变异程度高于16S rRNA基因;基于16S rRNA、rpoC1基因构建的系统发育NJ树拓扑结构基本一致,84株实验藻株分为2个属3个类群,其中仅F-351、F-904-2、F-1070和TJBC14-1藻株为螺旋藻,其余均为节旋藻;虽然2个基因都不能区分形态种和地理种,但rpoC1基因NJ树的置信度(100%)高于16S rRNA基因(99%),属内分群效果也明显优于16S rRNA基因。【结论】支持了螺旋藻、节旋藻为两个不同属的结论,且在属内分类时rpoC1基因比16S rRNA基因具有更高的区分度。     

Correlation between the distribution pattern of virulence genes and virulence of Aeromonas hydrophila strains

Nine strains of Aeromonas hydrophila isolated from diseased fish or soft-shelled tortoise were tested for the presence of three virulence genes including the genes encoding aerolysin, hemolysin, and extracellular serine protease (i.e., aerA, hlyA, and ahpA, respectively). These genes were investigated using polymerase chain reaction (PCR) with specific primers for each gene. And the pathogenicities to Carrassius auratus ibebio of these strains were also assayed. PCR results demonstrated that the distribution patterns of aerA, hlyA, and ahpA were different in these strains. 6/9 of A. hydrophila strains were aerA positive, 8/9 of strains hlyA positive, 7/9 of strains ahpA positive, respectively. However, the assay for pathogenesis showed that two strains (A. hydrophila XS91-4-1 and C2) were strong virulent, two strains (A. hydrophila ST78-3-3 and 58-20-9) avirulent and the rest middle virulent was to the fish. In conclusion, there are significant correlation between the distribution pattern of the three virulence genes and the pathogenicity to Carrassius auratus ibebio. All strong virulent A. hydrophila strains were aerA ⁺ hlyA ⁺ ahpA ⁺ genotype, and all aerA ⁺ hlyA ⁺ ahpA ⁺ strains were virulent. Strains with the genotype of aerA ⁻ hlyA ⁻ ahpA ⁺ have middle pathogenicity. In the present study, we found for the first time that all A. hydrophila isolated from the ahpA positive were virulent to Carrassius auratus ibebio. Additionally, there was a positive correlation between the virulence of A. hydrophila and the presence of aerA and ahpA. __________ Translated from Acta Scientiarum Naturalium Universitatis Sunyatseni, 2006, 45(1): 82–85 [译自: 中山大学学报 (自然科学版)]     

一株聚羟基脂肪酸酯合成菌的筛选及鉴定

【背景】传统石油基塑料产品给人类和环境带来的危害日益严重,聚羟基脂肪酸酯(polyhydroxyalknoates,PHA)作为新型可降解塑料原料越来越受到青睐。但PHA生产成本过高,使其推广应用严重受限。筛选适合大规模生产PHA的高产菌株是解决这一问题的重要途径。【目的】以挖掘合成PHA的菌种资源为目标,从极端环境筛选和鉴定新的高产PHA合成菌。【方法】通过尼罗蓝平板分离法和PCR法分离纯化菌株,采用16S rRNA基因鉴定并通过MEGA 6.0软件构建系统发育树,分析菌株的进化关系,最后通过尼罗红染色定性分析和气相色谱法定量测定该菌株在不同时期的PHA积累量。【结果】从盐碱地垃圾沉积物中分离得到了一株高产PHA的菌株,PhaC的PCR扩增结果证实了该菌株是PHA合成菌,经16S rRNA基因鉴定为Pseudomonas brassicacearum,将其命名为NP-2,进一步优化了菌株NP-2的培养条件,在培养48h时PHA积累量最大,达到3.78 mg/mL。【结论】NP-2属于Pseudomonas brassicacearum,能高产PHA。本研究为生产PHA提供了极端环境的...     

乌鳢源杀鱼爱德华菌的分离鉴定及药敏特性研究

[背景] 江苏省扬州市某乌鳢养殖场发生疾病,给养殖户造成了严重的经济损失。[目的] 确定病因并筛选出敏感药物,为乌鳢相关疾病的防治提供参考。[方法] 从患病乌鳢体内分离致病菌,并从形态、生理生化特征、16S rRNA和gyrB基因序列及特异性PCR检测等方面对分离菌株进行鉴定,同时开展人工感染试验分析其致病性,通过纸片扩散法进行药敏特性分析。[结果] 从患病乌鳢体内分离获得优势菌株SHL,经形态特征、理化特性、16S rRNA和gyrB基因序列及特异性PCR检测鉴定为杀鱼爱德华菌。进一步人工感染试验证实其对乌鳢有较强的致病性,LD₅₀为1.6×10⁵ CFU/g,发病症状与自然发病症状相似。药敏试验结果显示,该菌株对青霉素、氯霉素、四环素等28种抗菌药物高度敏感,对红霉素中度敏感,对苯唑西啉、克拉霉素、万古霉素等6种药物耐药。[结论] 引起江苏省扬州市某养殖场的乌鳢体表溃烂及死亡的病原菌为杀鱼爱德华菌,这是我国首次从淡水鱼类中检出致病性杀鱼爱德华菌,表明该菌的感染谱在扩大,需引起水产养殖领域的重视,在养殖过程中可根据药敏实验结果选用合适的国标渔药进行防治。