耐喹诺酮类抗生素的鱼源嗜水气单胞菌中内生质粒pAhW39的克隆及功能

为探究嗜水气单胞菌中内生质粒与菌株对喹诺酮类抗生素耐药表型的关系,实验克隆并分析了分离自湖北仙桃某渔场患病团头鲂的对喹诺酮类抗生素耐药的嗜水气单胞菌W39菌株中的质粒pAhW39.测序结果显示,质粒pAhW39的大小为6739 bp,GC含量为46.13％,低于目前已公布的嗜水气单胞菌染色体DNA的GC含量(60.1％...     

耐喹诺酮类药物嗜水气单胞菌的分离鉴定及电镜观察

本研究对吉林省内12个水域采集和送检的66尾病鱼进行细菌分离鉴定,共检出46株嗜水气单胞菌疑似株,通过生化试验和PCR扩增气溶素基因aer鉴定法,确定其中22株为嗜水气单胞菌。进一步采用纸片扩散法和双倍稀释法测定嗜水气单胞菌分离株对多种抗生素的耐药性,其中部分菌株存在不同程度的耐药性,22株嗜水气单胞菌中有4株对喹诺酮类药物耐药,并且为交叉耐药。其对喹诺酮类药物的抑菌圈均小于19mm。嗜水气单胞菌耐喹诺酮类药物的检出率为18 2%。将病料中分离的8株嗜水气单胞菌在电镜下观察发现,2株耐药菌表面发现均匀分布的球形结构,而6株敏感菌表面未见此结构。     

海南地区溶藻弧菌耐药性及其质粒多样性的研究

调查了海南省的30株溶藻弧菌环境菌株对24种常见抗生素的耐药性,并对菌株携带质粒及其多样性进行了研究,所有菌株对洁霉素、乙酰螺旋霉素、万古霉素、氯洁霉素、制霉菌素5种抗生素完全耐药,对24种抗生素的耐药率为20.8%～66.7%,表明该地区溶藻弧菌具有严重的多重耐药性;30株溶藻弧菌中有22株不携带质粒,8株携带1～3个质粒,质粒为1.20～12.5kb;8株携带质粒的菌株共形成了7种类型的质粒指纹图谱。试验结果表明,该地区溶藻弧菌的耐药性与其是否携带质粒无明显关系。分离菌株的耐药性可能主要是由染色体相关基因引起的。     

豫北地区主要淡水鱼类感染嗜水气单胞菌的流行病学调查

对豫北地区嗜水气单胞菌引起的细菌性败血病进行流行病学调查,分离鉴定致病性嗜水气单胞菌菌株,并对致病性嗜水气单胞菌菌株进行毒力验证和药敏试验。采集4个养殖场病鱼标本及水样,每月进行流行病学调查,利用生理生化及分子生物学方法检测嗜水气单胞菌的分布状况。结果显示,筛选出52株为嗜水气单胞菌,其中32株为致病性嗜水气单胞菌,20株为非致病性嗜水气单胞菌,且致病性嗜水气单胞菌主要分布在7—9月份,毒力验证试验表明,32株致病性菌株的毒力大小差异明显,筛选出强毒株XDMG(4),为后期试验的疫苗株做准备;药敏试验表明,不同菌株对同一种药物的药敏结果不同,但大部分药物的药敏结果基本一致,70%以上的致病性菌株对头孢哌酮、氟本尼考、菌必治、阿米卡星等抗菌药物表现为高度敏感,对青霉素类药物表现为高度耐药性,耐药率达100%,对氨基糖苷类(不包括阿米卡星)、磺胺类、四环素等药物呈现不同程度的耐药性,具有多重耐药性。本试验旨在丰富本地区的鱼类细菌性败血病的病原资料,并为该菌引起的人类疾病的防控提供科学依据。     

苏州市患病水生动物体内细菌的分离鉴定及药敏试验

为了解苏州市不同地区患病水生动物组织中感染细菌的种类及其耐药性,于2014年6～10月从不同地区采集的患病鱼、虾、蟹和中华鳖组织中共分离到14株细菌,通过16S rRNA基因分析对细菌进行了鉴定,并调查了他们对链霉素、恩诺沙星、强力霉素、庆大霉素硫酸盐和甲砜霉素等5种抗生素的抗性及最低抑菌浓度。结果显示:从不同地区水生动物分离到的同种细菌对同种抗生素的抗性有明显差异,其中,分离到的嗜水气单胞菌及摩根氏菌有多重耐药性;对14株细菌进行质粒抽提和琼脂糖凝胶电泳分析,发现8株细菌中存在分子量不同的质粒,且不同地区来源的同种细菌所含有的质粒的分子量不同,耐药性较强的嗜水气单胞菌和摩根氏菌均含有质粒;嗜水气单胞菌质粒的序列测定结果显示,该质粒与其抗药性无关;转化实验结果显示,从摩根氏菌分离的1.8 kb质粒与氨苄青霉素抗药性有关。     

27株嗜水气单胞菌致病性及ERIC—PCR指纹图谱分析

从福建省淡水养殖鱼类体内分离到27株嗜水气单胞菌,根据嗜水气单胞菌16SrDNA基因和气溶素基因（aerolysin）的保守序列,设计2对引物,对所分离到的27株嗜水气单胞菌进行双重PCR扩增,扩增结果表明,其中18株为含有Aer毒力基因的潜在致病性嗜水气单胞菌,占总菌数的66．67％。应用ERIC-PCR分型技术对27株嗜水气单胞菌菌株进行分析,以相似度54．00％为限,所有菌株可分为Ⅰ和Ⅱ两大聚类,以76．00％相似度为界,27株嗜水气单胞菌可分为11个聚类,同一个聚类中菌株分离区域基本相同。分析结果表明,分离的嗜水气单胞菌基因型的多样性和分离地域具有一定的关联,也表明ERIC-PCR可以有效应用于嗜水气单胞茵分子流行病学调查。     

异育银鲫源嗜水气单胞菌对磺胺类耐药性分析

为了解异育银鲫源嗜水气单胞菌对磺胺类药物的敏感性和磺胺类耐药基因的携带情况。采用纸片扩散法和微量肉汤稀释法测定了36株受试菌株对常见4种磺胺类药物的敏感性;应用PCR法检测菌株染色体DNA和质粒DNA磺胺类Sul1、Sul2和Sul3耐药基因。试验结果表明,试验菌株对磺胺嘧啶、磺胺二甲嘧啶、磺胺异噁唑、磺胺甲噁唑的耐药率分别为83.34%、72.22%、66.66%、69.45%;36株嗜水气单胞菌的染色体和质粒中均检测到Sul1和Sul2基因,Sul1基因在染色体和质粒中的检出率分别为30.56%和25.64%,Sul2基因在染色体和质粒中的检出率分别为83.33%和91.67%,Sul3基因在染色体和质粒中均未检出。异育银鲫源嗜水气单胞菌对磺胺类药物的耐药性不容乐观,其耐药性与Sul2和Sul1基因有较大相关性。     

一株从草鱼中分离的嗜水气单胞菌的病原学及基因组特征

为确定南昌地区某渔场草鱼出血性败血症的病原体及病原特征,从患病草鱼的肝脏病灶中分离出一株致病菌A1310。对分离菌进行了形态特征、理化特性等表型生物学检验、人工感染实验及对抗菌药物的敏感性实验,并对其进行了全基因组测序、基于多序列位点分型(multilocus sequence typing,MLST)的系统进化分析及毒力基因分析。结果显示,生理生化鉴定证明该菌为嗜水气单胞菌,当浓度达1.0×10⁶ CFU/mL时,对草鱼有致病性;对供试20种抗菌药物中的青霉素等7种耐药,对卡那霉素等5种敏感;A1310株基因组框架序列共包含96个与毒力和防御相关基因,其中多药耐药性外排泵基因占大多数,约为22%;与美国斑点叉尾鮰分离株(S15-242、S15-458、S15-591、S15-700)聚类为同一进化分支;比较基因组分析发现,A1310具有一个删减版的Ⅵ型分泌系统(type Ⅵ secretion system,T6SS),基因数量为标准Ⅵ型分泌系统的80%,缺少vgrG和vca0109基因的部分片段。综上所述,来源于草鱼的嗜水气单胞菌菌株A1310,与美国斑点叉尾鮰分离株具有亲缘关系,含有多个已报道的嗜水气单胞菌的毒力基因。本研究不仅丰富了嗜水气单胞菌的生物学性状内容,也为嗜水气单胞菌的有效检验、防治和深入研究提供一定的参考,对草鱼的疾病防控具有一定意义。     

高度耐药嗜水气单胞菌的定向诱导及其交叉耐药性分析

为了分析比较不同抗生素对耐药嗜水气单胞菌(Aeromonas hydrophila)的诱导效率,从五大类抗生素中各挑选出一种代表性的抗生素,在针对嗜水气单胞菌菌株AH10的防耐药突变浓度(mutant prevention concentration,MPC)的琼脂平板上,分别筛选出五株耐药菌株,并对筛选的耐药菌株进行交叉耐药性分析,制定出这些耐药菌株对常用抗生素的最小抑菌浓度(minimal inhibitory concentrations,MIC)图谱。结果显示:四环素、氟苯尼考、磺胺嘧啶、硫酸新霉素、诺氟沙星对AH10的MIC分别为:1.0、0.5、8.0、8.0、0.25μg/m L;MPC分别为4.0、3.0、128、88、2.0μg/m L,分离的耐药菌株对所使用抗生素的MIC均提高100倍以上。4℃划线平板冰箱保存条件下,一个月内个别耐药菌株耐药性下降。在药物筛选压力下,菌株耐药性随着传代次数增多而表现出递增的趋势。同属一类抗生素耐药相关性较高;不同大类抗生素压力下筛选出来的耐药菌株表现出不同的交叉耐药性。     

嗜水气单胞菌对氟苯尼考的耐药性获得与消失速率

为摸清淡水鱼类最主要的致病菌——嗜水气单胞菌对国家批准使用的新兽药——氟苯尼考的耐药性的获得与消失速率,提供科学使用该药的数据支撑,测定在低浓度的氟苯尼考压力下,3株嗜水气单胞菌对氟苯尼考的最小抑菌浓度(MIC)随时间的变化,以及3株菌对氟苯尼考的耐药性获得速率和消失速率。结果表明,菌株在低浓度的氟苯尼考下,4~5d其耐药浓度升高1.6~2.5倍。同时,菌株对氟苯尼考的耐药浓度随着药物浓度的逐渐增加可迅速上升15.9~40倍,但其消失较慢,28d的消失速率为1.58~2.54。因此,在使用氟苯尼考防治细菌性疾病时,连用时间不宜超过4d,再次使用的间隔不少于28d。     

对虾养殖场底泥中副溶血性弧菌的接合型质粒多样性及其与毒力之间的关系

为了阐明引起急性肝胰腺坏死病(acute hepatopancreatic necrosis disease, AHPND)副溶血性弧菌的接合型质粒在对虾养殖环境中的遗传多样性,实验从中国5个沿海省份的虾场收集了100个底泥样品,以质粒上编码接合转移蛋白的保守基因为目标,利用PCR法检测相关质粒的存在情况,并对质粒进行测序。结果显示,100个样品中有39个样品含有质粒的接合转移蛋白片段。从100个底泥样品中分离出15株副溶血性弧菌,其中13株含有1～2个质粒。质粒序列测序结果显示,这些质粒可分为8种类型/谱型,其中7种不携带pirAB,但均含有编码接合转移的基因簇。根据分离副溶血性弧菌携带质粒的8种谱型,分别选择8株副溶血性弧菌进行凡纳滨对虾攻毒实验,发现这些菌株对凡纳滨对虾的毒性有显著差异,实验虾死亡率为15%～100%。只有pirAB阳性菌株会对实验虾产生AHPND症状,死亡率为100%。对质粒组成进行分析表明,质粒之间遗传物质交换频繁,大部分质粒的遗传组成都来自一个183 kb的超大质粒pVP2HP。综上,本实验通过探究对虾养殖场底泥中结合性质粒的多样性,增强了人们对副溶血性弧菌...     

杂色鲍哈维弧茵耐药质粒的鉴定和分析

文章对引起杂色鲍（Hallotis diversicolor）肌肉萎缩症的病原菌哈维弧菌（Vibrio harveyi）质粒上的磺胺耐药基因进行研究,结果显示,该菌株对复方新诺明完全耐药,其质粒上含有sulⅡ耐药基因。接合转化试验显示该质粒具可转移性,可使受体菌对磺胺制剂复方新诺明产生耐药性,鉴定为耐药质粒,并测定了该耐药质粒的全基因组序列,序列总长为10 940 bp,初步分析显示有7个具有一定功能的ORF框。进一步构建重组表达质粒pR-SET-A-sulⅡ,表达了目的蛋白（31 kDa）。     

对虾病毒HHNBV DNA构建质粒的研究与分析

用ＰＵＣ１８构建了对虾病毒ＨＨＮＢＶＤＮＡ重组质粒，提取后经斑点杂交及酶切分析，证实质粒中插入片段为病毒ＤＮＡ，其中０５＃质粒的插入片段大于２Ｋｂ，与质粒的分子量相近。对０５＃质粒酶切后的Ｓｏｕｔｈｅｒｎ杂交亦取得了满意的结果。同时，还对０５＃质粒的插入片段进行了内切酶图谱分析，共进行了ＥｃｏＲｌ、Ｈｉｎｄｌｌｌ、Ｓｍａｌｌ、Ｐｓｔｌ、ＰＵＶｌ、Ｈｉｎｄｌｌ６种限制性内切酶分析，结果表明，只有Ｐｓｔｌ在插入片段的一端有一个酶切位点，酶切位点的确切位置有待于进一步确定。     

一种用PUC质粒高效克隆PCR产物的方法

ＰＵＣ质粒用Ｓａｍｌ酶切，经Ｔａｑ酶和ｄＴＴＰ处理，构建成ｄＴ载体。ＰＣＲ产物经低熔点琼脂糖凝胶纯化，与ｄＴ载体直接进行连接反应，再转化受体菌。运用该法对不同长度的病毒、细菌和动物细胞的ＤＮＡ片段进行了克隆试验，均获得高效率克隆。     

In vivo transfer of plasmid pRAS1 between Aeromonas salmonicida and Aeromonas hydrophila in artificially infected Cyprinus carpio L.

This study investigated the possible in vivo transfer of plasmid pRAS1 between Aeromonas salmonicida and A. hydrophila inhabiting two different organs of Cyprinus carpio L. To distinguish transconjugants from naturally occurring antibiotic resistant bacteria, twelve luminescent transposon‐tagged A. hydrophila strains using miniTn5luxCDABEKm2 transposon were generated. In conjugal transfer experiments, fish were conditioned with the donor bacteria and subsequently immersed in water containing the recipient strain. Bacteria were recovered from gills and intestines and isolated by growth on selective plates. Transconjugants were identified by their resistance to the pRAS1 encoded antimicrobials and by light emission. In vivo transfer frequencies ranged between 10^?3 and 10^?6 and were somewhat lower in intestines, compared to gills. Transfer frequencies were also smaller relative to those obtained in vitro. The minimal amount of donor and recipient bacteria needed to yield detectable transconjugants in vivo was 1 × 10⁴ CFU mL^?1. Implications of this plasmid transfer in natural settings and its possible consequences to human health are discussed.     

团头鲂tf和tfr1a基因启动子克隆及转录调控分析

为探索鱼类转铁蛋白基因tf和转铁蛋白受体基因tfr1a的转录调控机制,本实验以团头鲂为研究对象,在其全基因组数据库中获取tf和tfr1a基因序列,对2个基因候选启动子区转录因子结合位点及CpG岛进行预测,通过PCR方法克隆得到tf和tfr1a基因近端启动子区不同长度片段,连接至pGL3-Basic/pEGFP-1载体,瞬时转染入Hela细胞,并采用双荧光素酶报告基因检测系统进行检测。结果发现,团头鲂tf基因启动子区无CpG岛位点,而tfr1a基因启动子区有2个CpG岛位点。成功构建9个tf和10个tfr1a不同长度启动子片段的重组质粒,经双荧光素酶报告基因系统检测发现,tf启动子核心区域为-268^+56 bp,且-1 308^-1 102 bp片段可能存在正调控该基因表达的转录因子结合位点;tfr1a启动子核心区域为-224^+48 bp,且+48^+92 bp可能存在抑制该基因转录的负调控元件,而-1 229^-1 219 bp区域可能存在促进tfr1a基因表达的正调控转录因子结合位点。     

Development and validation of a quantitative PCR to detect Parvicapsula minibicornis and comparison to histologically ranked infection of juvenile Chinook salmon, Oncorhynchus tshawytscha (Walbaum), from the Klamath River, USA

Parvicapsula minibicornis is a myxosporean parasite that is associated with disease in Pacific salmon during their freshwater life history phase. This study reports the development of a quantitative (real-time) polymerase chain reaction (QPCR) to detect P. minibicornis DNA. The QPCR assay targets the 18S ribosomal subunit gene. A plasmid DNA control was developed to calibrate cycle threshold ( C _T) score to plasmid molecular equivalent (PME) units, a measure of gene copy number. Assay validation revealed that the QPCR was sensitive and able to detect 50 ag of plasmid DNA, which was equivalent to 12.5 PME. The QPCR assay could detect single P. minibicornis actinospores well above assay sensitivity, indicating a single spore contains at least 100 times the 18S DNA copies required for detection. The QPCR assay was repeatable and highly specific; no detectable amplification was observed using DNA from related myxozoan parasites. The method was validated using kidney tissues from 218 juvenile Chinook salmon sampled during the emigration period of March to July 2005 from the Klamath River. The QPCR assay was compared with histological examination. The QPCR assay detected P. minibicornis infection in 88.1% of the fish sampled, while histological examination detected infection in 71.1% of the fish sampled. Good concordance was found between the methods as 80% of the samples were in agreement. The majority of the disconcordant fish were positive by QPCR, with low levels of P. minibicornis DNA, but negative by histology. The majority of the fish rated histologically as having subclinical or clinical infections had high QPCR levels. The results of this study demonstrate that QPCR is a sensitive quantitative tool for evaluating P. minibicornis infection in fish health monitoring studies.     

青鱼和鲇肠道排泄物对养殖水体铜绿微囊藻休眠体复苏的影响及作用途径

为探讨养殖水体底栖鱼类肠道排泄物对铜绿微囊藻休眠体复苏的影响,将青鱼和鲇肠道排泄物与铜绿微囊藻休眠体用野外养殖水域沉积物(底泥)混匀包埋,在10、15和20°C梯度温度下进行休眠体复苏实验.结果显示,铜绿微囊藻休眠体主要复苏期为第3～15天,在10和15°C条件下,青鱼排泄物组(MP)、鲇排泄物组(SA)和青鱼-鲇排泄...     

Delivery of HUFA, probionts and biomedicine through bioencapsulated Artemia as a means to enhance the growth and survival and reduce the pathogenesity in shrimp Penaeus monodon postlarvae

One of the major problems in the shrimp culture industry is the difficulty in producing high-quality shrimp larvae. In larviculture, quality feeds containing a high content of highly unsaturated fatty acids (HUFA) and ingredients that stimulate stress and disease resistance are essential to produce healthy shrimp larvae. In the present study, Penaeus monodon postlarvae (PL15) were fed for 25 days on an unenriched Artemia diet (control; A) or on a diet of Artemia enriched with either HUFA-rich liver oil of the trash fish Odonus niger (B), probionts [Lactobacillus acidophilus (C1) or yeast-Saccharomyces cerevisiae (C2)] or biomedicinal herbal products (D) that have anti-stress, growth-promoting and anti-microbial characteristics. P. monodon postlarvae fed unenriched Artemia exhibited the lowest weight gain (227.9 ± 8.30 mg) and specific growth rate (9.95 ± 0.05%), while those fed the HUFA-enriched Artemia (B) exhibited the highest weight gain and specific growth rate (362.34 ± 12.56 mg and 11.77 ± 0.08%, respectively). At the end of the 25-day rearing experiment, the shrimp postlarvae (PL40) were subjected to a salinity stress study. At both low and high (0 and 50‰) salinities, the group fed the control diet (A) experienced the highest cumulative mortality indices (CMI) 935.7 ± 2.1 and 1270.7 ± 3.1, respectively. Those fed diet D showed the lowest stress-induced mortality, and CMI were reduced by 31.1 and 32.3% under conditions of low and high salinity stress, respectively. A 10-day disease challenge test was conducted with the P. monodon postlarvae (PL40–PL50) by inoculating the shrimp with the pathogen Vibrio harveyi at the rate of 10⁵–10⁷ CFU/ml in all rearing tanks. P. monodon postlarvae fed probiont-encapsulated Artemia diets (C1 and C2) exhibited the highest survival (94.3 and 82.3%, respectively) and lowest pathogen load (V. harveyi) in hepatopancreas (5.2 × 10² ± 9.0 × 10 and 4.6 × 10² ± 9.0 × 10 CFU g⁻¹, respectively) and muscle (2.0 × 10² ± 6 × 10 and 1.7 × 10² ± 8.6 × 10 CFU g⁻¹, respectively) tissues. The shrimp that were fed the unenriched Artemia (Control; A) showed the lowest survival (26.33%) and highest bacterial load in the hepatopancreas (1.0 × 10⁵ ± 5 × 10³ CFU g⁻¹) and muscle (3.6 × 10⁴ ± 6 × 10² CFU g⁻¹). The shrimp fed the herbal product (D)-enriched Artemia also exhibited enhanced survival and reduced V. harveyi load in the tissues tested compared to the control diet (A) group. The results are discussed in terms of developing a quality larval feed to produce healthy shrimp larvae.