荧光定量PCR法检测副溶血弧菌tdh基因的表达差异

以pvuA为内标基因,运用实时荧光定量PCR检测不同来源以及不同应激条件下副溶血弧菌热稳定直接溶血素基因tdh的表达量。pvuA和tdh基因的荧光定量PCR融解曲线分析表明,两者均为特异性扩增。尽管相同来源的不同菌株间tdh表达量存在显著差异,副溶血弧菌临床分离株的tdh mRNA平均表达量显著高于海产品分离株((57.2比13.8)。在pH4.0、0.5%和8%NaCl应激条件下,临床株ZJ2和海产品分离株FJ14A的tdh mRNA表达量显著高于对照组;另一海产品分离株KP34在8%NaCl条件下的表达量显著提高,而低pH应激时tdh mRNA的表达量显著降低。结果表明,不同副溶血弧菌分离株的tdh mRNA表达差异显著,临床分离株的tdh mRNA表达量总体上高于海产品分离株,副溶血弧菌在不同应激条件下主要表现为tdh mRNA表达上调。     

副溶血弧菌SHJLA株的分离鉴定及致病性分析

副溶血性弧菌（Vibrio parahaemolyticus）是海洋环境中常见的食源性致病茵。本研究从对虾中分离出1株细菌SHJLA,在TCBS和弧菌显色培养基上分别显示典型的蓝绿色和紫红色的菌落,且其生理生化特性具有典型副溶血弧菌的特性。以SHJLA菌株的基因组为模板。检测副溶血弧菌种特异性基因（tlh、toxR、groEL）均为阳性,gyrB基因序列分析表明SHJLA与副溶血弧茵的亲缘关系最近,同源性达98％～100％;检测副溶血弧菌主要毒力相关基因tdh和T3SS2（VopC2和vcrD2）均为阳性。该菌的神奈川溶血实验为阳性,对小鼠的半数致死量（LD50）为4．8×10^8 cfu／mL。结合SHJLA菌株的形态、生理生化、种特异性基因的检测、gyrB基因序列分析、毒力相关基因的检测以及小鼠半数致死量的测定结果,确定SHJLA是一株携带毒力基因的副溶血弧菌。     

广东省候鸟副溶血性弧菌的分离与鉴定

为了解广东省候鸟副溶血性弧菌及其携带的毒力基因、抗生素敏感性和遗传进化特征,收集广东省候鸟粪便,用国标法和toxR基因PCR检测的方法进行分离鉴定获得副溶血性弧菌;通过PCR对毒力基因进行鉴定、用Etset测定MIC值,通过ERIC-PCR对副溶血性弧菌进行遗传进化分析。结果共获得122株副溶血性弧菌,这些细菌仅含有tlh(不耐热溶血素基因),而tdh(耐热溶血素基因)与trh(耐热溶血素相关毒素基因)等均为阴性。通过ERIC分群发现福田红树林保护区与湛江的鹭源菌株和湛江雷州的鹭源菌株具有高度相似性,深圳华侨城湿地菌株分布在3群,与雷州的鹭源相似度较高,为95%;福田红树林保护区分离株与深圳华侨城湿地菌株相似度大于75%。广东省候鸟携带的副溶血性弧菌具有较为丰富的遗传多样性,毒力基因携带单一,保持着较高的药物敏感性。     

副猪嗜血杆菌临床分离株耐药性与耐药机制的研究

为研究副猪嗜血杆菌(HPS)的耐药性和耐药机制,本研究从临床疑似患多发浆膜炎和关节炎猪的病料样品中分离到30株致病菌,对其采用16S r RNA菌株鉴定方法和KRG琼脂扩散血清分型方法进行鉴定,并利用琼脂稀释法和PCR方法对分离株进行12种常用抗菌药物的最小抑菌浓度(MIC)测定和相应耐药基因或突变位点的检测。鉴定结果显示,30株分离株均为HPS,其血清型主要为5型(26.7%)、13型(26.7%)和不可分型(26.7%)。药物敏感性试验结果显示,30株HPS对氨苄西林、泰妙菌素、恩诺沙星和亚胺培南的耐药率较高,分别为50%、50%、46.7%和100%。利用PCR重点检测了介导喹诺酮类药物的耐药基因和基因突变情况,结果显示qnr A的检出率高达98.7%。根据MIC试验结果从中选出14株耐药性比较强的菌株,检测其gyr A/gyr B/par C/par E 4种基因的突变位点,结果显示gyr A基因突变位点主要为S83F和D87N,gyr B的突变位点均为P472G,par C的突变位点主要为L73S和A77E,par E基因的突变位点主要为R254H和A227T。本研究结果为临床选用合理抗菌药物防治HPS病提供了实验依据。     

89株副猪嗜血杆菌临床分离株血清型、基因型鉴定与分析

旨在明确副猪嗜血杆菌（HPS）临床分离株分离部位的组织分布,进而探讨其血清型和基因型的流行分布特点及相关性,为猪格氏病（Glässer's disease）的有效防控提供科学依据。针对临床分离鉴定的89株HPS,利用PCR技术鉴定血清型,统计各血清型HPS在不同分离部位的菌株分布数量;应用多位点序列分型（MLST）方法进行序列类型（ST）鉴定分析、位点多态性分析、BURST分群统计和UPGMA系统发育树聚类分析。89株HPS临床分离株共鉴定出9种血清型（1、2、4、5、7、11、12、13、14）以及未定型（NT）,血清4、13、7和5型为优势血清型,分别占比28.09%、22.47%、13.48%和10.11%,有64株HPS分离于肺,占比71.91%;24种ST型,ST267、ST268、ST387和ST365为优势基因型,分别占比26.97%、21.35%、8.99%和5.62%,每个基因位点存在3~13个等位基因,多态性位点从3（g3pd）到71（6pgd）不等,BURST分析中划分为2个单个ST型和11个克隆群（CC）,UPGMA系统发育树被分为4个分支,优势ST型处于2、3、4三个分支中,均对应具有毒力血清型的HPS分离株。HPS临床分离株流行血清型和基因型呈现多元化,具有明显的遗传异质性,同时,ST型与血清型存在一定的交叉性,且与HPS临床致病力相关。     

A型产气荚膜梭菌青海分离株tetA(P)耐药基因序列分析及蛋白结构预测

应用PCR技术对A型产气荚膜梭菌青海分离株的tetA(P)耐药基因进行扩增、测序,利用生物软件对tetA(P)基因进行序列分析与蛋白结构预测。结果表明,A型产气荚膜梭菌青海分离株的tetA(P)基因长度为1 263bp,编码420个氨基酸。与参考菌株EHE-NE18、Wakayama、CW92的核苷酸序列同源性依次为100%、99.8%和98.7%,氨基酸序列同源性依次为100%、100%和98.3%。分离株TetA(P)蛋白疏水性较强,具有10个跨膜区,含有4个N-糖基化位点,1个cAMP和cGMP依赖性蛋白激酶磷酸化位点,5个蛋白激酶C磷酸化位点,1个酪蛋白激酶Ⅱ磷酸化位点,1个酪氨酸激酶磷酸化位点,12个N-豆寇酰化位点,三级结构主要是由α-螺旋和无规则卷曲形成。研究结果可为A型产气荚膜梭菌tetA(P)基因功能研究提供依据。     

水生动物中分离的副溶血性弧菌菌株分子分型研究

本研究通过采用核糖体分型RT、脉冲场凝胶电泳PFGE、多位点序列分型MLST三种方法,对从水生动物中分离到的59株副溶血性弧菌进行分子分型研究。结果将59株副溶血性弧菌分为43个RT型,9个大的聚类群,DI为0.9754;51个PFGE型,13个大的聚类群,DI为0.9988;53个ST型,包括107个新的等位基因、46个新的ST型,DI为0.9982。结果显示PFGE的分辨力最高,MLST较PFGE稍低,RT的分辨力最低。总体来看59株副溶血性弧菌菌株间亲缘关系较远,从同一年份、同一地区、同类样品中分离到的菌株被分为不同的型,表现出较大的遗传多样性。     

鸭疫里默氏杆菌贵州分离株OmpA及16S rRNA基因序列分析

为了解鸭疫里默氏杆菌(Riemerella anatipestifer,RA)贵州分离株外膜蛋白A(Omp A)和16 S r RNA基因序列的相关性以及与RA血清型之间的关系,对12株RA贵州分离株通过PCR分别扩增Omp A和16 S r RNA基因,并对其构建系统进化树和分析其系统进化关系。结果显示,12株RA分离株Omp A基因分为2个群,其核苷酸和氨基酸序列同源性分别为96.9%~100.0%和98.4%~100.0%;Omp A蛋白中有规律的变异位点为100(R/G)、143(S/P)、145(T/I)和268(S/P)。12株RA分离株16 S r RNA基因同属1个群,其核苷酸序列同源性为99.4%~100.0%。结果表明,12株RA贵州分离株Omp A基因和16 S r RNA基因序列所属的基因群无明显相关性,并且与RA血清型的分型也无直接关联。     

盐碱水体副溶血弧菌的分离和鉴定

为了明确副溶血弧菌在内陆淡水水体中的分布情况，本试验从查干湖水体中分离得到1株革兰阴性杆菌，命名为A201805S2,对其进行形态观察、生理生化鉴定，并利用PCR方法对其16S rDNA基因序列进行扩增和测序，将测得结果于NCBI上进行BLAST同源性比较。结果显示，该菌株在硫化硫酸盐柠檬酸盐胆盐蔗糖琼脂培养基(TCBS)上为绿色菌落，革兰染色呈红色，两端钝圆，为革兰阴性杆菌；生理生化鉴定结果与副溶血弧菌特性相一致；分离菌与副溶血弧菌16S rDNA基因序列同源性达99%。结果表明，本试验首次从内陆苏打盐碱型淡水水体中分离出副溶血弧菌，为淡水水体副溶血弧菌防控提供数据支持。     

猪链球菌2型江苏分离株溶血素基因的检测及序列分析

根据猪链球菌2型(strep tococcus su is type 2)溶血素基因(sly)设计和合成了一对可扩增其完整阅读框的引物,对HA 9801等6株猪链球菌2型江苏分离株、1株德国分离株SS2-D及猪链球菌C群参考株ATCC 35246的核酸进行PCR扩增,结果显示HA 9801等6株江苏分离株及德国株SS2呈阳性,ATCC 35246呈阴性。HA 9801株PCR产物纯化后测序,序列分析结果表明该DNA片段与猪链球菌2型1933株的sly基因同源性为99%。     

豫陕两省14株鸡杆菌的gyrB、16S rRNA和rpoB基因序列比较分析

分析河南、陕西分离的14株鸡杆菌(Gallibacterium)之间的进化关系,及gyrB基因序列比较在该菌进化分析中的作用。PCR扩增鸡杆菌分离株的gyrB、16SrRNA和rpoB3个看家基因,PCR产物纯化后直接测序。将鸡杆菌分离株、国外参考株的3个看家基因序列进行比较分析,用Phylip 3.67软件构建进化树。结果表明,14株鸡杆菌与鸭源鸡杆菌(Gallibacterium anatis)模式株间的相似性为96.3%～98.0%(gyrB)、97.7%～99.6%(16SrRNA)和97.7%～99.0%(rpoB);14株鸡杆菌与鸡杆菌复合群1(Gallibacterium genomosp.1)参考株间的相似性为88.8%～89.9%(gyrB)、96.2%～97.5%(16SrRNA)和92.6%～93.6%(rpoB)。基于3个看家基因序列的进化分析,均显示14株鸡杆菌和鸭源鸡杆菌模式株形成单独的一个群。14株鸡杆菌分离株均属于鸭源鸡杆菌种;在3个看家基因位点,鸡杆菌河南株与陕西株之间、鸡杆菌输卵管炎病鸡分离株与健康鸡分离株之间均无明显遗传上的差异;gyrB基因序列分析可用于鸡杆菌分离株的种类鉴定,且对14株鸡杆菌与复合群1参考株的区别能力优于另外2个看家基因。     

Clonal relationships among Clostridium perfringens of porcine origin as determined by multilocus sequence typing

Clostridium perfringens is ubiquitous in the environment and the intestinal tracts of most mammals, but this organism also causes gas gangrene and enteritis in human and animal hosts. While expression of specific toxins correlates with specific disease in certain hosts, the other factors involved in commensalism and host pathogenesis have not been clearly identified. A multilocus sequence typing (MLST) scheme was developed for C. perfringens with the aim of grouping isolates with respect to disease presentation and/or host preference. Sequence data were obtained from one virulence and seven housekeeping genes for 132 C. perfringens isolates that comprised all five toxin types and were isolated from 10 host species. Eighty sequence types (STs) were identified, with the majority (75%) containing only one isolate. eBURST analysis identified three clonal complexes, which contained 59.1% of the isolates. Clonal complex (CC) 1 contained 31, predominantly type A isolates from diverse host species. Clonal complex 2 contained 75% of the bovine type E isolates examined in this study. Clonal complex 3 consisted predominantly of porcine type A and type C isolates. Interestingly, these porcine isolates (n=32) all carried consensus cpb2 and cna genes, encoding beta2 toxin and CpCna, a collagen binding protein, respectively. This compares to carriage of both these genes by only 3.6% of porcine isolates not present in clonal complex 3 (n=28). The data obtained indicates that MLST may be used to identify host species relationships with respect to these C. perfringens isolates.     

Multi-locus Sequence Analysis (MLSA) of Edwardsiella tarda isolates from fish

Edwardsiella tarda is an enteric fish pathogen that has caused significant economic losses in a range of fish species residing in diverse ecological conditions. Several molecular methods relying on DNA fingerprinting (RAPD, RFLP and ERIC-PCR) and the gyrB gene marker have been used to characterize E. tarda isolates. However, all had drawbacks in resolving power and reproducibility. The present study was aimed at developing a novel Multi-locus Sequence Analysis (MLSA) scheme for genetic characterization of E. tarda isolates originating from multiple sources. MLSA has been described as an effective molecular tool with superior discriminatory power and reproducibility for exploring intra-species genetic diversity of several bacterial species. Nucleotide sequence fragments of eight protein coding housekeeping genes (gyrB, mdh, adk, dnaK, phoR, metG, pyrG and aroE2) were obtained from 23 fish pathogenic E. tarda isolates of different geographical origins, one human isolate and 3 reference strains. The phylogenetic relationships between isolates in individual gene analyses were not consistent, although some common patterns were apparent. Phylogenetic analysis based on concatenated sequences of seven gene loci, however, buffered the conflicting phylogenetic signals and resolved isolates according to their geographical origin and/or fish host. The MLSA revealed two major genetically diverging clusters in E. tarda isolates examined, one cluster representing isolates from fish and the other representing (in the main) human isolates, with E. ictaluri cluster situated in between. The results suggest, therefore, that the fish pathogenic E. tarda isolates may have been previously misclassified and probably represent one or more as yet unrecognized taxa within the genus Edwardsiella. The MLSA described here was robust enough in discriminating E. tarda isolates not only with respect to their geographical origins but also within different hosts from the same geographical location, high-lighting its potential application in tracing the source of infection and understand the epidemiological relationships among isolates of environmental, fish, other domestic animals or human origins.     

Prevalence of plasmid-mediated quinolone resistance qnr genes in poultry and swine clinical isolates of Escherichia coli

The prevalence of qnr genes was investigated in veterinary clinical isolates of Escherichia coli in Guangdong province, China, and the aac (6')-Ib gene and the mutations in QRDRs of gyrase and topoisomerase IV were examined in qnr-positive strains. A total of 232 E. coli strains isolated from pig and poultry were screened for the presence of the qnrA, qnrB and qnrS genes by PCR and sequencing. The aac (6')-Ib gene was detected in qnr-bearing strains by PCR and sequencing. For all strains carrying qnr, MICs for six quinolones were determined. Mutations within the gyrase and topoisomerase were analyzed by PCR and sequencing for all the QRDRs of gyrA, gyrB, parC and parE. Among 232 E. coli isolates, 14 (6%) isolates were positive for the qnr gene, including one for qnrB, 13 for qnrS, but no qnrA was identified in this population. Detection of the aac (6')-Ib gene showed that one qnrS-positive isolate from pig and one qnrB-positive isolate from duck carried aac (6')-Ib gene, and both were the cr variant allele of aac (6')-Ib. All of the 14 isolates had MICs of ciprofloxacin more than 0.25 mg/L. Mutations in the QRDR of gyrA mutations were observed in 5 (35.7%) of the 14 strains. Three fluoroquinolone-resisting strains showed one mutation S83L of gyrA, while one S83I. One high-level resistance strains harboured gyrA S83L and A87N of gyrA. A singe mutation in site 58 of parC was detected in 3 (21.4%) strains. None mutations were found in QRDRs of gyrB and parE. The emergence of qnr genes in veterinary clinical E. coli isolates is described for the first time. This is also the first report of aac (6')-Ib-cr gene in E. coli isolates from food-producing animals.     

四川白三叶根瘤菌遗传多样性及系统发育研究

为阐明四川部分地区野生白三叶根瘤菌的遗传多样性及系统发育地位,对分离自四川雅安、康定、泸定、西昌、成都和乐山6个地区白三叶根瘤的69株菌进行系统研究。采用16S rDNA限制性片段长度多态性分析(restriction fragment length polymorphism, RFLP)和16S rDNA基因、持家基因(recA、atpD、glnII)、结瘤基因(nodC)、固氮基因(nifH)系统发育分析的方法进行了研究。结果表明,16S rDNA PCR-RFLP中所有供试菌株产生了4种酶切图谱类型,表现出较为丰富的遗传多样性。持家基因与16S rDNA基因系统发育分析结果基本一致,9株代表菌株主要分布在α-变形菌纲(Alpha-Proteobacteria)的根瘤菌属(Rhizobium),并与豌豆根瘤菌三叶草生物型(R. leguminosarum bv. trifolii) ATCC 14480^T的亲缘关系较近。PCR可扩增出nodC和nifH基因片段,但从属于土壤杆菌属(Agrobacterium)的菌株LS1105中则扩增不出这两个基因。所有供试菌株被鉴定到了种的水平,证实了68株为白三叶根瘤菌,并通过不同采样地点菌株之间的比较,发现白三叶与根瘤菌的共生关系因地理分布不同而具有多样性,对于丰富白三叶根瘤菌资源及其开发利用具有重要意义。     

Infectivity, pathogenicity, and genetic characteristics of mammalian gastric Cryptosporidium spp. in domestic ruminants.

Farm ruminants were infected experimentally with four mammalian gastric Cryptosporidium, namely Cryptosporidium andersoni LI03 originated from cattle and three isolates of Cryptosporidium muris from brown rat (isolate RN66), Bactrian camel (isolate CB03) and firstly characterized isolate from East African mole rat (isolate TS03). Sequence characterizations of the small-subunit rRNA gene showed that the LI03 isolate was C. andersoni and the other three isolates belonged to C. muris, although the TS03 isolate showed unique sequence variations (one single nucleotide change and four nucleotide insertions). C. andersoni LI03 was infectious for calves only, whereas lambs and kids were susceptible to C. muris CB03. C. muris TS03 and RN66 were not infectious for any farm ruminants. Infection dynamics including prepatent and patent period and infection intensity of the isolates used differed depending on the host species, but no clinical signs of cryptosporidiosis were observed in any of experimentally infected hosts. Cryptosporidium developmental stages were only detected in infected animals in the abomasum region. Histopathological changes were characterized by dilatation and epithelial metaplasia of infected gastric glands with no significant inflammatory responses in the lamina propria.     

Bacteriological investigation on “Mauro” sold in Catania

Seaweeds known as “Mauro” are traditionally used fresh or to prepare omelettes in Sicily (Italy). Twenty samples sold in Catania between May 2005 and September 2007 were analyzed for Escherichia coli, Total Enterobacteria, Aeromonas spp., Pseudomonas spp., Vibrio spp., and Salmonella spp. Thirty Vibrio strains were examined for the presence of the virulence genes, toxR, toxRS, tl, tdh, and trh in the genomes of the isolates. Total Enterobacteria ranged between 2.23 and 6.85 log CFU/g, and in six samples, E. coli ranged between 0.70 and 2.74 log CFU/g. Aeromonas spp. was present in samples between 1.0 and 5.90 log CFU/g, while Pseudomonas spp. ranged between 2.70 and 7.27 log CFU/g. Vibrio spp. was present in 75% of samples at values between 1.30 and 4.60 log CFU/g. The most frequently isolated species was V. alginolyticus (76.66% of isolates), of which 82.60% were positive for toxR and the remaining 17.40% of strains were positive for toxRS. V. parahaemolyticus was identified in 13.33% of strains; all were positive for toxR and, in one case, for both toxR and toxRS. V. coralliitycus was isolated in 6.66% of strains (all positive for toxR), and one was identified as V. mimicus (positive for toxRS). The results of this study suggest that there is need for stringent quality control during harvesting and distribution of Mauro.

     

Microarray-based genotyping of Staphylococcus aureus isolates from camels

Staphylococcus aureus is a common cause of mastitis and other diseases in camels. In order to obtain data on population structure as well as on the carriage of toxin genes and resistance markers, a collection of 45 isolates from dromedaries of Dubai, United Arab Emirates, were genotyped. These isolates belonged to clonal complexes CC6 (twenty isolates; 44.44%), CC30 (sixteen isolates; 35.56%), CC188 (five isolates; 11.11%), CC152 (1 isolate, 2.2%) and to a previously un-described sequence type (ST1755: arcc-18, aroe-115, glpf-6, gmk-2 pta-109, tpi-50 and yqil-2; three isolates; 6.67%). Resistance genes proved to be rare. Only three out of 45 isolates (6.67%) carried the beta-lactamase operon. The tetracycline resistance gene tetK was also detected in three isolates (6.67%). Neither the mecA gene, defining MRSA, nor other resistance genes were found. Common virulence markers included leukocidin genes lukD+lukE (in twenty-five isolates; 55.56%), the staphylokinase gene sak (twenty-two isolates; 48.89%), the enterotoxin gene cluster egc (fifteen isolates; 33.33%), and a distinct variant of the enterotoxin A gene (sea-320E, GenBank AY196686.1; thirteen isolates; 28.89%). One CC152 isolate was positive for genes encoding the Panton-Valentine leukocidin (lukF-PV+lukS-PV). This study provides first genotyping data on the population structure and the presence of toxin genes and resistance markers of S. aureus strains in Middle Eastern camels.