光唇鱼源无乳链球菌的分离鉴定及遗传特征

【目的】为查明浙江养殖光唇鱼大量死亡的病原,了解病原的遗传特征。【方法】本工作对患病光唇鱼进行病原分离,结合形态特征、生理生化特性和16S rRNA基因序列同源性,对分离菌株进行鉴定;采用人工回感试验确定其病原性,并对分离株的血清型、多位点序列分型(multilocus sequence typing,MLST)、毒力基因型和表面蛋白抗原基因型等遗传特征进行分析;此外,还测试了菌株的药敏特性。【结果】从患病光唇鱼体中分离得到优势菌株ACRO-0708,为革兰氏阳性球菌,不溶血,分子与生化鉴定为无乳链球菌(Streptococcus agalactiae);人工感染试验证实其对光唇鱼有较强的致病性,LD50为6.47×10~3CFU/g,属于血清型Ⅰb和MLST型ST261,毒力基因型为sip~+bibA~+cfb~+hylB~+iagA~+fbsA~+fbsB~+bac~–bca~–cylE~–scpB~–lmb~–,不携带所检测的6种表面蛋白基因。药敏试验结果显示,对青霉素、氨苄西林等8种药物较敏感,对氯霉素、复方新诺明等7种药物耐药。【结论】引起浙江养殖光唇鱼死亡的病原菌为无乳链球菌,其分子特征与水产动物主要流行的无乳链球菌株具有显著差异,生产中可选用氨苄西林、氟苯尼考等药物进行防治。     

病原微生物基因多位点序列分型的研究进展

多位点序列分型(Multilocus sequence typing,MLST)是近年来发展很快的分子生物学分析方法,具有很高的分辨能力,既适于分子流行病学研究,也可用于分子进化学的研究。通过分析多个管家基因(Housekeeping gene)450bp左右的核心片段的核酸序列,从而对菌株的等位基因进行多样性的比较,不同的菌株对应不同的序列型(Se-quence type)。所以MLST越来越多的被作为能进行国际间菌株比较的常用工具,建立一种更为准确的分型系统方法。并且应用于研究出现的不同的抗生素抵抗株,毒力或抗原的相关特殊基因型,及新的变异株引起的疾病流行等流行病学分析,进行生物进化和种群结构的研究。     

Genetic Diversity and Population Structure of the Xanthomonas campestris pv. campestris Strains Affecting Cabbages in China Revealed by MLST and Rep-PCR Based Genotyping

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot for cruciferous vegetables worldwide, especially for the cole crops such as cabbage and cauliflower. Due to the lack of resistant cabbage cultivars, black rot has brought about considerable yield losses in recent years in China. Understanding of the pathogen features is a key step for disease prevention, however, the pathogen diversity, population structure, and virulence are largely unknown. In this study, we studied 50 Xcc strains including 39 Xcc isolates collected from cabbage in 20 regions across China, using multilocus sequence genotyping (MLST), repetitive DNA sequence-based PCR (rep-PCR), and pathogenicity tests. For MLST analysis, a total of 12 allelic profiles (AP) were generated, among which the largest AP was AP1 containing 32 strains. Further cluster analysis of rep-PCR divided all strains into 14 DNA groups, with the largest group DNA I comprising of 34 strains, most of which also belonged to AP1. Inoculation tests showed that the representative Xcc strains collected from diverse regions performed differential virulence against three brassica hosts compared with races 1 and 4. Interestingly, these results indicated that AP1/DNA I was not only the main pathotype in China, but also a novel group that differed from the previously reported type races in both genotype and virulence. To our knowledge, this is the first extensive genetic diversity survey for Xcc strains in China, which provides evidence for cabbage resistance breeding and opens the gate for further cabbage-Xcc interaction studies.     

Wolbachia-mediated persistence of mtDNA from a potentially extinct species

Drosophila quinaria is polymorphic for infection with Wolbachia, a maternally transmitted endosymbiont. Wolbachia-infected individuals carry mtDNA that is only distantly related to the mtDNA of uninfected individuals, and the clade encompassing all mtDNA haplotypes within D. quinaria also includes the mtDNA of several other species of Drosophila. Nuclear gene variation reveals no difference between the Wolbachia-infected and uninfected individuals of D. quinaria, indicating that they all belong to the same interbreeding biological species. We suggest that the Wolbachia and the mtDNA with which it is associated were derived via interspecific hybridization and introgression. The sequences in the Wolbachia and the associated mtDNA are ≥6% divergent from those of any known Drosophila species. Thus, in spite of nearly complete species sampling, the sequences from which these mitochondria were derived remain unknown, raising the possibility that the donor species is extinct. The association between Wolbachia infection and mtDNA type within D. quinaria suggests that Wolbachia may be required for the continued persistence of the mtDNA from an otherwise extinct Drosophila species. We hypothesize that pathogen-protective effects conferred by Wolbachia operate in a negative frequency-dependent manner, thus bringing about a stable polymorphism for Wolbachia infection.     

Typing of Campylobacter jejuni and Campylobacter coli isolated from live broilers and retail broiler meat by flaA-RFLP, MLST, PFGE and REP-PCR

We analyzed 100 Campylobacter spp. isolates (C. jejuni and C. coli) from Grenada, Puerto Rico and Alabama, which were collected from live broilers or retail broiler meat. We analyzed these isolates with four molecular typing methods: restriction fragment length polymorphism of the flaA gene (flaA-RFLP), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and automated repetitive extragenic palindromic polymerase chain reaction (REP-PCR) using the DiversiLab system. All methods performed similarly for the typing of C. jejuni and C. coli. The DNA extraction method appears to influence the results obtained with REP-PCR. This method was better for the typing of C. jejuni than C. coli, however both REP-PCR and flaA-RFLP generated types that were indistinguishable between C. jejuni and C. coli and appeared to be random, without any relationship to species, location, or source of isolates. PFGE and MLST generated typing results that had a better correlation with the geographic location of the isolates and showed higher concordance with the Wallace coefficient. The adjusted Rand coefficient did not show higher concordance among the methods, although the PFGE/MLST combination exhibited the highest concordance. PFGE and MLST revealed a better discriminatory power for C. coli isolates than REP-PCR or flaA-RFLP. The use of readily available online tools to calculate the confidence interval of the Simpson's index of diversity and the adjusted Rand and Wallace coefficients helped estimate the discriminatory power of typing methods. Further studies using different C. jejuni and C. coli strains may expand our understanding of the benefits and limitations of each of these typing methods for epidemiological studies of Campylobacter spp.     

47株空肠弯曲菌湖北禽源株的多位点序列分型

【目的】为了解湖北地区家禽空肠弯曲菌的流行状况及其分子特征,应用多位点序列分型方法对2013–2014年的47株禽源空肠弯曲菌湖北分离株进行分子分型研究。【方法】以空肠弯曲菌的7个管家基因aspA、glnA、gltA、glyA、pgm、tkt和uncA为目的基因,提取样本基因组后PCR扩增,测序和分析。将测序结果上传数据库进行比对,制作成多位点序列分型(Multilocus sequence typing,MLST)遗传进化树。【结果】分离株共有38个ST型,10个克隆群,其中最多的克隆群为ST-353CC和ST-464CC,发现2个新的等位基因编号和25个新的ST型。遗传进化树显示,不同家禽宿主中空肠弯曲菌序列型存在一定的差异,不同地区和来源的空肠弯曲菌呈现出遗传多样性。【结论】本研究对湖北分离的47株禽源空肠弯曲菌进行了MLST分析,其结果显示菌株多样性较为丰富,将为我国家禽空肠弯曲菌的流行病学调查提供科学的数据。     

Evolution of pathogenicity in the Bacillus cereus group

The Bacillus cereus group of bacteria comprises soil-dwelling saprophytes but on occasion these bacteria can cause a wide range of diseases in humans, including food poisoning, systemic infections and highly lethal forms of anthrax. While anthrax is almost invariably caused by strains from a single evolutionary lineage, Bacillus anthracis, variation in the virulence properties of strains from other lineages has not been fully addressed. Using multi-locus sequence data from 667 strains, we reconstructed the evolutionary history of the B. cereus group in terms of both clonal inheritance and recombination. The strains included 155 clinical isolates representing B. anthracis, and isolates from emetic and diarrhoeal food poisoning, septicaemia and related infections, wound, and lung infections. We confirmed the existence of three major clades and found that clinical isolates of B. cereus (with the exception of emetic toxin-producing strains) are evenly distributed between and within clades 1 and 2. B. anthracis in particular and emetic toxin-producing B. cereus show more clonal structure and are restricted to clade 1. Our characterization of the patterns of genetic exchange showed that there exist partial barriers to gene flow between the three clades. The pathogenic strains do not exhibit atypically high or low rates of recombination, consistent with the opportunistic nature of most pathogenic infections. However, there have been a large number of recent imports in clade 1 of strains from external origins, which is indicative of an on-going shift in gene-flow boundaries for this clade.     

多位点序列分型分析空肠弯曲菌华东动物源分离株

【目的】研究空肠弯曲菌菌株间的分子特征,对不同宿主来源的空肠弯曲菌进行分子分型研究。【方法】选择空肠弯曲菌的7个看家基因gltA、aspA、glnA、glyA、pgm、tkt和uncA作为目的基因,对2006-2008年间华东地区分离的42株空肠弯曲菌样本进行PCR扩增后测序。将测序结果软件分析并上传到数据库进行比对,将结果制作多位点序列分型(multilocus sequence typing,MLST)遗传进化树并进行分析。【结果】与数据库已有类型比对,发现了24个新的ST型,通过进化树得到其遗传关系。【结论】MLST方法对于研究空肠弯曲菌的菌株群体基因差异与进化趋势具有重要意义。     

日化产品中洋葱伯克霍尔德氏菌复合群(Bcc)的分类和神秘伯克霍尔德氏菌的耐药性研究

【目的】对从2020–2022年不同日化产品中分离的29株洋葱伯克霍尔德氏菌复合群(Burkholderia cepacia complex,Bcc)进行分类和分型,另将2020年前来源于日化产品中6株被鉴定为Burkholderia lata的菌株进行分类更正。探究神秘伯克霍尔德氏菌(Burkholderia aenigmatica)的耐药性。【方法】本文主要应用多位点分型研究方法(multilocus sequence typing,MLST),PCR扩增atpD、gltB、gyrB、recA、lepA、phaC和trp B 7个管家基因片段,将测序结果与MLST数据库中的数据比对分析,获得菌株各管家基因的编号和ST型(sequence type),对本检测中心分离自日化产品的Bcc进行分型;利用多位点序列分析(multilocus sequence analysis,MLSA),结合MLST中等位基因的核苷酸序列构建进化树,从而对Bcc进行系统发育分析和鉴定。利用最小抑菌浓度法(minimum inhibitory concentration,MIC)测定Bcc对常见防腐剂(1,...