弗朗西斯菌的分离方法和基因分类研究

发布时间：2018-05-17 14:16

本文选题：弗朗西斯菌属 + 头孢吡肟　；参考：《天津医科大学》2015年硕士论文

【摘要】：目的弗朗西斯菌属为一类革兰染色阴性、营养要求苛刻、无动力、严格需氧性球杆菌,可存在于多种自然环境中。该菌可胞内寄生,对脊椎动物和无脊椎动物均可致病,现已发现大自然界中高达百种以上的动物可感染此菌。土拉热菌病是一种人畜共患性疾病,致死率高,其病原体为属模式菌种土拉热弗朗西斯菌,其传播途径多样、易扩散、毒性强,被美国疾病控制预防中心列为A类生物恐怖制剂之一。现属内有6个有效命名种及6个亚种,除了土拉热弗朗西斯菌以外,研究发现西班牙弗朗西斯菌和蜃楼弗朗西斯菌也与人类疾病有关,但是该菌分离培养困难,导致国内相关调查研究极少。本研究的目的:尝试建立一种可靠、高效的从环境水样中分离弗朗西斯菌的方法,以初步了解我国环境水系中弗朗西斯菌的主要分布菌型,对分离所得菌株运用系统分类学技术分析其系统发育地位,进而命名发现的新种。方法弗朗西斯菌的药物敏感性以及自制选择性培养基的效果研究复苏实验室保存的蜃楼弗朗西斯菌、广州弗朗西斯菌和嗜肺军团菌标准菌株,选用头孢吡肟抗生素纸片对三种菌株进行药物敏感性试验(K-B法),依据CLSI M100-A24药敏试验解释标准进行对比分析。复苏实验室保存的其他标准菌株,调配菌悬液,记录麦氏浊,同时涂布于哥伦比亚血平板、BCYEα-GVPCC培养基和CHAB-PACCV培养基孵育培养,对比分析三种培养基上菌落生长情况,验证自制培养基的选择性。蜃楼弗朗西斯菌、广州弗朗西斯菌两株标准菌株酸抵抗性研究借鉴军团菌分离流程里的酸处理操作对两标准菌株进行酸耐受强度试验,测定在恒定p H=2.2的KCL-HCL缓冲液下不同培养基上不同时间点的菌落数目,绘制生存曲线,找出生存率明显改变的时间点,确定水样的酸处理时间。培养基的分离效果验证研究运用两标准菌株自制模拟水样,加抽滤浓缩、酸处理因素后BCYEα-GVPCC培养基和CHAB-PACCV培养基,观察是否能成功分离到目的菌株。采集环境水样,经抽滤浓缩、酸处理后涂布接种BCYEα-GVPCC培养基CHAB-PACCV培养基,观察是否能成功分离到目的菌株,保存分离菌株。对19株分离株进行16S r DNA、sdh A、mdh、rpo B基因测序分析,构建系统发育树,对菌株鉴定和分群,发现疑似新种,结合水质和地理信息探讨我国弗朗西斯菌多样性。运用DNA-DNA杂交技术对疑似新种进行DNA-DNA相关性分析,以确证发现的新种。结果头孢吡肟敏感性:蜃楼弗朗西斯菌和广州弗朗西斯菌耐药,嗜肺军团菌敏感。的对头孢吡肟的抑菌圈分别是6mm、6mm、38mm,查阅CLSI M100-A24中收录的所有有关头孢吡肟的解释标准后发现,前二者对头孢吡肟为耐药,嗜肺军团菌为敏感。两种培养基上基本无杂菌生长或有极少量存在。BCYEα-GVPCC和CHAB-PACCV两种培养基上蜃楼弗兰西斯菌和广州弗朗西斯菌在p H=2.2的酸性条件下0-20min内生存率变化轻微,20min以后其生存率显著降低。BCYEα-GVPCC和CHAB-PACCV两种培养基均能自模拟水样和环境水样中成功分离到目的菌,弗朗西斯菌于前者为灰白湿润圆形较小菌落,杂菌生长较小且少;于后者为灰绿色圆形凸起稍大菌落,非目的菌生长较多。自环境水样中共分离得到目的菌株19株,保存于-80℃冰箱。16S rRNA、sdhA、mdh、rpo B系统发育分析表明发现19株分离株主要以蜃楼弗朗西斯菌为主(57.90%,11/19),疑似新种次之(36.84%,7/19),疑似弗朗西斯菌科候选属(5.26%,1/19);主要分布于海水中(47.37%,9/19),中央空调水次之(26.32%,5/19),咸水为(21.05%,4/19),淡水分布极少(5.26%,1/19)。5.10HL1970,10HP457,10HP82-10,10HL1938与08HL01032菌株的杂交率分别为25.59%,29.26%,25.61%,31.54%.结论弗朗西斯菌对头孢吡肟耐药,对酸有一定的抵抗性,头孢吡肟和酸处理可用以祛除杂菌干扰,提高弗朗西斯菌的检出率。弗朗西斯菌在BCYEα-GVPCC与CHAB-PACCV均可生长,酸处理、抽滤水样的前处理可有效用于环境水样中弗朗西斯菌的分离培养。本次研究分离的弗朗西斯菌主要以蜃楼弗朗西斯菌为主,其次为大量的潜在新种,多样性程度较高,有潜在爆发流行的趋势。10HP457、10HP82-10、10HL1970、10HL1938四株经DNA-DNA杂交试验确证为弗朗西斯新种,结合其水样信息将其暂命名为冷却水弗朗西斯菌,模式种暂定为10HL1970T。
[Abstract]:Objective the genus Francis is a class of gram-negative Gram-negative bacteria, which has severe nutritional requirements, no power and strict Bacillus aerobic, which can exist in a variety of natural environments. The bacteria can parasitism in vertebrates and invertebrates. It has been found that more than 100 species of animals in the natural world can infect the bacteria. There are 6 effective nomenclature and 6 subspecies of the disease control and prevention center of the American Center for Disease Control and prevention. There are 6 effective nomenclature species and 6 subspecies. In addition to the fever of Francis, the research has been found. Francis Rand and mirage Francis Rand in Spain are also related to human diseases, but the isolation and culture of the bacteria is difficult, which leads to few domestic related investigations. The purpose of this study is to establish a reliable, efficient method for separating Francis Rand from environmental water samples to understand Francis in our environmental water system. Mainly distributed bacteria type, the phylogenetic status of the isolated strains was analyzed by systematic taxonomy, and the new species were named. The drug sensitivity of Francis bacteria and the effect of self-made selective medium were used to study the mirage of Francis, Francis and Legionella pneumophila in the resuscitation laboratory. The strain, drug sensitivity test (K-B method) was performed on three strains of cefepime antibiotic (cefepime), according to the standard of CLSI M100-A24 drug sensitivity test. Other standard strains were preserved in the resuscitation laboratory, and the bacteria suspension was used to record the malt, and it was coated on the Columbia blood plate, BCYE a -GVPCC medium and CHAB-PACCV. Culture medium incubation, comparison and analysis of the growth of colonies on the three medium, verifying the selectivity of the self-made medium. The acid resistance of two standard mirage Francis bacteria and two standard strains of Guangzhou Francis strain for reference to the acid treatment operation of the Legionella isolation process for two standard strains, and the determination of the constant P H=2. The number of colonies at different time points on different medium of 2 KCL-HCL buffer solution, draw the survival curve, find the time point that the birth rate is obviously changed, determine the acid treatment time of the water sample. The isolation effect of the medium is verified by the use of the two standard strains to simulate water samples, and the BCYE alpha -GVPCC medium and CHA after the acid treatment factors. B-PACCV culture medium, observe whether can successfully isolate the target strain. Collect environmental water samples, through the filtration concentration, acid treatment, coated with BCYE alpha -GVPCC culture medium CHAB-PACCV medium, observe whether the isolate can be successfully isolated to the target strain and preserve the isolated strains. 19 isolates were sequenced, 16S R DNA, SDH A, MDH, RPO B genes were sequenced and analyzed. Phylogeny tree, identification and grouping of strains, discovery of suspected new species, combined with water quality and geographical information to explore the diversity of Francis bacteria in China. Using DNA-DNA hybridization technique to analyze the suspected new species by DNA-DNA correlation analysis, to confirm the new species found. Results of cefepime sensitivity: mirage Francis Rand and Francis resistant Guangzhou, drug resistance, eosinophilia, The Bacteriostasis of Legionella pneumonii was sensitive. The Bacteriostasis of cefepime was 6mm, 6mm, 38mm, and all the related cefepime interpretation criteria included in CLSI M100-A24 found that the first two were resistant to cefepime and were sensitive to Legionella pneumophila. Two kinds of culture medium were basically no heterozygous growth or a very small amount of.BCYE alpha -GVPCC and CHAB-PACCV two Under the acid condition of P H=2.2, the survival rate of Francis bacteria and Francis bacteria in P H=2.2 was slight. After 20min, the survival rate of.BCYE alpha -GVPCC and CHAB-PACCV could be significantly reduced by two cultures of.BCYE and CHAB-PACCV, and the former was gray and humid in the former. Small round colonies, small and small heterozygous growth, the latter was a gray green circular bulge and a little larger colony, and the non purpose bacteria grew more. 19 strains of the target strains were isolated from the environmental water samples. The phylogenetic analysis of.16S rRNA, sdhA, MDH and RPO B in the refrigerator at -80 C showed that the main 19 isolates were mainly mirage Francis bacteria (57.90%, 11/19), a suspected new species (36.84%, 7/19), suspected to be a candidate for the Francis family (5.26%, 1/19); mainly distributed in sea water (47.37%, 9/19), central air conditioning water (26.32%, 5/19), salt water (21.05%, 4/19), rare fresh water (5.26%, 1 /19).5.10HL1970,10HP457,10HP82-10,10HL1938 and 08HL01032 strains 25.59%, 29.2, respectively, 29.2. 6%, 25.61%, 31.54%. concluded that Francis bacteria were resistant to cefepime and had a certain resistance to acid. Cefepime and acid treatment could be used to remove the interference from the heterozygous bacteria and improve the detection rate of Francis bacteria. Francis bacteria can grow in BCYE alpha -GVPCC and CHAB-PACCV. Acid treatment, and the pretreatment of filtered water samples can be used effectively in the environmental water samples. The separation and culture of Francis Francis Rand, the main separation of Francis Rand mainly with mirage Francis bacteria, followed by a large number of potential new species, high degree of diversity, there is a potential outbreak of epidemic trend.10HP457,10HP82-10,10HL1970,10HL1938 four strains confirmed by DNA-DNA hybridization test as a new species of Francis, combined with its water sample information It was tentatively named as Francis 10HL1970T. in cooling water.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

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