本文選題：弗朗西斯菌屬 + 頭孢吡肟��； 參考：《天津醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的弗朗西斯菌屬為一類(lèi)革蘭染色陰性、營(yíng)養(yǎng)要求苛刻、無(wú)動(dòng)力、嚴(yán)格需氧性球桿菌,可存在于多種自然環(huán)境中。該菌可胞內(nèi)寄生,對(duì)脊椎動(dòng)物和無(wú)脊椎動(dòng)物均可致病,現(xiàn)已發(fā)現(xiàn)大自然界中高達(dá)百種以上的動(dòng)物可感染此菌。土拉熱菌病是一種人畜共患性疾病,致死率高,其病原體為屬模式菌種土拉熱弗朗西斯菌,其傳播途徑多樣、易擴(kuò)散、毒性強(qiáng),被美國(guó)疾病控制預(yù)防中心列為A類(lèi)生物恐怖制劑之一�，F(xiàn)屬內(nèi)有6個(gè)有效命名種及6個(gè)亞種,除了土拉熱弗朗西斯菌以外,研究發(fā)現(xiàn)西班牙弗朗西斯菌和蜃樓弗朗西斯菌也與人類(lèi)疾病有關(guān),但是該菌分離培養(yǎng)困難,導(dǎo)致國(guó)內(nèi)相關(guān)調(diào)查研究極少。本研究的目的:嘗試建立一種可靠、高效的從環(huán)境水樣中分離弗朗西斯菌的方法,以初步了解我國(guó)環(huán)境水系中弗朗西斯菌的主要分布菌型,對(duì)分離所得菌株運(yùn)用系統(tǒng)分類(lèi)學(xué)技術(shù)分析其系統(tǒng)發(fā)育地位,進(jìn)而命名發(fā)現(xiàn)的新種。方法弗朗西斯菌的藥物敏感性以及自制選擇性培養(yǎng)基的效果研究復(fù)蘇實(shí)驗(yàn)室保存的蜃樓弗朗西斯菌、廣州弗朗西斯菌和嗜肺軍團(tuán)菌標(biāo)準(zhǔn)菌株,選用頭孢吡肟抗生素紙片對(duì)三種菌株進(jìn)行藥物敏感性試驗(yàn)(K-B法),依據(jù)CLSI M100-A24藥敏試驗(yàn)解釋標(biāo)準(zhǔn)進(jìn)行對(duì)比分析。復(fù)蘇實(shí)驗(yàn)室保存的其他標(biāo)準(zhǔn)菌株,調(diào)配菌懸液,記錄麥?zhǔn)蠞?同時(shí)涂布于哥倫比亞血平板、BCYEα-GVPCC培養(yǎng)基和CHAB-PACCV培養(yǎng)基孵育培養(yǎng),對(duì)比分析三種培養(yǎng)基上菌落生長(zhǎng)情況,驗(yàn)證自制培養(yǎng)基的選擇性。蜃樓弗朗西斯菌、廣州弗朗西斯菌兩株標(biāo)準(zhǔn)菌株酸抵抗性研究借鑒軍團(tuán)菌分離流程里的酸處理操作對(duì)兩標(biāo)準(zhǔn)菌株進(jìn)行酸耐受強(qiáng)度試驗(yàn),測(cè)定在恒定p H=2.2的KCL-HCL緩沖液下不同培養(yǎng)基上不同時(shí)間點(diǎn)的菌落數(shù)目,繪制生存曲線,找出生存率明顯改變的時(shí)間點(diǎn),確定水樣的酸處理時(shí)間。培養(yǎng)基的分離效果驗(yàn)證研究運(yùn)用兩標(biāo)準(zhǔn)菌株自制模擬水樣,加抽濾濃縮、酸處理因素后BCYEα-GVPCC培養(yǎng)基和CHAB-PACCV培養(yǎng)基,觀察是否能成功分離到目的菌株。采集環(huán)境水樣,經(jīng)抽濾濃縮、酸處理后涂布接種BCYEα-GVPCC培養(yǎng)基CHAB-PACCV培養(yǎng)基,觀察是否能成功分離到目的菌株,保存分離菌株。對(duì)19株分離株進(jìn)行16S r DNA、sdh A、mdh、rpo B基因測(cè)序分析,構(gòu)建系統(tǒng)發(fā)育樹(shù),對(duì)菌株鑒定和分群,發(fā)現(xiàn)疑似新種,結(jié)合水質(zhì)和地理信息探討我國(guó)弗朗西斯菌多樣性。運(yùn)用DNA-DNA雜交技術(shù)對(duì)疑似新種進(jìn)行DNA-DNA相關(guān)性分析,以確證發(fā)現(xiàn)的新種。結(jié)果頭孢吡肟敏感性:蜃樓弗朗西斯菌和廣州弗朗西斯菌耐藥,嗜肺軍團(tuán)菌敏感。的對(duì)頭孢吡肟的抑菌圈分別是6mm、6mm、38mm,查閱CLSI M100-A24中收錄的所有有關(guān)頭孢吡肟的解釋標(biāo)準(zhǔn)后發(fā)現(xiàn),前二者對(duì)頭孢吡肟為耐藥,嗜肺軍團(tuán)菌為敏感。兩種培養(yǎng)基上基本無(wú)雜菌生長(zhǎng)或有極少量存在。BCYEα-GVPCC和CHAB-PACCV兩種培養(yǎng)基上蜃樓弗蘭西斯菌和廣州弗朗西斯菌在p H=2.2的酸性條件下0-20min內(nèi)生存率變化輕微,20min以后其生存率顯著降低。BCYEα-GVPCC和CHAB-PACCV兩種培養(yǎng)基均能自模擬水樣和環(huán)境水樣中成功分離到目的菌,弗朗西斯菌于前者為灰白濕潤(rùn)圓形較小菌落,雜菌生長(zhǎng)較小且少;于后者為灰綠色圓形凸起稍大菌落,非目的菌生長(zhǎng)較多。自環(huán)境水樣中共分離得到目的菌株19株,保存于-80℃冰箱。16S rRNA、sdhA、mdh、rpo B系統(tǒng)發(fā)育分析表明發(fā)現(xiàn)19株分離株主要以蜃樓弗朗西斯菌為主(57.90%,11/19),疑似新種次之(36.84%,7/19),疑似弗朗西斯菌科候選屬(5.26%,1/19);主要分布于海水中(47.37%,9/19),中央空調(diào)水次之(26.32%,5/19),咸水為(21.05%,4/19),淡水分布極少(5.26%,1/19)。5.10HL1970,10HP457,10HP82-10,10HL1938與08HL01032菌株的雜交率分別為25.59%,29.26%,25.61%,31.54%.結(jié)論弗朗西斯菌對(duì)頭孢吡肟耐藥,對(duì)酸有一定的抵抗性,頭孢吡肟和酸處理可用以祛除雜菌干擾,提高弗朗西斯菌的檢出率。弗朗西斯菌在BCYEα-GVPCC與CHAB-PACCV均可生長(zhǎng),酸處理、抽濾水樣的前處理可有效用于環(huán)境水樣中弗朗西斯菌的分離培養(yǎng)。本次研究分離的弗朗西斯菌主要以蜃樓弗朗西斯菌為主,其次為大量的潛在新種,多樣性程度較高,有潛在爆發(fā)流行的趨勢(shì)。10HP457、10HP82-10、10HL1970、10HL1938四株經(jīng)DNA-DNA雜交試驗(yàn)確證為弗朗西斯新種,結(jié)合其水樣信息將其暫命名為冷卻水弗朗西斯菌,模式種暫定為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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R446.5

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