本文選題：解脲脲原體 + 淋球菌　； 參考：《東南大學(xué)》2006年碩士論文

【摘要】： 目的:(1)調(diào)查江蘇省局部地區(qū)解脲脲原體的耐藥狀況并提出用藥建議;(2)研究江蘇省局部地區(qū)解脲脲原體的耐藥機(jī)制(包括對四環(huán)素類、氟喹諾酮類和大環(huán)內(nèi)酯類藥物);(3)根據(jù)解脲脲原體兩個(gè)不同基因型的基因組差別建立和評價(jià)新的基因分型方法,并進(jìn)行初步的臨床應(yīng)用;(4)研究江蘇省淋球菌對氟喹諾酮類藥物的耐藥機(jī)制。 研究方法:(1)用法國梅里埃支原體鑒定試劑盒進(jìn)行解脲脲原體(Uu)與人型支原體(Mh)的初步分離和鑒定,并對解脲脲原體培養(yǎng)陽性物進(jìn)一步經(jīng)過濾培養(yǎng)和特異性PCR試驗(yàn)進(jìn)行確認(rèn)。(2)應(yīng)用微量肉湯稀釋法檢測八種抗生素(四環(huán)素、米諾環(huán)素、紅霉素、克拉霉素、阿齊霉素、環(huán)丙沙星、氧氟沙星和左旋氧氟沙星)對確認(rèn)的84株解脲脲原體的最小抑菌濃度(Minimum inhibitory concentration,MIC)。(3)按藥敏結(jié)果分層,從84株解脲脲原體菌株中隨機(jī)抽取34株,用PCR擴(kuò)增四環(huán)素耐藥基因tetM;隨機(jī)抽取28株,采用PCR擴(kuò)增氟喹諾酮耐藥基因gyrA的氟喹諾酮耐藥決定區(qū)(QRDR),并對擴(kuò)增產(chǎn)物進(jìn)行測序分析,采用Blast比對法比較臨床菌株序列與標(biāo)準(zhǔn)菌株序列的差異。(4)基于解脲脲原體14個(gè)血清型的23S rRNA基因序列的差異,采用primer premier5.0軟件設(shè)計(jì)PCR引物和選取限制性內(nèi)切酶,自行建立解脲脲原體的基因分型方法,通過與標(biāo)準(zhǔn)菌株和“金標(biāo)準(zhǔn)”鑒定的臨床菌株比較,對其進(jìn)行評價(jià)并初步應(yīng)用于臨床菌株進(jìn)行基因分型和分析。(5)根據(jù)淋球菌藥敏結(jié)果,從已經(jīng)鑒定為淋球菌的95株菌株中,分層隨機(jī)抽取54株,采用PCR擴(kuò)增氟喹諾酮耐藥基因gyrA和parC的QRDR,并進(jìn)行DNA測序,分析其突變位點(diǎn)與藥敏結(jié)果MIC的相關(guān)關(guān)系。(6)結(jié)合實(shí)驗(yàn)結(jié)果和流行病學(xué)調(diào)查資料,采用卡方檢驗(yàn)、方差分析、秩和檢驗(yàn)和精確概率法對數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析。 研究結(jié)果:(1)解脲脲原體對米諾環(huán)素、四環(huán)素、克拉霉素、阿齊霉素、紅霉素、環(huán)丙沙星、氧氟沙星和左旋氧氟沙星的MIC50(50%的菌株被抑制的濃度)分別為0.0625、0.125、0.25、1、2、8、4和2mg/L;MIC90(90%的菌株被抑制的濃度)分別為0.125、1、1、4、8、64、16和8mg/L。(2)大環(huán)內(nèi)酯類抗生素(克拉霉素、阿齊霉素和紅霉素)的耐藥水平(包括MIC幾何均數(shù)和耐藥率)在有Mh合并感染組與無Mh合并感染組間有統(tǒng)計(jì)學(xué)差異(P0.05),伴隨Mh感染的解脲脲原體對大環(huán)內(nèi)酯類抗生素的MIC水平和耐藥率都較高。(3)在34株非四環(huán)素耐藥的解脲脲原體菌株中,有7株檢測到tetM基因。tetM基因陽性組的四環(huán)素和米諾環(huán)素的MIC水平都比tetM陰性組高,且有統(tǒng)計(jì)學(xué)意義(四環(huán)素:t=-4.34,P=0.0001;米諾環(huán)素:t=-5.90,P0.0001)。在28株解脲脲原體菌株中,經(jīng)測序分析發(fā)現(xiàn)gyrA基因出現(xiàn)三種突變形式:①302位堿基C→T的突變,導(dǎo)致101位(相當(dāng)于大腸桿菌84位)丙氨酸改變?yōu)槔i氨酸;②336位堿基C→A的突變,導(dǎo)致112位(相當(dāng)于大腸桿菌95位)天冬氨酸改變?yōu)楣劝彼?③267位堿基T的缺失。發(fā)生gyrA基因突變的主要是耐藥菌株和中度敏感菌株,在敏感菌株沒有發(fā)現(xiàn)gyrA基因突變;所有有基因改變的菌株其氟喹諾酮類藥物的MIC水平明顯高于沒有突變的菌株組。(4)在標(biāo)準(zhǔn)菌株中,PCR-RFLP(PCR為基礎(chǔ)的限制性酶切多態(tài)性分析)方法分型能力為100%,同時(shí)分型結(jié)果有較好的特異性和可重復(fù)性,電泳圖譜清晰易于分辨和解釋。在通過“金標(biāo)準(zhǔn)”鑒定的臨床菌株中,該分型方法的靈敏度為97%,特異度為89%,重復(fù)試驗(yàn)一致率為98%。PCR陽性擴(kuò)增模板兩次獨(dú)立分型實(shí)驗(yàn)的一致率為90.62%;PCR-RFLP分型結(jié)果與測序分型結(jié)果的一致率為100%。(5)有生殖道感染病史和合并Mh感染的解脲脲原體基因型主要表現(xiàn)為生物1型,而合并霉菌感染的解脲脲原體主要為混合型。不同型別的解脲脲原體對四環(huán)素類、大環(huán)內(nèi)酯類和氟喹諾酮類藥物的敏感性存在統(tǒng)計(jì)學(xué)差異,其MIC的平均水平均表現(xiàn)為T960型高于生物1型。解脲脲原體基因型與氟喹諾酮類藥物耐藥相關(guān)的gyrA基因的氨基酸改變有較強(qiáng)的相關(guān)性,出現(xiàn)氨基酸Asp95Glu改變的菌株均屬于T960型,而無此改變的菌株均不屬于T960型。(6)分離的95株淋球菌對環(huán)丙沙星100%的耐藥;通過測序分析,在54株淋球菌中檢測到gyrA和parC的18種突變形式。環(huán)丙沙星的MIC水平在不同parC基因突變組之間差異有統(tǒng)計(jì)學(xué)意義(P=0.006),parC基因突變位點(diǎn)越少其MIC相對較低,突變位點(diǎn)越多其MIC相對較高。 研究結(jié)論:(1)在江蘇局部地區(qū)第三代以前的氟喹諾酮類藥物和紅霉素MIC水平較高,耐藥嚴(yán)重,此類藥物不宜作為治療解脲脲原體感染的一線藥物,而四環(huán)素類藥物可以重新作為治療解脲脲原體感染的一
[Abstract]:Objective: (1) the status of drug resistance investigation of Ureaplasma urealyticum in some areas of Jiangsu province and put forward recommendations; (2) the resistant mechanisms in some areas of Jiangsu province of Ureaplasma urealyticum (including tetracyclines, fluoroquinolones and macrolides); (3) according to the genomic difference of Ureaplasma urealyticum in two different genes the establishment and evaluation of a new genotyping method and preliminary clinical application; (4) the mechanism of fluoroquinolone resistance of Neisseria gonorrhoeae in Jiangsu province.
Research methods: (1) Ureaplasma urealyticum Mycoplasma in France bioMerieux identification Kit (Uu) and human Mycoplasma (Mh) preliminary isolation and identification of Ureaplasma urealyticum, and positive culture further filtered culture and specificity of PCR test were confirmed. (2) using broth dilution method to detect eight kinds of antibiotics (tetracycline, minocycline, erythromycin, clarithromycin, azithromycin, ciprofloxacin, ofloxacin and levofloxacin) the minimum inhibitory concentration of 84 strains of Ureaplasma urealyticum (Minimum inhibitory confirmed by concentration, MIC). (3) stratified by drug sensitivity test, were randomly selected from 84 strains of Ureaplasma urealyticum strains in 34 strains. Amplification of tetracycline resistant gene tetM with PCR; randomly selected 28 strains of fluoroquinolone resistance by PCR amplification of fluoroquinolone resistance gene gyrA (QRDR), and determine the area of amplified products were sequenced by Blast analysis, comparison method comparison The difference of clinical strains and standard strains sequence sequence. (4) the difference of 23S rRNA gene sequence of Ureaplasma urealyticum serotype 14 based on primer, using premier5.0 software to design PCR primers and selected restriction enzymes, to establish Ureaplasma urealyticum genotyping method through the comparison of clinical strains and standard strains and gold the standard "identification, evaluate and preliminary clinical application of strain type and gene analysis. (5) according to the results of drug sensitivity of Neisseria gonorrhoeae, from have already identified 95 strains of Neisseria gonorrhoeae, were randomly selected from 54 strains of fluoroquinolone resistance and gyrA gene amplification by parC with PCR and QRDR. The sequence of DNA, to explore the relationship between the mutation and susceptibility of MIC. (6) according to the experimental results and epidemiological data, using the chi square test, analysis of variance, statistical data and rank test and exact probability method Analysis.
Results: (1) of Ureaplasma urealyticum to tetracycline, minocycline, clarithromycin, azithromycin, erythromycin, ciprofloxacin, ofloxacin and levofloxacin (MIC50 concentration of 50% strains was inhibited by 0.0625,0.125,0.25,1,2,8,4 and 2mg/L respectively); MIC90 (concentration of 90% strains inhibited) were 0.125,1,1,4,8,64,16 and 8mg/L. (2) macrolide antibiotics (clarithromycin, azithromycin and erythromycin resistance (MIC) level including geometric mean and resistance rate) in Mh infection group and Mh infection group had statistically significant difference (P0.05), with Mh of Ureaplasma urealyticum infection of macrolide antibiotics the level of MIC and the resistance rate is higher. (3) in Ureaplasma urealyticum strains 34 strains of non tetracycline resistance in 7 strains were detected tetM gene.TetM gene positive group of tetracycline and minocycline MIC levels than tetM negative group, and There was statistical significance (t=-4.34, P=0.0001; tetracycline, minocycline: t=-5.90, P0.0001). In the 28 strains of Ureaplasma urealyticum strains, by sequencing the gyrA gene mutation of three mutated forms: 302 base C to T, resulting in 101 (equivalent to Escherichia coli 84) alanine to valine change; the 336 base mutation of C to A, resulting in 112 (equivalent to Escherichia coli 95) aspartate to glutamic acid; lack of the 267 base T. GyrA gene mutation is the main drug resistant strains and moderately sensitive strains in the sensitive strains, no mutation of the gyrA gene; all the gene change the strain of the fluoroquinolone MIC level is significantly higher than that without strain group mutation. (4) in the standard strains, PCR-RFLP (restriction enzyme polymorphism analysis PCR based typing method cut) was 100%, the specificity is good at the same type of results And repeated, clear and easy to distinguish and explain the electrophoresis. In clinical strains through the "gold standard" identification, sensitivity of the typing method was 97%, the specificity was 89%, the coincidence rate is 98%.PCR positive repeat test two independent template classification experiments the concordance rate was 90.62%; the consistent rate of PCR-RFLP the results and sequencing typing results for 100%. (5) with reproductive tract infection of Ureaplasma urealyticum genotypes with Mh infection history and mainly for biological type 1, and the combination of Ureaplasma urealyticum fungal infection is mainly mixed type. Different types of Ureaplasma urealyticum to tetracyclines, there was significant difference in sensitivity of ring macrolides and fluoroquinolones, the average level of MIC were higher than the T960 type biological type 1. Amino acid of gyrA gene of Ureaplasma urealyticum genotypes associated with fluoroquinolone resistance is stronger than the phase change The correlation of the amino acid change of Asp95Glu strains belong to the T960 type, but not change the strain does not belong to T960 type. (6) of 95 strains of Neisseria gonorrhoeae isolates resistant to ciprofloxacin 100%; through sequencing, 18 mutations gyrA and parC was detected in 54 strains of Neisseria gonorrhoeae to ciprofloxacin. The level of MIC in different parC mutation group have statistically significant difference (P=0.006), parC gene mutation in the less the MIC is relatively low, the more the MIC mutation is relatively high.
Research conclusions: (1) in the third Jiangsu local area before the generation of fluoroquinolones and erythromycin MIC high level of resistance is serious, that these drugs should not be used as first-line treatment of Ureaplasma urealyticum infection, and tetracycline treatment can be used as a primary infection of Ureaplasma urealyticum

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R181.3

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