本文選題：腸炎沙門菌 + 最小抑菌濃度(Minimal��； 參考：《南方醫(yī)科大學》2015年碩士論文

【摘要】：研究背景：沙門菌是革蘭氏陰性桿菌,屬腸桿菌科,在自然界有廣泛宿主分布,存在于大多數(shù)動物及人的胃腸道中；也常常存在于水、蛋及蛋制品、肉類及其他食品中。根據(jù)O抗原(菌體抗原)及H抗原(鞭毛抗原)的抗原特性進行分類,沙門菌有2500多種血清型；其中鼠傷寒沙門菌、腸炎沙門菌是人獸共患最常見的血清型�；颊吒腥境３霈F(xiàn)腹瀉、腹痛、嘔吐等癥狀；沙門菌感染多為自限性疾病,患者可自愈,但敗血癥等并發(fā)癥需要抗生素治療。目前,臨床上治療沙門菌病時,主要采用喹諾酮類和三代頭孢菌素類抗生素,隨著使用時間和使用頻次的積累,已有相當一部分耐藥菌株產(chǎn)生。據(jù)美國國家抗生素耐藥監(jiān)測系統(tǒng)(national antimicrobial resistance monitoring system, NARMS)最近的報道顯示,22.5%的非傷寒沙門菌至少耐一種抗生素。腸炎沙門菌出現(xiàn)了多重耐藥(multidrug resistance,MDR)現(xiàn)象。質(zhì)粒介導(Plasmid-Mediated Quinolone Resistance,PMQR)是沙門菌耐藥的重要機制。目前有3類報道較多的PMQR質(zhì)�；�,分別是qnr(編碼Qnr蛋白)、aac(6')-Ib-cr(編碼AAC(6')Ib-cr蛋白)、qepA(編碼QepA蛋白)。PMQR主要介導低水平的耐藥,但也可累積突變,引起高水平的耐藥。PMQR陽性菌主要以多耐藥基因同時出現(xiàn)為主,呈多重耐藥表型,且不同地區(qū)也有不同的分布。沙門菌污染食品致腹瀉暴發(fā)由以前的點源性集中暴發(fā),越來越多地轉(zhuǎn)變?yōu)榭绲貐^(qū)跨州(省)的散在暴發(fā)形式出現(xiàn),使污染源及傳播途徑的發(fā)現(xiàn)更需要分子分型手段的輔助及確認。分子水平上的分型方法一般包括三種：基于DNA序列多態(tài)性分析的分型方法、基于擴增特定基因產(chǎn)物的PCR分型方法、以及基于細菌DNA的限制性內(nèi)切酶分析的分型方法。脈沖場凝膠電泳(pulsed field gel electrophoresis,PFGE)是20世紀80年代中期發(fā)展起來的用于分離大分子量線性DNA分子的一種電泳技術(shù),被譽為細菌分子生物學分型技術(shù)的“金標準”。其原理是在瓊脂糖凝膠上外加方向、時間與電流大小交替改變的脈沖電場,從而使得DNA分子得到有效地分離。通過比較DNA分子電泳圖譜來確認菌株之間的關(guān)聯(lián)。菌株間由于酶切位點突變、插入序列不同等原因會產(chǎn)生不同圖譜,一般認為電泳條帶差異越少則基因差異越小,菌株親緣關(guān)系則越近；反之則菌株間無相關(guān)性,但對親緣關(guān)系較遠的菌株分型效果不佳。PFGE適于研究一個暴發(fā)事件中,菌株之間的來源關(guān)系。多位點序列分型技術(shù)(multi-locus sequence typing,MLST)基于核酸序列測定技術(shù),通過測定基因序列的變化反映菌株之間的進化關(guān)系,在推論菌株間遺傳進化關(guān)系和種群相關(guān)性等方面有其他分型方法無可比擬的優(yōu)勢。其具有每種細菌的等位基因信息可通過互聯(lián)網(wǎng)使其全球標準化,具有快速、實驗結(jié)果可比性高和分辨水平高等優(yōu)點。由于他們遺傳穩(wěn)定性,他們不能為短期的流行病學研究提供有力的幫助。故MLST適于研究在一個大群體內(nèi),一段時期內(nèi),菌株之間的親緣關(guān)系。本研究旨在對廣東地區(qū)開展腸炎沙門菌耐藥監(jiān)測和耐藥菌株的分子分型研究,對2007-2013年GSS的腸炎沙門菌臨床分離株進行藥物敏感性檢測(微量肉湯稀釋法),并針對目前臨床上治療沙門菌感染主要使用喹諾酮類藥物和頭孢類藥物,特別是Ⅱ代頭孢菌素類以及Ⅲ代喹諾酮類的耐藥菌株進行耐藥基因PMQR的檢測和PFGE、MLST分子分型,有利于我們對于臨床高頻用藥的監(jiān)測追蹤,將重點監(jiān)測結(jié)果及時反饋,可以更好地指導臨床合理用藥。獲取的耐藥菌株的核酸指紋基礎(chǔ)數(shù)據(jù),可以分析本省監(jiān)測點醫(yī)院腹瀉病例臨床分離株之間的遺傳學關(guān)聯(lián)性,了解其分子遺傳特征,為發(fā)現(xiàn)腸炎沙門菌可能引起的暴發(fā)提供本底發(fā)病水平,為實驗室監(jiān)測、鑒定溯源及防控預警提供技術(shù)支持和數(shù)據(jù)參考,提高食源性疾病的檢測和防控能力。研究目的：1、了解2007年-2013年廣東省人群中感染腸炎沙門菌的耐藥情況,繪制耐藥譜,分析多重耐藥特點。為指導臨床醫(yī)生合理選擇使用抗生素,同時為預防和控制腸炎沙門菌引起的感染性腹瀉的發(fā)生和流行提供科學依據(jù)。2、根據(jù)耐藥結(jié)果選擇耐氟喹諾酮類和頭孢類藥物的菌株進行PMQR基因檢測,了解耐藥基因在廣東省腸炎沙門菌中的分布,發(fā)現(xiàn)其流行病學規(guī)律。3、對耐藥腸炎沙門菌菌株進行PFGE分型和MLST分型,對菌株帶型的聚集和分布規(guī)律進行分析,掌握廣東省流行株型別與全球流行現(xiàn)況,以發(fā)現(xiàn)帶型的流行病學規(guī)律,為監(jiān)測和暴發(fā)研究提供基礎(chǔ)。研究方法：1、采用CLSI推薦的微量肉湯稀釋法對廣東省2007-2013年腸炎沙門菌進行抗生素敏感性實驗,測定菌株的最小抑菌濃度(MIC值)。使用WHONET 5.3進行抗生素敏感性試驗數(shù)據(jù)分析,并將數(shù)值導入SPSS 13.0軟件進行統(tǒng)計分析,對2007-2013年GSS系統(tǒng)腸炎沙門菌的陽性檢出率狀況、三間分布特點,以及抗生素敏感性和多重耐藥的特點進行描述性統(tǒng)計分析。2、對耐頭孢類和氟喹諾酮的菌株進行PMQR質(zhì)�；騫nrA、qnrC、 qnrD、qurS、qepA、 oqxAB、aac(6')-Ib-cr)檢測,分析介導腸炎沙門菌對氟喹諾酮耐藥的質(zhì)�；蛟趶V東省的分布情況及其對MIC值的影響。MIC幾何均數(shù)值的組間比較采用變量變換后的非配對t檢驗,計數(shù)資料的組間比較采用卡方檢驗或Fisher確切概率法。檢驗水準α=0.05,以P0.05為差異有統(tǒng)計學意義。3、參照國際PulseNet的沙門菌PFGE分子分型標準操作方案,對上述耐頭孢類和氟喹諾酮類的腸炎沙門菌進行分子分型。菌株先經(jīng)限制性內(nèi)切酶消化,脈沖場凝膠電泳后,使用凝膠成像分析系統(tǒng)生成圖像條帶,BioNumerics軟件對圖像條帶進行識別、處理,H9812菌株作為標準分子量進行校準。選擇非加權(quán)配對算術(shù)平均法進行條帶的聚類分析,根據(jù)分析結(jié)果使用統(tǒng)一命名規(guī)則對條帶進行編號,建立耐藥腸炎沙門菌的PFGE分子分型數(shù)據(jù)庫。4、參考PulseNet推薦的沙門菌7個位點(aroC、dnaN、hemD、hisD、purE、sucA、 thrA)MLST標準操作方案,對反應(yīng)體系和檢測步驟進行相關(guān)的優(yōu)化,測序后根據(jù)分析結(jié)果使用既定命名規(guī)則對ST型別進行編號,建立數(shù)據(jù)庫。結(jié)合菌株的流行病學資料,對MLST型別的親緣進化關(guān)系、分布規(guī)律以及與耐藥譜的關(guān)系進行分析,同時與PFGE分型進行比較,綜合評價MLST的分型能力和實際應(yīng)用價值。研究結(jié)果：1、2007-2013年共收集散發(fā)腹瀉病例63687例,分離得到腸炎沙門菌菌株386例(檢出率6.06%0)。菌株分離主要來源于廣州、東莞,并以珠三角城市群為中心,呈散發(fā)趨勢,各地區(qū)檢出率無顯著性差異。在分離的386株散發(fā)病例腸炎沙門菌中,男女比例為1：0.698；各個年齡段均有感染的可能性,總體以1-4歲的兒童病例為主,占總數(shù)的35.23%(136/386),中青年組(18-60歲)病例排行第二,占總數(shù)的23.83%(92/386)。全年均可檢出,高峰期為6-10月。2、MIC結(jié)果顯示,腸炎沙門菌對八種抗生素均可能產(chǎn)生耐藥。喹諾酮類藥物中以第一代喹諾酮類藥物萘啶酸(NAL)耐藥率最高85.23%(329/386),MIC50值為128μg/ml;以第二代頭孢菌素類的頭孢西丁耐藥率最低(0.52%,2/386),MIC50值為2μg/ml,此外,四環(huán)素(TET)的耐藥率也較高(30.83%,119/386),MIC50值為2μg/ml。386株菌共有27個耐藥譜,以單一耐萘啶酸(NAL)(43.78%,169/386)為主,其次耐四環(huán)素加萘啶酸(TET+NAL)(15.03%,58/386)。有63株菌株對3種及3種以上的抗生素耐藥,占菌株總數(shù)的16.32%(63/386)。3、根據(jù)MIC藥敏結(jié)果篩選出71株耐氟喹諾酮類藥物和耐頭孢類藥物的腸炎沙門菌的PMQR基因檢測結(jié)果為：總體質(zhì)粒攜帶率為63.38%(45/71),其中35株僅攜帶一種PMQR基因(49.30%,35/71)；9株攜帶兩種PMQR基因(12.68%,9/71)；1株攜帶四種PMQR基因(1.41%,1/71)。oqxAB檢出率最高,達40.85%(29/71),其次為qnrC(9.86%,7/71),qepA未檢出。攜帶PMQR質(zhì)粒與未攜帶PMQR質(zhì)粒的菌株在氯霉素(CHL)和磺胺類藥物(TMP/SMZ)的MIC幾何均數(shù)值有統(tǒng)計學差異,攜帶PMQR質(zhì)粒組多重耐藥的比例(75.56%,34/45)高于未攜帶PMQR質(zhì)粒的菌株比例。4、71株耐藥腸炎沙門菌Xba I酶切的聚類分析可分為兩個大聚類,PFGE圖譜的相似值為56%-100%,共分為PFGE-Xba I 1-35型。最大的型別有8株菌,最小的為1株。5、71株耐藥腸炎沙門茵的MLST分型均為同一型別,等位基因序為aroC5、dnaN2、 hemD3、hisD7、purE6、sucA6、thrA11;等位基因譜為ST11型,屬于eBG4型。這說明廣東省耐藥腸炎沙門菌的優(yōu)勢序列型均為ST11型。結(jié)論：1、腸炎沙門菌為廣東省第二大優(yōu)勢血清型,主要易感人群為14歲的幼兒,夏秋季高發(fā),病例集中分布于珠三角地區(qū)。藥敏結(jié)果顯示萘啶酸(NAL)耐藥率最高,多重耐藥率為16.32%。2、對于耐藥菌株的PMQR質(zhì)�；驒z測結(jié)果說明攜帶PMQR質(zhì)粒的菌株對比未攜帶質(zhì)粒的菌株耐藥性更高,且更易發(fā)生多重耐藥現(xiàn)象。3、MLST的分子分型結(jié)果表明,在沙門菌同一血清型內(nèi)各菌株看家基因堿基序列的高度保守,而PFGE則提示腸炎沙門菌的分化程度不高；對于腸炎沙門菌來說,MLST和PFGE的分辨力均較低,還需用其他分辨力更高的分子分型方法。
[Abstract]:Research background: Salmonella is gram-negative bacilli, a family of Enterobacteriaceae, widely distributed in nature and exists in most animals and human gastrointestinal tract; it also exists in water, egg and egg products, meat and other foods. Classification of O antigen (mycelium antigen) and H antigen (flagellum antigen) antigen characteristics, Salmonella There are more than 2500 serotypes. Among them, Salmonella typhimurium and Salmonella enteritis are the most common serotypes of human zoonosis. Patients often have symptoms such as diarrhea, abdominal pain, vomiting and other symptoms; Salmonella infection is a self limiting disease, and the patients can heal themselves, but the complications such as sepsis need antigreen treatment. At present, the main clinical treatment of Salmonella disease is mainly in clinical treatment. Using the quinolones and three generation cephalosporins, a number of resistant strains have been produced with the accumulation of time and frequency of use. According to the recent reports of national antimicrobial resistance monitoring system (NARMS) in the United States, 22.5% of non typhoid Salmonella is at least one resistance. Multidrug resistance (MDR) has appeared in Salmonella enteritis. Plasmid mediated (Plasmid-Mediated Quinolone Resistance, PMQR) is an important mechanism for the drug resistance of Salmonella. There are 3 kinds of PMQR plasmids which are reported to be qnr (encoded Qnr protein) and AAC (6'). EpA protein).PMQR mainly mediates low level of resistance, but it can also accumulate mutation, causing the high level of drug resistant.PMQR positive bacteria mainly to appear mainly with multidrug-resistant genes and multidrug-resistant phenotype, and there are different distribution in different regions. In the form of outbreaks across regions and provinces, the discovery of pollution sources and channels of transmission requires the assistance and confirmation of molecular typing methods. The classification methods on the molecular level generally include three types: the typing based on DNA sequence polymorphism analysis, the PCR typing method based on the amplification of the specific gene products, and the DN based on the bacteria. A's restriction endonuclease analysis method. Pulsed field gel electrophoresis (PFGE) is an electrophoretic technique developed for the separation of large molecular weight linear DNA molecules in the mid 1980s. It is known as the "gold standard" for bacterial molecular biological typing. The principle is on agarose gel. A pulsed electric field, alternately changing the size of time and current, makes the DNA molecules effectively separated. The correlation between strains is confirmed by comparing the DNA molecular electrophoretic map. The smaller the difference, the closer the strain relationship was, and conversely, there was no correlation between the strains, but the poor genotyping effect of the far relative strains.PFGE was suitable for the study of the source relationship between the strains. The multiple point sequence typing (multi-locus sequence typing, MLST) was based on nucleic acid sequencing technology and through the determination of the basis. The evolution relationship between strains is reflected by the variation of the sequence, and there are unparalleled advantages in other classification methods, such as genetic evolution relationship and population correlation among the strains, and the allelic information of each bacterium can be standardized through the Internet, fast, high experimental results and high resolution. Advantages. Because of their genetic stability, they can not provide strong help for short-term epidemiological studies. Therefore, MLST is suitable for studying the relationship between strains in a large group, for a period of time. This study aims to study the molecular typing of Salmonella enteritis in Guangdong and the molecular typing of resistant strains for 2007-2013 years. GSS clinical isolates of Salmonella enteritis were tested for drug sensitivity (micro broth dilution), and the detection of drug-resistant gene PMQR and PFGE, MLST molecules for the main use of quinolones and cephalosporins, especially the second generation cephalosporins and third generation quinolones, were detected in the clinical treatment of Salmonella infection. The classification is beneficial to the monitoring and tracking of clinical high frequency drugs and the timely feedback of the key monitoring results. It can guide the clinical rational use of drugs better. The basic data of nucleic acid fingerprint of the resistant strains obtained can be used to analyze the genetic association between the clinical isolates of diarrhea cases in the provincial monitoring points and understand its molecular genetic specificity. In order to find the possible background of the outbreak of Salmonella enteritis, it provides technical support and data reference for laboratory monitoring, identification and prevention and control early warning. The purpose of this study is to improve the detection and control ability of food borne diseases. 1. 1. To understand the drug resistance of Salmonella enteritis in Guangdong Province in 2007. In order to guide the clinicians to choose antibiotics reasonably and provide scientific basis for the prevention and control of the occurrence and epidemic of infectious diarrhea caused by Salmonella enteritis,.2, PMQR gene detection was carried out to select the strains resistant to fluoroquinolones and cephalosporins to understand the resistance genes. The distribution of Salmonella enteritidis in Guangdong Province, we found its epidemiological law.3, PFGE typing and MLST typing of Salmonella resistant strains of Salmonella enteritis, analysis of the distribution and distribution of strain band type, grasp the epidemic situation of the epidemic plant type and the global epidemic situation in Guangdong Province, in order to find the pattern of epidemiology, and provide the monitoring and outbreak research. Basic and research methods: 1, the antibiotic susceptibility test of Salmonella enteritis in 2007-2013 years in Guangdong province was carried out by the method of micro broth dilution recommended by CLSI. The minimum inhibitory concentration (MIC value) of the strain was measured. WHONET 5.3 was used to analyze the antibiotic sensitivity test data, and the number of values was introduced into the SPSS 13 software for statistical analysis, to 2007-2013 The positive rate of Salmonella enteritis in GSS system, three distribution characteristics, and the characteristics of antibiotic sensitivity and multidrug resistance were analyzed by.2. The PMQR plasmid gene qnrA, qnrC, qnrD, qurS, qepA, oqxAB, AAC (6') -Ib-cr) for the strains resistant to cephalosporins and fluoroquinolones were detected, and the fluorine was analyzed and mediated by Salmonella enteritis. The distribution of quinolone resistant plasmid gene in Guangdong province and the effect of the MIC value on the value of.MIC were compared with the non paired t test after variable transformation. The comparison of the count data was compared to the chi square test or the Fisher exact probability method. The level of alpha =0.05 was tested, and the P0.05 was statistically significant.3, referring to international Pul. SeNet of Salmonella PFGE molecular classification standard operation scheme, the molecular classification of the above Salmonella enteritis resistant cephalosporins and fluoroquinolones. The strain was digested by restriction endonuclease first, and after pulsed field gel electrophoresis, the image strip was generated by the gel imaging analysis system. The BioNumerics software identified, processed, and H9812 bacteria. The plant was calibrated as a standard molecular weight. The unweighted pairing arithmetic mean method was selected to cluster analysis of strip. According to the results, the PFGE molecular typing database of Salmonella enteritis was numbered and.4 was set up. 7 sites of Salmonella (aroC, dnaN, hemD, hisD, purE, sucA, th) recommended by PulseNet were referred to. RA) MLST standard operation scheme, optimizing the reaction system and detection steps. After sequencing, the ST type is numbered and the database is set up according to the results of the analysis. The relationship of the phylogenetic evolution of the MLST type, the distribution rule and the relationship with the drug resistance spectrum are analyzed with the epidemiological data of the strain. PFGE classification was compared to evaluate the classification ability and practical application value of MLST. Results: 63687 cases of sporadic diarrhea cases were collected in 12007-2013 years, 386 cases of Salmonella enteritis were isolated (detection rate 6.06%0). The isolation of strains was mainly from Guangzhou, Dongguan, and was distributed in the Pearl River Delta city group as the center. There was no significant difference in the detection rate. In the 386 sporadic cases of Salmonella enteritis, the proportion of men and women was 1:0.698, the possibility of infection in each age group was 1-4 years old, accounting for 35.23% (136/386) of the total number of children, and second in the young and middle-aged group (18-60 years old), accounting for 23.83% (92/386). All year all could be detected, high The peak period was 6-10 months.2, MIC results showed that Salmonella enteritis could be resistant to eight antibiotics. The highest quinolones drug resistance rate was 85.23% (329/386), MIC50 value was 128 micron, and second generation cephalosporin (0.52%, 2/386) and 2 g/ml, with the second generation cephalosporin. In addition, the resistance rate of tetracycline (TET) was also higher (30.83%, 119/386), and the MIC50 value of 2 micron g/ml.386 strains had 27 resistance profiles, with single naphthidine (NAL) (43.78%, 169/386), followed by tetracycline and naphthidine acid (TET+NAL) (15.03%, 58/386). 63 strains were resistant to 3 and more than 3 antibiotics, accounting for 16.32% (63/386).3 of the total strain. The results of PMQR gene detection of 71 strains of fluoroquinolone resistant and cephalosporin resistant Salmonella were screened according to the MIC drug sensitivity results: the overall plasmid carrying rate was 63.38% (45/71), 35 of which only carried one PMQR gene (49.30%, 35/71); 9 strains carried two PMQR genes (12.68%, 9/71), and 1 carrying four PMQR genes (1.41%, 1/7). 1) the highest detection rate of.OqxAB was 40.85% (29/71), followed by qnrC (9.86%, 7/71), and qepA was not detected. The PMQR plasmid and the MIC without PMQR plasmid had a statistical difference between the MIC geometry of chloramphenicol (CHL) and the sulfonamides (TMP/SMZ), and the proportion of the multidrug resistance (75.56%, 34/45) carrying the PMQR mass group (75.56%, 34/45) was higher than that of the bacteria without the plasmid carrying plasmid. The cluster analysis of Xba I enzyme cut of Salmonella enteritis resistant to.4,71 strain can be divided into two large clusters. The similarity value of PFGE atlas is 56%-100%, which is divided into PFGE-Xba I 1-35. The largest type has 8 strains, and the minimum of 1 strains of.5,71 strain of Salmonella is the same type, and the allele sequence is aroC5, dnaN2, hemD3. RE6, sucA6, thrA11; the allele gene spectrum is ST11 type, which belongs to eBG4 type. This shows that the dominant sequence of drug resistant Salmonella enteritis in Guangdong province is ST11 type. Conclusion: 1, Salmonella enteritis is the second dominant serotype in the province, the main susceptible population is 14 year old children, in summer and autumn, the case is concentrated in the Pearl River Delta region. The drug sensitivity results show that the case is concentrated in the Pearl River Delta region. The drug resistance of nnidium acid (NAL) was the highest and the multidrug resistance rate was 16.32%.2. For the PMQR plasmid DNA detection results of the resistant strains, the strains carrying PMQR plasmids were more resistant to the strains without plasmid and more susceptible to multidrug resistance.3. The molecular typing of MLST showed that each strain of Salmonella was in the same serotype. Because of the highly conserved base sequence, PFGE suggests that the differentiation degree of Salmonella enteritis is not high; for Salmonella enteritis, the resolution of MLST and PFGE is low, and other molecular typing with higher resolution is needed.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R446.5

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