山東省食品中單核細(xì)胞增生性李斯特氏菌耐藥性與分子分型研究
發(fā)布時(shí)間：2018-12-27 13:08
【摘要】：[研究背景] 單核細(xì)胞增生性李斯特氏菌(Listeria monocytogenes,以下簡(jiǎn)稱單增李斯特氏菌),為革蘭氏陽(yáng)性無(wú)芽孢桿菌,是一種重要的食源性人畜共患病原菌。據(jù)報(bào)道,單增李斯特氏菌在歐洲及美國(guó)的發(fā)病率為0.2-0.8/100000,但其病死率卻高達(dá)20-70%,國(guó)際各衛(wèi)生研究機(jī)構(gòu)已經(jīng)對(duì)此給予了高度關(guān)注。單增李斯特氏菌在自然界中分布廣泛,人類主要通過(guò)接觸即時(shí)食品和冷凍食品發(fā)生感染。人類感染后的臨床癥狀主要表現(xiàn)為敗血癥,腦膜炎,單核細(xì)胞增多及自發(fā)性晚期流產(chǎn)等,孕婦、新生兒、老人及免疫缺陷者為易感人群。因單增李斯特氏菌污染而引起的食源性疾病暴發(fā)在全球范圍內(nèi)屢有報(bào)道,加之其較高的死亡率,為公共衛(wèi)生健康造成了巨大的危害。美國(guó)馬薩諸塞州于1983年因食用該菌污染的牛奶導(dǎo)致49人患病,14人死亡；加利福尼亞州于1985年因食用污染的奶酪造成142人發(fā)病,其中29人死亡；2002年多個(gè)州又因單增李斯特氏菌污染的火雞導(dǎo)致46人患病,7人死亡,3人流產(chǎn)。1992年因食用該菌污染的豬舌頭造成法國(guó)279人患病。我國(guó)從1964-2003年因感染單增李斯特氏菌的病例為150例,死亡39例。從1988年首次報(bào)道單增李斯特氏菌對(duì)四環(huán)素產(chǎn)生耐藥以來(lái),從不同環(huán)境、食品及臨床病例中分離出的菌株對(duì)各種抗生素的耐藥及多重耐藥現(xiàn)象愈發(fā)嚴(yán)重。隨著單增李斯特氏菌耐藥性的日益加重,臨床藥物選擇壓力也逐漸加重,這進(jìn)一步增加了疾病的死亡率。 單增李斯特氏菌已成為中國(guó)嚴(yán)重的公共衛(wèi)生問(wèn)題。近年來(lái)雖有不少學(xué)者開展了食品中單增李斯特氏菌的研究,但多數(shù)局限于對(duì)該菌污染率的主動(dòng)性監(jiān)測(cè)。因此,本研究對(duì)山東省2003-2010年食品中單增李斯特氏菌分別運(yùn)用耐藥性試驗(yàn)、血清學(xué)分型和脈沖場(chǎng)凝膠電泳分型(PFGE)三種方法進(jìn)行綜合的流行病學(xué)研究,探討不同菌株之間的遺傳相關(guān)性及不同分型方法之間的聯(lián)系,為有效地控制和預(yù)防食源性疾病提供科學(xué)依據(jù)。 [研究目的] 1.研究山東省不同食品中單增李斯特氏菌的耐藥性,分析山東省單增李斯特氏菌的耐藥趨勢(shì),為抗生素耐藥的危險(xiǎn)性評(píng)估和管理提供理論基礎(chǔ)。 2.研究不同食品中單增李斯特氏菌的血清學(xué)型別,分析優(yōu)勢(shì)血清型菌株的分布情況。 3.研究單增李斯特氏菌PFGE型別的分布特點(diǎn),分析不同菌株之間的遺傳相關(guān)性,建立山東省單增李斯特氏菌的數(shù)據(jù)庫(kù),為單增李斯特氏菌的分布特點(diǎn)、菌株關(guān)聯(lián)及分子流行病學(xué)研究提供平臺(tái)。 4.比較耐藥性試驗(yàn)、血清學(xué)分型和脈沖場(chǎng)凝膠電泳分型三種方法的優(yōu)劣,并分析不同分型方法之間的關(guān)系。 [研究方法] 1.采用CLSI推薦的微量肉湯稀釋法進(jìn)行耐藥性試驗(yàn)。 2.采用玻片凝集法、試管凝集法對(duì)單增李斯特氏菌進(jìn)行血清學(xué)分型。 3.按照PulseNet標(biāo)準(zhǔn)方法進(jìn)行脈沖場(chǎng)凝膠電泳分型。 [研究結(jié)果] 1.252株單增李斯特氏菌對(duì)15種抗生素的平均耐藥率為15.87%,可分為21種耐藥型別。其中,對(duì)亞胺培南的耐藥率最高(3.97%),其次為四環(huán)素和環(huán)丙沙星(均為1.59%),對(duì)慶大霉素、氨芐西林和青霉素尚未出現(xiàn)耐藥菌株。多重耐藥率為4.37%。不同樣品、采樣年份和采樣地點(diǎn)中分別以生豬肉中(22.09%),2007年(38.89%),臨沂(29.41%)的菌株耐藥率最高。 2.252株單增李斯特氏菌分為6種血清型,以1/2c型菌株最多,出現(xiàn)了致病力最強(qiáng)的4b型菌株,且4b型菌株的耐藥率最高(22.74%)。不同樣品、采樣年份和采樣地點(diǎn)中分別以生雞肉中(優(yōu)勢(shì)血清型為1/2a型,59.15%),2005年(優(yōu)勢(shì)血清型為1/2c型,40.54%),濟(jì)南地區(qū)(優(yōu)勢(shì)血清型為1/2c型,38.71%)的菌株血清型種類最多,分別包括6種血清型。 3. PFGE分型將252株單增李斯特氏菌分為73個(gè)型別,同源性在50.43-100.00%之間,優(yōu)勢(shì)型別為24型(15.48%),不同食品來(lái)源、采樣年份和采樣地點(diǎn)的優(yōu)勢(shì)型別分別為33型(23.94%)、24型(22.97%)及24型(15.48%)。 4.三種方法的辛普森分辨指數(shù)分別為：耐藥性試驗(yàn)D=0.7048,血清學(xué)分型D=0.7023, PFGE分型D=0.9371,可見(jiàn)PFGE分型方法具有較好的分辨能力。PFGE分型結(jié)果與血清學(xué)分型結(jié)果存在較為密切的聯(lián)系,與耐藥譜分型結(jié)果無(wú)明顯聯(lián)系。 [結(jié)論] 1.山東省不同種類食品中存在單增李斯特氏菌的污染,應(yīng)當(dāng)加強(qiáng)對(duì)食品衛(wèi)生的監(jiān)管力度。 2.首次運(yùn)用耐藥性試驗(yàn)、血清學(xué)分型和PFGE分型相結(jié)合的方法對(duì)山東省食品中單增李斯特氏菌進(jìn)行研究。不同菌株存在一定的耐藥性,但尚未出現(xiàn)對(duì)臨床一線藥物耐藥的菌株。山東省單增李斯特氏菌的血清學(xué)型別分布較廣,應(yīng)加強(qiáng)對(duì)致病型菌株的監(jiān)測(cè),尤其應(yīng)警惕4b型菌株的擴(kuò)散和新型耐藥型別的出現(xiàn)。山東省食品中單增李斯特氏菌來(lái)源于不同的克隆株,但部分菌株存在不同程度的親緣關(guān)系。PFGE型別與菌株的樣品來(lái)源不存在明顯相關(guān)性。PFGE型別與血清型別聯(lián)系密切,與耐藥譜無(wú)明顯聯(lián)系。 3.通過(guò)三種方法之間的比較,PFGE分型是分辨力最高的分型方法(辛普森分辨指數(shù)D=0.9371)。在進(jìn)行食源性致病菌的流行病學(xué)研究時(shí),建立以PFGE型別為主,結(jié)合血清型別和耐藥譜的數(shù)據(jù)庫(kù),可以為掌握食源性致病菌流行趨勢(shì)、調(diào)查食源性疾病暴發(fā)及追蹤傳染源提供依據(jù)。
[Abstract]:[Study Background] Listeria monocytogenes (hereinafter referred to as Listeria monocytogenes), which is a Gram-positive bacillus-free bacterium, is an important food-derived human animal It is reported that the incidence of Listeria monocytogenes in Europe and the United States is 0.2-0.8/ 100000, but the case fatality rate is high by 20-70%. Note: Listeria monocytogenes are widely distributed in nature, human being mainly through contact with instant food and frozen food The clinical symptoms after human infection are mainly characterized by septicemia, meningitis, increased mononucleosis and spontaneous late-stage abortion. The outbreak of foodborne disease caused by the contamination of Listeria monocytogenes has been reported worldwide, and the higher mortality rate has caused great danger for public health and health in 1983, 49 people were sick and 14 died in Massachusetts, Massachusetts, in 1983; in 1985, 142 people were killed by eating tainted cheese, of which 29 were dead. in many states in 2002, 46 people were affected by a single increase of Listeria monocytogenes. Seven people were killed and three were miscarriages. The pig's tongue, which was contaminated with the bacteria in 1992, caused 279 people in France. The number of Listeria monocytogenes in China from 1964 to 2003 is 150, and death 39 Example: Since the first report of the drug resistance of Listeria monocytogenes to tetracycline in 1988, the strains isolated from different environment, food and clinical cases have become more resistant to the drug resistance and multiple drug resistance of various antibiotics. Heavy. As the drug resistance of Listeria monocytogenes is increasing, the pressure of clinical drug selection is also gradually increased, which further increases the death of the disease. The rate of Listeria monocytogenes has become a serious public health guard in China In recent years, many scholars have carried out the study of Listeria monocytogenes in food, but most of them are limited to the activity of the bacteria pollution rate. In this study, the drug resistance test, serological typing and pulse-field gel electrophoresis (PFGE) method were used to make a comprehensive epidemic of Listeria monocytogenes in the food of Shandong Province from 2003 to 2010. To study the relationship between different strains and the relationship between different types of different strains, and to provide the effective control and prevention of foodborne diseases. The basis of the study.[Study Objective] 1. To study the drug resistance of Listeria monocytogenes in different foods in Shandong Province, and to analyze the drug resistance of Listeria monocytogenes in Shandong Province. To provide a theoretical basis. 2. To study the serological type of Listeria monocytogenes in different foods and to analyze the dominant blood. Distribution of the Qing-type strain. 3. The distribution of PFGE-type of Listeria monocytogenes was studied. The genetic relationship among different strains was analyzed, and the database of Listeria monocytogenes was established. Sub-epidemiological studies provide the platform. 4. Compare the advantages and disadvantages of drug resistance test, serological typing and pulse-field gel electrophoresis. Analysis of different types The relationship between the methods.[Study method] 1. CLSI is used The drug resistance test was carried out by the recommended microbroth dilution method. Serological typing of Listeria monocytogenes by tube agglutination test. lseNet Standard method for pulse-field gel electrophoresis typing.[Results] 1. 252 strains of Listeria monocytogenes were used for 15 antigenics The average drug-resistance rate was 15.87%, which could be divided into 21 drug-resistant types, among which, the drug-resistance rate of imipenem was the highest (3.97%), followed by tetracycline and ciprofloxacin (both 1.59 and 1.59).%), for gentamicin, aminophene There were no drug-resistant strains in the forest and the penicillins. The multiple drug-resistance rate was 4.37%. In different samples, the sampling year and the sampling location were in raw pork (22.09%), 2007 (3 The strains of Listeria monocytogenes (8.89%) and Linyi (29. 41%) were the highest. The resistance rate of the 4b-type strain was the highest (22.74%). In different samples, in the sample year and the sampling place, in the raw chicken (the dominant serotype is 1/ 2a, 55.9%), in 2005 (the dominant serotype is 1/ 2c, 40. 54%), the Jinan region (the dominant serotype is 1/ 2c type, The type of strain of the strain of 38. 71%) was the most, including six serotypes. 3. PGE type, 252 strains of Listeria monocytogenes were divided into 73 types, with the homology of 50. 43-100. 00%, the dominant type was 24 (15.48%), and the advantages of different food sources, sampling year and sampling place were 33 (23 (94%), 24-type (22. 97%) and 24 (15.48%). 4. The Simpson-resolved indexes of the three methods were: drug resistance test D = 0.7048, serologic type D = 0.7023, PFG E-type D = 0. 9371, it can be seen that the PFGE classification method has better resolving power. PFGE typing results and serology There was a close relationship between the typing results and there was no significant association with the results of the drug resistance spectrum typing.[Conclusion] 1. Shandong The contamination of Listeria monocytogenes in different kinds of food in the province should strengthen the supervision of food hygiene. The method of combination of serological typing and PFGE's typing in the food of Shandong province is Listeria monocytogenes. The strains of Listeria monocytogenes have not yet been resistant to the first-line drug. The serological type of Listeria monocytogenes in Shandong Province has a wide distribution. The monitoring of the pathogenic strains should be strengthened, especially the spread of the 4b-type strain and the emergence of a new drug-resistant type. Listeria monocytogenes in the food of the province are derived from different clones, but the part There is a different degree of genetic relationship among the strains. PFGE-type is not the same as that of the strain. There was no significant correlation between the source of the product. The PFGE type was not in close contact with the serotype, and there was no significant association with the resistance spectrum. The type of PFGE was the highest-resolution method (Simpson-resolved index, D = 0.97371). In the case of the epidemiological study of food-borne pathogenic bacteria, PFGE-type was the main type, which was combined with serotype and drug-resistance spectrum.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R155.5
本文編號(hào)：2393117
[Abstract]:[Study Background] Listeria monocytogenes (hereinafter referred to as Listeria monocytogenes), which is a Gram-positive bacillus-free bacterium, is an important food-derived human animal It is reported that the incidence of Listeria monocytogenes in Europe and the United States is 0.2-0.8/ 100000, but the case fatality rate is high by 20-70%. Note: Listeria monocytogenes are widely distributed in nature, human being mainly through contact with instant food and frozen food The clinical symptoms after human infection are mainly characterized by septicemia, meningitis, increased mononucleosis and spontaneous late-stage abortion. The outbreak of foodborne disease caused by the contamination of Listeria monocytogenes has been reported worldwide, and the higher mortality rate has caused great danger for public health and health in 1983, 49 people were sick and 14 died in Massachusetts, Massachusetts, in 1983; in 1985, 142 people were killed by eating tainted cheese, of which 29 were dead. in many states in 2002, 46 people were affected by a single increase of Listeria monocytogenes. Seven people were killed and three were miscarriages. The pig's tongue, which was contaminated with the bacteria in 1992, caused 279 people in France. The number of Listeria monocytogenes in China from 1964 to 2003 is 150, and death 39 Example: Since the first report of the drug resistance of Listeria monocytogenes to tetracycline in 1988, the strains isolated from different environment, food and clinical cases have become more resistant to the drug resistance and multiple drug resistance of various antibiotics. Heavy. As the drug resistance of Listeria monocytogenes is increasing, the pressure of clinical drug selection is also gradually increased, which further increases the death of the disease. The rate of Listeria monocytogenes has become a serious public health guard in China In recent years, many scholars have carried out the study of Listeria monocytogenes in food, but most of them are limited to the activity of the bacteria pollution rate. In this study, the drug resistance test, serological typing and pulse-field gel electrophoresis (PFGE) method were used to make a comprehensive epidemic of Listeria monocytogenes in the food of Shandong Province from 2003 to 2010. To study the relationship between different strains and the relationship between different types of different strains, and to provide the effective control and prevention of foodborne diseases. The basis of the study.[Study Objective] 1. To study the drug resistance of Listeria monocytogenes in different foods in Shandong Province, and to analyze the drug resistance of Listeria monocytogenes in Shandong Province. To provide a theoretical basis. 2. To study the serological type of Listeria monocytogenes in different foods and to analyze the dominant blood. Distribution of the Qing-type strain. 3. The distribution of PFGE-type of Listeria monocytogenes was studied. The genetic relationship among different strains was analyzed, and the database of Listeria monocytogenes was established. Sub-epidemiological studies provide the platform. 4. Compare the advantages and disadvantages of drug resistance test, serological typing and pulse-field gel electrophoresis. Analysis of different types The relationship between the methods.[Study method] 1. CLSI is used The drug resistance test was carried out by the recommended microbroth dilution method. Serological typing of Listeria monocytogenes by tube agglutination test. lseNet Standard method for pulse-field gel electrophoresis typing.[Results] 1. 252 strains of Listeria monocytogenes were used for 15 antigenics The average drug-resistance rate was 15.87%, which could be divided into 21 drug-resistant types, among which, the drug-resistance rate of imipenem was the highest (3.97%), followed by tetracycline and ciprofloxacin (both 1.59 and 1.59).%), for gentamicin, aminophene There were no drug-resistant strains in the forest and the penicillins. The multiple drug-resistance rate was 4.37%. In different samples, the sampling year and the sampling location were in raw pork (22.09%), 2007 (3 The strains of Listeria monocytogenes (8.89%) and Linyi (29. 41%) were the highest. The resistance rate of the 4b-type strain was the highest (22.74%). In different samples, in the sample year and the sampling place, in the raw chicken (the dominant serotype is 1/ 2a, 55.9%), in 2005 (the dominant serotype is 1/ 2c, 40. 54%), the Jinan region (the dominant serotype is 1/ 2c type, The type of strain of the strain of 38. 71%) was the most, including six serotypes. 3. PGE type, 252 strains of Listeria monocytogenes were divided into 73 types, with the homology of 50. 43-100. 00%, the dominant type was 24 (15.48%), and the advantages of different food sources, sampling year and sampling place were 33 (23 (94%), 24-type (22. 97%) and 24 (15.48%). 4. The Simpson-resolved indexes of the three methods were: drug resistance test D = 0.7048, serologic type D = 0.7023, PFG E-type D = 0. 9371, it can be seen that the PFGE classification method has better resolving power. PFGE typing results and serology There was a close relationship between the typing results and there was no significant association with the results of the drug resistance spectrum typing.[Conclusion] 1. Shandong The contamination of Listeria monocytogenes in different kinds of food in the province should strengthen the supervision of food hygiene. The method of combination of serological typing and PFGE's typing in the food of Shandong province is Listeria monocytogenes. The strains of Listeria monocytogenes have not yet been resistant to the first-line drug. The serological type of Listeria monocytogenes in Shandong Province has a wide distribution. The monitoring of the pathogenic strains should be strengthened, especially the spread of the 4b-type strain and the emergence of a new drug-resistant type. Listeria monocytogenes in the food of the province are derived from different clones, but the part There is a different degree of genetic relationship among the strains. PFGE-type is not the same as that of the strain. There was no significant correlation between the source of the product. The PFGE type was not in close contact with the serotype, and there was no significant association with the resistance spectrum. The type of PFGE was the highest-resolution method (Simpson-resolved index, D = 0.97371). In the case of the epidemiological study of food-borne pathogenic bacteria, PFGE-type was the main type, which was combined with serotype and drug-resistance spectrum.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R155.5
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