豬分離沙門氏菌特異性PCR引子之發展、脈衝式膠體電泳、逢機擴增多形性DNA 、質體圖譜及抗藥性基因片匣之分析

Salmonella enterica serovar Choleraesuis is one of the major bacterial pathogens for swine. The Salmonella serovar has been often isolated from diarrheagenic and septicemia swine in Taiwan. In this study, pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis were used for the subtyping of 95 S. Choleraesuis strains isolated from swine with diarrhea and systemic infection during 2000~2002. The swine were grown in the swine fields in southern Taiwan. For PFGE, XbaI, AvrⅡ, SpeI and NotI restriction enzymes were used for chromosomal DNA digestion. The results showed that, for these 95 Salmonella strains, 86 PFGE patterns combinations were found. Of them, pattern X1A1S1N1, was the major subtype since 11 strains belong to this pattern. Strains of this pattern may be the most epidemic strains. On the other hand, RAPD analysis generated 74 patterns for these 95 isolated strains. Comparison of the PFGE and RAPD patterns, RAPD could subdivide strains within a PFGE type. Thus, RAPD was a better discriminative tool for subtyping of S. Choleraesuis, if suitable primer was selected for typing. Salmonella enterica serovar Choleraesuis may cause swine salmonellosis and human infection. Since conventional method for the detection of this Salmonella serovar may take 3-5 days, in this study, PCR method was developed. By comparison of the sequence of phase-1 flagellin (fliC) gene of S. Choleraesuis with those of other Salmonella serovars and the sequences of other bacteria spp. available in GenBank, two PCR primers, i.e, flinC-F and flinC-R, were designed. Using these primers, all the 97 S. Choleraesuis strains assayed generated expected PCR products with molecular weight equal to 963 bp. Except of S. Paratyphi C, Salmonella isolates other than S. Choleraesuis, and non-Salmonella isolates including strains of Enterobacteriaceae, all generated negative PCR results. S. Paratyphi C could be differentiated from S. Choleraesuis through the use of primers designed from viaB gene. When S. Choleraesuis in swine stool, pork, liver, feed, and human whole-blood samples were assayed, with a pre-enrichment step, as low as 100 CFU per g or ml of the sample could be detected. Antimicrobial susceptibility test was also preformed for the 95 S. Choleraesuis strains collected from the swine field in Southern Taiwan. A total of 18 antibiotics were tested. Results showed that 46 antibiograms were obtained. All these strains were multidrug resistant strains. PCR was used to screen the gene encoding the resistance to ampicillin (TEM, and PSE group), streptomycin-spectinomycin (aadA), kanamycin (aphA), sulfadiazine (sulI, and sul3), trimethoprim (dfrA12), tetracycline (tetA), streptomycin (strA, and strB), chloramphenicol (cmlA), gentamycin (aac(3)Ⅳ), fluoroquinolone (gyrA, parC, and parE), and that class I integron. Results showed that 32.6% of the 95 strains of Salmonella Choleraesuis are with the class I integron. The presense of antibiotic resistance genes on integron cassette was further analyzed by PCR mapping. Four types of patterns were detected. They were dfrA12-aadA2, aadA5-aadA2, dfrA12-aad2-su1l, and dfrA12-aadA5. Our data demonstrated that the problem of antibiotic resistance in Salmonella Choleraesuis need strains to be paid with attention. Certain Salmonella serovars have virulence plasmid, including serovars Typhimurium, Choleraesuis, Dublin, and Enteritidis. In this study, plasmid profile analysis of 95 swine S. Choleraesuis isolates showed that S. Choleraesuis isolates harbored 1~3 large virulence plasmids. These plasmids sizes ranging from 40 kb to 190 kb, a total of 14 types plasmid profiles were obtained. Forty-two strains of S. Choleraesuis belong to a major type which containing 50 kb and 90 kb plasmids. When using spvR gene specific primers were used for PCR, all the 95 swine isolates generated the expected PCR product. The results indicted that spvR gene may be present in plasmid with size large than 50 kb. The variation of plasmids sizes may be caused by high frequency gene exchange. Since microarray method has become an important technology for detection of mass genes. This method was rapid, highly specific, and sensitive. To develop a microarray method for detecting different antibiotic resistant genes, identification of bacterial strains and their virulence genes, in this study, a total of 32 gene specific PCR products were printed on the microarray, including the products from 22 antibiotic resistant genes, 3 virulence genes, 5 specific genes for Salmonella, and the 16S rDNA gene for positive control. Genomic DNAs from swine isolates were fluorescently labeled for array hybridization. Three different multi-drug resistant S. Choleraesuis isolates SCV28, SCV62, and SCV27a were tested. The results showed that all tested strains were detected by the microarray as expected. Our results suggested that our microarray detected the antibiotic resistant genes, virulence genes, and specific genes and may be used as a platform for S. Choleraesuis isolates. Furthermore, this microarray might also be useful for the detection of antibiotic resistant genes for other serovar Salmonella or closely related bacteria. Salmonella enterica serovar Choleraesuis was one of the highly invasive serotypes to animal and human. In this study, we attempt to elucidate the relationship between their molecular subtypes and their invasiveness to different epithelial celltypes. Meanwhile, the invasiveness for different serotypes of Salmonella were also compared. Twenty-three strains representing randomly selected PFGE subtypes were studied for their invasiveness into Intestine 407 cells. Four strains were tested of the invasiveness to different celltypes. On the other hand, serovar Typhimurium, Derby, Enteritidis, Paratyphi C, and Choleraesuis strains were compared for their invasiveness into Intestine 407 cells and LLC-PK1 cells. The results showed that there were no significant differences in invasiveness between different molecular subtypes. The results also showed that Intestine 407 cells, HEp-2 cells, and LLC-PK1 cells were variable in susceptibility to Salmonella invasion. Comparison with different serotypes of Salmonella for invasion, S. Choleraesuis strain showed higher invasion rate to Intestine 407 cell, but low invasion rate to LLC-PK1 cells.在台灣地區，豬霍亂沙門氏菌為腹瀉及敗血症豬隻中所分離之主要細菌病原菌。本研究利用脈衝式膠體電泳（PFGE）及逢機擴增多形性DNA（RAPD）方法針對95株2000至2002年間由南台灣地區腹瀉及全身性敗血症感染豬隻所分離之豬霍亂沙門氏菌進行次分型分析。脈衝式膠體電泳方法中使用XbaI、AvrⅡ、SpeI及NotI四種酵素對於染色體DNA進行作用，結果顯示，95株豬霍亂沙門氏菌菌株可得86種PFGE結合的分型。這些分型中，X1A1S1N1為主要次分型，有11株分離株屬於此分型，此型之菌株可能為最盛行之菌株分型。另一方面，利用RAPD方法分析95株分離株，可得74種分型。比較PFGE及RAPD兩種方法之分型，RAPD方法能將PFGE方法同一分型的菌株再給予次分型。因此，如果選擇適合之分型引子，RAPD方法針對株霍亂沙門氏菌次分型為一有效解析工具。 豬霍亂沙門氏菌能引起豬隻沙門氏菌症及人類的感染。傳統的沙門氏菌檢測方法須費時3-5天，因此本研究即在於發展PCR方法檢測。藉由比對基因庫中豬霍亂沙門氏菌、其他血清型沙門氏菌及其他菌種第一相鞭毛基因（fliC）之序列，設計出flinC-F及flinC-R引子對。以所設計之引子對分析97株豬霍亂沙門氏菌，皆可得預期分子量為963 bp之PCR產物。所有其他血清型沙門氏菌及非沙門氏菌菌株皆無PCR產物，但S. Paratyphi C菌株除外。利用先前文獻所發表之viaB基因檢測可將S. Paratyphi C及豬霍亂沙門氏菌區別。經過預増殖步驟後，於豬糞便、豬肉、豬肝臟、飼料及人類血液樣品中檢測豬霍亂沙門氏菌，可檢測之菌數為每克或每毫升100 CFU。 針對95株由南台灣養豬場所分離之豬霍亂沙門氏菌進行18種抗生素敏感性測試，結果顯示可得到46種抗生素圖譜，且所有菌株皆具多重抗藥性。利用PCR方法篩選抗藥性基因，ampicillin (TEM, and PSE group)、streptomycin- spectinomycin (aadA)、kanamycin (aphA)、sulfadiazine (sulI及sul3)、trimethoprim (dfrA12)、tetracycline (tetA)、streptomycin (strA及strB)、chloramphenicol (cmlA)、gentamycin (aac(3)Ⅳ)、fluoroquinolone (gyrA、parC及 parE)及class I integron。結果顯示95株豬霍亂沙門氏菌中有32.6%檢測出class I integron。更進一步檢測integron上抗藥基因種類，可得四種基因片匣組合，為dfrA12-aadA2、aadA5-aadA2、dfrA12-aad2-su1l及dfrA12-aadA5。由結果證明豬霍亂沙門氏菌抗藥性的問題相當值得注意。 部分沙門氏菌血清型具有毒性質體，這些血清型包括Typhimurium、 Choleraesuis、Dublin及Enteritidis等。本研究中針對95株豬霍亂沙門氏菌分離株進行質體分析，結果顯示豬霍亂沙門氏菌分離株中具有1~3個大的毒性質體。這些質體大小介於40 kb~190 kb，可分成14種型態。42株的分離株屬於主要的型態，含有50 kb及90 kb之質體。當利用PCR方法檢測spvR基因時，所有95株分離株皆有預期PCR產物。這結果也顯示spvR基因可能存在於大於50 kb之質體上。質體多樣化的演化可能為高頻率之基因交換所造成。 Microarray方法已變成一重要的技術用以檢測大量的基因，並且非常快速、具高度特異性及靈敏度。本研究目的即在發展一microarray方法同時檢測抗藥性基因、毒性基因及沙門氏菌特異性基因。共32基因佈放於microarray上，包括22個抗藥基因、3個毒性基因、5個沙門氏菌特異性基因及16S rDNA基因，由豬分離菌株DNA利用螢光標誌以作為進行雜合作用。取三株不同多重抗藥性豬霍亂沙門氏菌菌株進行檢測，分別為SCV28、SCV62及SCV27a。結果顯示所有受測菌株皆可得預期的結果。本研究所得結果證實以microarray檢測豬霍亂沙門氏菌分離株之抗藥性基因、毒性基因及沙門氏菌特異性基因為一實用平台。更進一步，此microarray可能應用於其他血清型沙門氏菌或親源相近之菌株檢測。 豬霍亂沙門氏菌對於動物及人類為高侵入性之血清型。本研究中，目的在於研究不同分子分型與侵入的關係、沙門氏菌侵入不同表皮細胞株的影響及不同血清型沙門氏菌的侵入性。隨機選取23株由PFGE所得不同分子分型之菌株與Intestine 407細胞進行侵入作用，取4株分離株針對不同細胞株進行試驗。另一方面，將不同血清型菌株Typhimurium、Derby、Enteritidis、Paratyphi C及Choleraesuis菌株對Intestine 407及LLC-PK1細胞株進行侵入試驗。結果顯示，不同分子分型分離株其侵入並無顯著不同，但結果也顯示，Intestine 407、HEp-2及 LLC-PK1細胞株對於沙門氏菌侵入則呈現不同的敏感性。比較不同血清型之沙門氏菌侵入性，豬霍亂沙門氏菌對於Intestine 407細胞株具有較高之侵入性，但對於LLC-PK1細胞株侵入性則較低。目次 頁數 表次………………………………………….…….……………………...….………Ⅰ 圖次……………………………………………..……………………………………Ⅲ 中文摘要………………………………………..……………………………………Ⅵ 英文摘要……………………………………………………..…………...……….…Ⅸ 文獻整理 1.沙門氏菌起源…………………………………………………………….………1 2.沙門氏菌屬之命名………………………………………………….……………1 3.豬霍亂沙門氏菌特性………………………………………………….…………2 4.生化反應…………………………………………………………….……………4 5.宿主特異性………………………………………………………….……………4 6.毒性因子與病原性…………………………………………………….…………6 7.豬霍亂沙門氏菌之臨床病學………………………………………...…………11 8.抗藥性……………………………………………………………………...……13 9.沙門氏菌病原性小島（Salmonella pathogenicity islands, SPIs）……...…..……14 10.毒性質體…………………………………………………..…………...………19 11.豬隻中沙門氏菌的散播及感染劑量……………………….………..……….22 12.分離與鑑定………………………………………………..…………...………22 13.研究目的…………………………………………………..…………...………23 參考文獻……………………………………………………..……………...…… 26 第一章 豬霍亂沙門氏菌之分子檢測方法研究 1-1. 前言……………..………………..…………..……………………………38 1-2. 材料與方法 1-2-1.儀器…………..………………………………..………………………39 1-2-2.電腦軟體……………………………….……………...………………40 1-2-3.藥品…………………………………………………..…..……………40 1-2-4.菌株……………………………………………..……..………………42 1-2-5.PFGE菌膠之製作……………………….………….…………………43 1-2-6.限制酶之剪切…………………………..……………......……………43 1-2-7.脈衝式電場膠體電泳…………………………………....……………44 1-2-8.Image analysis之方法………………………….………...……………45 1-2-9.RAPD-PCR條件…………………………….…….……..……………45 1-2-10.分型效能指數之計算……………………..…………………………46 1-3. 結果 1-3-1.PFGE…………………………………………………….….…………46 1-3-2.RAPD……………………………………………………..……………48 1-3-3.結合PFGE及RAPD分型方法…………..……………..……………48 1-4. 討論……………………………………………………….……………….49 參考文獻………………………………………………………………………..52 第二章 發展PCR方法檢測Salmonella enterica serovar Choleraesuis鞭毛基因fliC 2-1.前言………………………………………………………..….….…………70 2-2.材料方法 2-2-1.儀器……………………………………………………..……..………71 2-2-2.藥品……………………………………………………..…..…………72 2-2-3.菌株來源………………………………………………………………73 2-2-4.Primers之設計……………………………………………..…….……74 2-2-5.PCR增幅…………………………………………………...….………75 2-2-6.利用PCR檢測人類全血樣本中之S. enterica serovar Chloeraesuis…75 2-2-7.利用PCR檢測豬糞便樣本中之S. enterica serovar Chloeraesuis……75 2-2-8.利用PCR檢測豬肉、豬肝樣本中之S. enterica serovar Chloeraesuis..76 2-2-9.利用PCR檢測飼料樣本中之S. enterica serovar Chloeraesuis…....…76 2-2-10.利用傳統方法檢測食品樣本中之Salmonella屬……………..……77 2-3. 結果與討論 2-3-1.Primer特異性及檢測之靈敏度……………………….………………77 2-3-2.於多樣之樣本檢測S. enterica serovar Choleraesuis……...…….……79 2-4. 結論…………………………………………………………….…….……81 參考文獻…………………………………………………………….…….……82 第三章 豬霍亂沙門氏菌抗藥性及integron之探討 （A）部份：豬霍亂沙門氏菌抗藥性分析 3-1-1.抗藥性之由來…………………………..………………..……………96 3-1-2.畜產用抗生素………………………..…………..……………..……100 3-1-3.各類抗生素藥物之抗藥機制………..……..………………..………101 3-2. 材料與方法 3-2-1.菌種…………………………………………...…………...…………111 3-2-2.藥品…………………………………………..….………...…………111 3-2-3.培養基………………………………………...…………...…………112 3-2-4.緩衝液及試劑………………………….…..……………...…………113 3-2-5.儀器…………………………………..……………………....………113 3-2-6.電腦軟體…………………………..…………….…………...………114 3-2-7. 方法： （1）紙錠敏感性測試…………………………………………..………115 （2）PCR引子組………………………………………………..………115 （3）PCR條件……………………………………….…………..………116 （4）定序…………………………………………….………...…………118 3-3. 結果 3-3-1.Salmonella enterica serovar Choleraesuis的抗生素耐性測試…...…118 3-3-2.PCR增幅抗藥性基因結果……………….…………….……………120 3-3-3.各類抗生素抗性機制結果……………..……………………………120 3-4. 討論………………………………………………………………………124 （B）部份：豬霍亂沙門氏菌分離株integron之分析 3-5. 前言……………………………………………………………...…….…127 3-6. 方法 3-6-1.PCR方法檢測integron…………..…….…………………….………132 3-7. 結果 3-7-1.Integron分析結果……………………..…….……….………………133 3-7-2.Integron定序結果………………………………...………….………133 3-8. 討論…………………………………………………………….……..…134 參考文獻…………………………………………..………………….…...…..138 第四章 台灣地區豬霍亂沙門氏菌毒性質體之研究 4-1. 前言………………………………………………………..………..……186 4-2. 材料與方法 4-2-1.藥品…………………………………..…..……………..……………188 4-2-2.儀器…………………………………..………..………………..……190 4-2-3.菌株……………………………………..……………………....……190 4-2-4.質體製備………………………………..……………………....……190 4-2-5.PCR方法確認spv gene……………….…..………………….………191 4-3. 結果與討論…………………………………………..…………..………192 參考文獻………………………………………………………………....……194 第五章 豬霍亂沙門氏菌抗藥性生物晶片之發展 5-1. 前言………………………………………………………………..…..…199 5-2. 材料與方法 5-2-1.儀器………………………………..…….……………………………201 5-2-2.藥品……………………………………………………….…..………202 5-2-3.Microarray probe之製備……………..…..……………….……..……202 5-2-4.DNA labeling流程……………………….………………………...…203 5-2-5.雜合與分析……………………………………....………………...…203 5-3. 結果………………………………………………………………....……204 5-4. 討論……………………………………………………………....………205 參考文獻………………………………………………………………..………208 第六章 豬霍亂沙門氏菌in vitro細胞試驗之研究 6-1. 前言…………………………………………….….…………..…………222 6-2. 材料與方法 6-2-1.藥品………………………………………….……………..…………223 6-2-2.設備……………………………………………..………….....………225 6-2-3.菌株…………………………………………….…………..…………225 6-2-4.細胞株……………………………………………...……..……..……225 6-2-5.侵入性試驗…………………………………………...…..……..……226 6-2-6.不同血清型之細胞侵入性比較試驗…………..…...………..………227 6-2-7.細胞侵入後之顯微鏡觀察………………..………………….………227 6-2-8.Ceftriaxone 致死分析試驗……………….………………….....……228 6-3. 結果………………………………………………………………...….…228 6-4. 討論…………………………………………………………...…….……229 參考文獻……………………………………….…….…………..…….………23

番茄表面沙門氏菌及腸桿菌之檢測

Salmonella species have been regarded as important foodborne pathgens mainly come from the contaminated animal-sourced egg, poultry and meat food products. More recently, salmonellosis has been linked to tomatoes, seed sprouts, cantaloupe, apple juice, an沙門氏菌一向被視為是主要存在於動物來源之肉類及蛋類食品中的食品病原菌，但近年來的食品中毒案例顯示，番茄、香瓜、果及柳橙等蔬菜及水果中也可能存在有沙門氏菌。本研究之目的即在於檢測市售番茄樣品中沙門氏菌可能之污染及分佈情形。於民國93年四月至六月期間由臺中市八個行政區域共採集32個聖女小番茄樣品，分別使用UP broth (Universal pre-enrichment broth)與Lactose broth沖洗表面所得到的洗液，先進行增殖培養24小時後，利用選擇性培養基（BS、HE及XLD agar）