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Studies on vibrio parahaemolyticus by PCR during the four seasons in the south coast of taiwan

Studies on vibrio parahaemolyticus by PCR during the four seasons in the south coast of taiwan. 指導老師 : 張福林副教授 研究生 : 陳念群. Introduction. 致病三大弧菌. V.vulnificus V.cholerae V. parahaemolyticus 霍 亂 弧 菌 與 腸 炎 弧 菌 是 食 品 界 及 防 疫 單 位 最 重 視 的 食 品 中 毒 菌 ( 資料來源 : 行政院衛生署 ).

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Studies on vibrio parahaemolyticus by PCR during the four seasons in the south coast of taiwan

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  1. Studies on vibrio parahaemolyticus byPCR during the four seasons in the south coast of taiwan 指導老師:張福林副教授 研究生:陳念群

  2. Introduction

  3. 致病三大弧菌 V.vulnificus V.cholerae V. parahaemolyticus • 霍 亂 弧 菌 與 腸 炎 弧 菌 是 食 品 界 及 防 疫 單 位 最 重 視 的 食 品 中 毒 菌 (資料來源:行政院衛生署)

  4. TCBS V. parahaemolyticus :直徑1~2mm、有光澤、綠色 V. cholerae :直徑2~4mm 、平滑、黃色、中心不透明、周圍透明 • 嗜鹽性試驗(0% 、1% 、6% 、8% 、10%) V. parahaemolyticus :6% 、8%生長良好 V. cholerae :0% 、 1%生長良好

  5. Vibrio parahaemolyticus is one of the most important food-borne pathogens in Taiwan, Japan and other coastal countries. • Vibrio parahaemolyticus is a gram-negative, halophilic bacterium that occurs naturally in estuarine environments world-wide. (Environmental Microbiology 5(8),706-710 ,2003)

  6. 致病因子 • Thermostable direct hemolysin (TDH) • TDH/ • TDH-related haemolysin (TRH) • Urease positive • Lethal toxin • 附著因子

  7. Pathogenic V.parahaemolyticus generally produces a thermostable direct hemolysin (TDH). • More than 90% of clinical V.parahaemolyticus isolates, but fewer than 1% of food or environmental strains produce TDH. (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2003)

  8. Aim 橫跨四季有系統地蒐集﹑保存與研究台灣南部海域中的腸炎弧菌，並且進行一些基本特性分析，進而由分析結果來評估腸炎弧菌在台灣南部海域的分佈情形，而最後再來加以更深入的探討。

  9. 研究架構 海水、有機樣本收集 (水質測量) 利用PCR偵測樣本中 致病性腸炎弧菌(tdh/ trh)及分析KP和 血清型 傳統方法培養 腸炎弧菌 利用腸炎弧菌ToxR基因專一性 引子進行最確數-聚合酵素連鎖 反應(MPN-PCR),偵測樣本中全 部的腸炎弧菌密度 數據整理與分析

  10. 採樣地點 安平漁港 興達漁港 前鎮漁港

  11. 採樣方法 • 水樣:利用採樣筒丟入海水中採得水樣後放入無菌塑膠試管中 • 有機物質:刮取岸邊岩石上的藻類、貝類或水中魚類後放入無菌塑膠試管中 • 保存於室溫3小時內帶回實驗室分析

  12. 水質測量 • PH (METTLER TOLEDO) • 比重 • 鹽度 • 溫度 (VWR 1551-004) • 透視度 • 濁度 (HACH 46500-00) • 綜合水質DO、TDS、 EC (DR/2500 Prot HACH5900) • COD (HACH COD Reactor) 、(HACH ODYSSEY)

  13. Sample collection (3水樣3有機樣本) APW 每管APW各取1loop接種於TCBS 抽取DNA挑選典型綠色菌落分別各接種於TSB-2及TSA-2 PCR(toxR)由TSB-2 抽取DNA PCR (toxR) toxR(+): toxR(-): PCR (tdh) PCR (16S rRNA)toxR(+) PCR (trh) 致病性分析 生化鑑定 菌體抗原 PCR (tdh) KP試驗 PCR (trh)

  14. toxR • 對腸炎弧菌的專一性非常強 • L-GTCTTCTGACGCAATCGTTG R-ATACGAGTGGTTGCTGTCATG • (JOURNAL OF CLINICAL MICROBIOLOGY,Apr. 1999, p. 1173–1177)

  15. TDH & TRH • 通常致病性腸炎弧菌都會具有的致病因子 • TDH: L-GGTACTAAATGGCTGACATC R-CCACTACCACTCTCATATGC • TRH: L-GGCTCAAAATGGTTAAGCG R-CATTTCCGCTCTCATATGC (Applied and Environmental Microbiology, Nov. 2004, P.6401-6406)

  16. 16S rRNA • 所有的菌都具有16s rRNA ,於是選擇保守的序列來做為primer ,這樣就可知道樣本中的DNA是否有毀損. • L-ACGGCGCAGACTCCTACGGGAGGC R-GGGTTGCGCTCGTTGCGGCACTTA (Journal of Clinical Microbiology, Aug. 1994, P.1911-1917)

  17. 生化鑑定

  18. KP 使用 Wagatsuma agar 加以培養，若有 β溶血 ( 在培養基上形成透明圈 ) 現象則稱為 Kanagawa phenomenon ( KP 陽性 )，很可能就是致病菌株.

  19. 命 名 系 統 • （一）方式： 西元年/月/地點/樣本編號/菌株編號 • （二）說明： 西元年2005 →取後二碼 05 月份：01、02、03、04、05、06、07、08、09、10、11、12 地點：安平港→A 興達港→B 前鎮港→C • 樣本編號 : • 菌株編號：自01開始編號 • 例如 : 0508A1302

  20. RESULTS

  21. Table 1.安平港水質結果

  22. Table 2.興達港水質結果

  23. Table 3.前鎮港水質結果

  24. Fig1.Amplification of the toxR gene by PCR 1 2 3 4 5 6 The primers amplified a 368bp fragment Lane1…….100bp marker Lane2…….CCRC 10806 Lane3…….negative control Lane4…….(+)sample Lane5…….(-)sample Lane6…….100bp marker 368bp

  25. 原液 10倍 100倍 1000倍 10ml原液 90ml緩衝液 90ml緩衝液 90ml緩衝液 每稀釋檢液各接種3支(稱三階三支），並於35~37℃，培養16~18小時

  26. Table 4.判定為腸炎弧菌陽性者，利用最確數表（如附表），推算出腸炎弧菌之最確數(MPN/g或MPN/ml )。

  27. Table 5. Vibrio parabaemolyficus densities in different samples from three areas Data Temp. pH MPN (MPN/ml或g) MPN-PCR(toxR) (MPN/ml或g) 0508Awater1 32 7.62 < 3.6 < 3.6 0508Awater2 30 8.38 15 93 0508Awater3 32 7.58 < 3.6 < 3.6 0508Aorganics1 32 7.62 160 > 1100 0508Aorganics2 30 8.38 35 > 1100 0508Aorganics3 32 7.58 290 > 1100 0508Bwater1 28 6.89 3.6 93 0508Bwater2 28 7.41 < 3.6 240 0508Bwater3 29 7.08 6.2 9.4 0508Borganics1 28 6.89 < 3.6 > 1100 0508Borganics2 28 7.41 3.0 > 1100 0508Borganics3 29 7.08 28 > 1100 0508Cwater1 29 8.27 < 3.6 3 0508Cwater2 29 8.27 < 3.6 < 3.6 0508Cwater3 29 8.27 < 3.6 < 3.6 0508Corganics1 29 8.27 3.6 460 0508Corganics2 29 8.27 < 3.6 > 1100 0508Corganics3 29 8.27 < 3.6 64

  28. Table 6. Vibrio parabaemolyficus densities in different samples from three areas Data Temp. pH MPN (MPN/ml或g) MPN-PCR(toxR) (MPN/ml或g) 0509Awater1 29 7.93 15 9.2 0509Awater2 29 7.93 7.4 9.2 0509Awater3 29 7.94 3.6 23 0509Aorganics1 29 7.93 7.2 > 1100 0509Aorganics2 29 7.93 < 3.6 > 1100 0509Aorganics3 29 7.94 < 3.6 > 1100 0509Bwater1 30 7.84 3.0 3.0 0509Bwater2 30 7.91 3.6 43 0509Bwater3 30 7.89 15 > 1100 0509Borganics1 30 7.84 3.0 1100 0509Borganics2 30 7.91 15 290 0509Borganics3 30 7.89 6.1 > 1100 0509Cwater1 29 8.11 < 3.6 3.6 0509Cwater2 29 8.14 < 3.6 < 3.6 0509Cwater3 29 8.16 < 3.6 < 3.6 0509Corganics1 29 8.11 < 3.6 3.6 0509Corganics2 29 8.14 3.6 23 0509Corganics3 29 8.16 < 3.6 23

  29. Table 7. Vibrio parabaemolyficus densities in different samples from three areas Data Temp. pH MPN (MPN/ml或g) MPN-PCR(toxR) (MPN/ml或g) 0510Awater1 30.1 7.12 3 93 0510Awater2 30.1 7.76 3 >1100 0510Awater3 30.1 7.85 9.2 36 0510Aorganics1 30.1 7.12 7.4 >1100 0510Aorganics2 30.1 7.76 <3.6 9.2 0510Aorganics3 30.1 7.85 <3.6 >1100 0510Bwater1 31.1 8.02 9.2 93 0510Bwater2 30.8 7.83 6.1 >1100 0510Bwater3 30.8 7.94 9.2 43 0510Borganics1 31.1 8.02 3.0 >1100 0510Borganics2 30.8 7.83 15 93 0510Borganics3 30.8 7.94 6.2 >1100 0510Cwater1 30.2 8.18 <3.6 <3.6 0510Cwater2 30.2 8.23 3.0 75 0510Cwater3 30.2 8.17 <3.6 3.6 0510Corganics1 30.2 8.17 <3.6 <3.6 0510Corganics2 30.2 8.23 <3.6 23 0510Corganics3 30.2 8.17 <3.6 23

  30. Table 8. Detection of Vibrio parahaemolyticus from water and organic samples V. parahaemolyticus detected by Sample (n) Culture method toxR-PCR Water (n= 27) 14 (51.9) 20 (74.1) Organics (n = 27) 18 (66.7) 26 (96.3) Total (n= 54) 32 (59.3) 46 (85.2)

  31. Fig2.Amplification of the 16S rRNA gene by PCR 1 2 3 4 5 6 7 8 9 10 11 12 763bp The primers amplified a 763bp fragment Lane1…..marker Lane2…..0508C32 Lane3…..0508C33 Lane4…..0508C34 Lane5…..0508C35 Lane6…..0508C36 Lane7…..0508C45 Lane8…..0508C47 Lane9…..0508C48 Lane10….. negative control Lane11…no sample Lane12…...no sample

  32. Fig 3. tdhFig 4. trh 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 251bp 250bp The primers amplified a 251bp fragment Lane1……marker Lane2……0508A41 Lane3…… 0508A42 Lane4……0508A43 Lane5……0508A44 Lane6……0508A45 Lane7……0508A46 Lane8……0508A47 Lane9……0508A48 Lane10….0508A49 Lane11……vp1 Lane12…negative control The primers amplified a 250bp fragment Lane1……marker Lane2……0508A41 Lane3…… 0508A42 Lane4……0508A43 Lane5……0508A44 Lane6……0508A45 Lane7……0508A46 Lane8……0508A47 Lane9……0508A48 Lane10….0508A49 Lane11……vp2 Lane12…negative control

  33. Fig 5. Amplification of the tdh gene by PCR The primers amplified a 251bp fragment Lane1…..marker Lane2…..0508B44 Lane3…..0508B45 Lane4…..0508B46 Lane5…..0508B47 Lane6…..0508B48 Lane7…..0508B49 Lane8…..0508B50 Lane9…..0508B51 Lane10…..0508B52 Lane11…..positive control Lane12…..negative control 1 2 3 4 5 6 7 8 9 10 11 12 300bp 200bp

  34. Fig 6. Amplification of the trh gene by PCR The primers amplified a 250bp fragment. Lane1…..marker Lane2…..0508B53 Lane3…..0508B54 Lane4…..0508B55 Lane5…..0508B56 Lane6…..0508B57 Lane7…..0508B58 Lane8…..0508B59 Lane9…..0508B60 Lane10…0508B61 Lane11…..positive control Lane12…..negative control 1 2 3 4 5 6 7 8 9 10 11 12 300bp 200bp

  35. Table 9. Detection of haemolysin genes (tdh) from water and organic samples tdh detected by Sample (n) Culture method tdh-PCR Water (n= 27) 0 (0) 0 (0) Organics (n = 27) 0 (0) 4 (14.8) Total (n= 54) 0 (0) 4 (7.4)

  36. Table 10. Detection of haemolysin genes (trh) from water and organic samples trh detected by Sample (n) Culture method trh-PCR Water (n= 27) 0 (0) 0 (0) Organics (n = 27) 1 (3.7) 5 (27.8) Total (n= 54) 1 (1.9) 5 (13.9)

  37. Table 11. Serotyping of the 101 strains isolated O1………………………………………………21 O2………………………………………………20 O3………………………………………………16 O4………………………………………………5 O5………………………………………………8 O6………………………………………………0 O7………………………………………………3 O8………………………………………………3 O9………………………………………………1 O10……………………………………………23 O11……………………………………………..1 血清型 數量

  38. Fig 7. percentage of serotyping from three areas % serotyping

  39. Conclusion • 研究中所發現的tdh(7.4)及trh(13.9)大多是藉由MPN-PCR所發現(MPN只發現1.9%trh). • 在MPN與MPN-PCR兩者間比較的話,可以發現藉由MPN-PCR所偵測到的腸炎弧菌數是較多的. • 在三個採樣點中可以發現,其中前鎮港所分離出的菌量都是很少量的,其次是安平港.

  40. END Thank for your attention

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