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.

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)

Thermostable direct hemolysin (TDH) cholera toxin ( CT ) 水（為主），食物及生活接觸
V. parahaemolyticus V.cholerae 1950年首次發現 1854年首次發現 H、O、K 01群(稻葉型霍亂弧菌及小川型霍亂弧菌)及非01群(0-139 霍亂弧菌 ) Thermostable direct hemolysin　(TDH) cholera toxin ( CT ) 水（為主），食物及生活接觸 2-48小時的潛伏期 24-48小時的潛伏期會伴隨輕度發燒病人通常不會發燒呈水性且黏液狀、15%之患者含血便有時有些黏液，不含血便，略帶甜味似洗米過後的水不會有低血壓低血壓攝氏75度時，15分鐘以上攝氏100度時，3分鐘兩者在生化試驗上的區別:TCBS及嗜鹽性試驗

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

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), ,2003 )

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

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)

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。

研究架構海水、有機樣本收集 (水質測量) 數據整理與分析傳統方法培養腸炎弧菌利用腸炎弧菌ToxR基因專一性利用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

採樣地點安平漁港興達漁港前鎮漁港

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

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

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

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)

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 )

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 )

生化鑑定試驗正反應負反應腸炎弧菌的反應斜面黃色紅色－ TSI 底部＋產氣斷裂硫化氫黑色非黑色 MTM 混濁狀
澄清狀革蘭氏染色陽性陰性 O/129敏感性 10μg 生長不生長 150μg Oixdase 深紫色非深紫色 ONPG 原色 Arginine 紫色 Lysine Halophilism 0%及10%Nacl 可生長不生長或緩慢 6%及8%Nacl 42℃ 混濁澄清 HLGB 歐普氏

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 .

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

RESULTS

Table 1.安平港水質結果水樣一水樣二水樣三 8 9 10 溫度 ( ℃) 31.56 29 30.1 30.29 31.86
濁度 (NTU) 9.07 2.79 4.45 8.45 2.82 4.04 2.74 2.77 比重 1.022 1.018 1.023 1.017 1.019 1.021 鹽度 (ppt) 35.61 29.02 29.42 34.79 28.07 31.58 30.37 33.46 透視度 (15cm) 28 19.2 15.6 25 14.2 13 23.1 22.4 22 PH ( ) 7.62 7.93 7.12 8.38 7.76 7.58 7.94 7.85 DO (>5.0mg/l) 16.33 5.02 4.98 10.06 5.73 5.94 7.49 4.93 4.31 TDS (56 ms / cm) 66.6 61.66 58.32 71.14 60.98 56.46 60.66 61.42 69.7 EC 48,000uS ~ 50,000uS(26℃) 2 49198 49252 56825 48285 47155 52597 48866 58613 COD (mg/l) 1650 1054 1430 1647 710 1563 1737 1036 1607

Table 2.興達港水質結果水樣一水樣二水樣三 8 9 10 溫度 ( ℃) 28 30 31.1 30.8 28.5
濁度 (NTU) 20.6 2.92 5.72 6.81 2.81 2.29 1.7 1.17 1.84 比重 1.014 1.022 1.023 1.018 1.021 1.025 鹽度 (ppt) 23.26 34.79 36.55 28.62 33.04 36.13 39.23 透視度 (15cm) 17.2 25 26.8 23.3 23.8 21.2 27.2 26.6 10.8 PH ( ) 6.89 7.84 8.02 7.41 7.91 7.83 7.08 7.89 7.94 DO (>5.0mg/l) 6.36 6.02 6.15 7.22 6.28 5.12 6.49 5.28 5.24 TDS (56 ms / cm) 45.28 67.1 70.74 56.22 67.92 74.8 63.44 73.32 74.2 EC 48,000uS ~ 50,000uS(26℃) 35046 51086 59923 43238 51643 62429 48920 55932 61852 COD (mg/l) 1384 1455 1536 1590 1605 1598 over 1346 1678

Table 3.前鎮港水質結果水樣一水樣二水樣三 8 9 10 溫度 ( ℃) 29 30.2 濁度 (NTU) 4.93 4.45
4.37 18.4 2.53 3.19 39.4 2.27 比重 1.022 1.024 1.025 1.021 1.023 鹽度 (ppt) 34.38 37.05 38.81 33.04 36.13 37.47 透視度 (15cm) 16 11 20.5 19.2 27.6 20.4 10.2 22 PH ( ) 8.27 8.11 8.18 8.14 8.23 8.16 8.17 DO (>5.0mg/l) 5.34 4.99 7.44 5.17 6.66 6.34 4.61 5.89 6.3 TDS (56 ms / cm) 72.8 73.54 73.66 72.48 73.92 72.92 72.86 73.7 71.58 EC 48,000uS ~ 50,000uS(26℃) 58467 56524 62761 58068 56673 61029 57814 56594 59572 COD (mg/l) over 1571 1168 1423

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

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
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

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 )。
正反應試管數 MPN/ml (g) 95%信賴界限 0.10mL 0.01mL 0.001mL 下限上限 <3.6 -- 9.5 2 21 4.5 42 1 3.0 0.15 9.6 28 8.7 94 11 35 6.1 1.2 18 3 29 6.2 36 9.4 3.6 38 23 4.6 0.17 110 7.2 1.3 64 17 180 43 9 7.4 20 75 200 120 37 420 160 40 15 93 16 150 9.2 1.4 210 430 14 290 90 1,000 240 3.7 460 2,000 1100 4,100 27 >1100

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) 0508Awater < < 3.6 0508Awater 0508Awater < < 3.6 0508Aorganics > 1100 0508Aorganics > 1100 0508Aorganics > 1100 0508Bwater 0508Bwater < 0508Bwater 0508Borganics < > 1100 0508Borganics > 1100 0508Borganics > 1100 0508Cwater < 0508Cwater < < 3.6 0508Cwater < < 3.6 0508Corganics 0508Corganics < > 1100 0508Corganics <

Data Temp pH MPN (MPN/ml或g) MPN-PCR(toxR) (MPN/ml或g) 0509Awater 0509Awater 0509Awater 0509Aorganics > 1100 0509Aorganics < > 1100 0509Aorganics < > 1100 0509Bwater 0509Bwater 0509Bwater > 1100 0509Borganics 0509Borganics 0509Borganics > 1100 0509Cwater < 0509Cwater < < 3.6 0509Cwater < < 3.6 0509Corganics < 0509Corganics 0509Corganics <

Data Temp pH MPN (MPN/ml或g) MPN-PCR(toxR) (MPN/ml或g) 0510Awater 0510Awater >1100 0510Awater 0510Aorganics >1100 0510Aorganics < 0510Aorganics < >1100 0510Bwater 0510Bwater >1100 0510Bwater 0510Borganics >1100 0510Borganics 0510Borganics >1100 0510Cwater < <3.6 0510Cwater 0510Cwater < 0510Corganics < <3.6 0510Corganics < 0510Corganics <

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

Fig 2. Amplification of the 16S rRNA gene by PCR
763bp The primers amplified a 763bp fragment Lane1…..marker Lane2…..0508C32 Lane3…..0508C Lane4…..0508C34 Lane5…..0508C Lane6…..0508C36 Lane7…..0508C Lane8…..0508C47 Lane9…..0508C Lane10….. negative control Lane11…no sample Lane12…...no sample

Fig 3. tdh Fig 4. trh 251bp 250bp The primers amplified a 251bp fragment Lane1……marker Lane2……0508A41 Lane3…… 0508A Lane4……0508A43 Lane5……0508A Lane6……0508A45 Lane7……0508A Lane8……0508A47 Lane9……0508A Lane10….0508A49 Lane11……vp Lane12…negative control The primers amplified a 250bp fragment Lane1……marker Lane2……0508A41 Lane3…… 0508A Lane4……0508A43 Lane5……0508A Lane6……0508A45 Lane7……0508A Lane8……0508A47 Lane9……0508A Lane10….0508A49 Lane11……vp Lane12…negative control

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 300bp 200bp

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 300bp 200bp

Sample (n) Culture method tdh-PCR Water (n= 27) 0 (0) 0 (0)
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) Organics (n = 27) (0) (14.8) Total (n= 54) (0) (7.4)

Sample (n) Culture method trh-PCR Water (n= 27) 0 (0) 0 (0)
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) Organics (n = 27) (3.7) (27.8) Total (n= 54) (1.9) (13.9)

Table 11. Serotyping of the 101 strains isolated
血清型數量

Fig 7. percentage of serotyping from three areas
% serotyping

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.

END Thank for your attention

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.

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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.

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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.

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