活的非可培養(yǎng)狀態(tài)溶藻膠弧菌復(fù)蘇后的致病性及基因表達(dá)

姜熒安,陳吉祥＊＊,賈俊濤,郭倩茹,趙明君

(1.中國海洋大學(xué),山東青島266003;2.山東出入境檢驗(yàn)檢疫局,山東青島266002;3.廣東海洋大學(xué),廣東湛江524088)

研究簡(jiǎn)報(bào)

活的非可培養(yǎng)狀態(tài)溶藻膠弧菌復(fù)蘇后的致病性及基因表達(dá)

姜熒安1,陳吉祥1＊＊,賈俊濤2,郭倩茹3,趙明君1

(1.中國海洋大學(xué),山東青島266003;2.山東出入境檢驗(yàn)檢疫局,山東青島266002;3.廣東海洋大學(xué),廣東湛江524088)

溶藻膠弧菌廣泛分布于海洋環(huán)境,是海洋生物及人類的條件性致病菌。溶藻膠弧菌在不良環(huán)境進(jìn)入活的非可培養(yǎng)狀態(tài),當(dāng)條件適宜時(shí)復(fù)蘇為可培養(yǎng)形式。本文研究了溶藻膠弧菌由活的非可培養(yǎng)狀態(tài)復(fù)蘇為可培養(yǎng)狀態(tài)時(shí)的生理特征及對(duì)斑馬魚的致病性,結(jié)果發(fā)現(xiàn)復(fù)蘇為可培養(yǎng)狀態(tài)的溶藻膠弧菌對(duì)紫外輻射、熱激、冷激的抵抗力沒有明顯改變。用RT-PCR未檢測(cè)到VBNC狀態(tài)溶藻膠弧菌tox S和tox R基因的表達(dá),而復(fù)蘇后的細(xì)胞檢測(cè)到這些基因的表達(dá)。復(fù)蘇后的溶藻膠弧菌對(duì)斑馬魚的LD50為6.25×106CFU/尾,與野生菌株(LD50為4.80×106CFU/尾)沒有顯著差別。

溶藻膠弧菌;活的非可培養(yǎng)狀態(tài);復(fù)蘇;致病性;基因表達(dá)

溶藻膠弧菌(Vibrio alginolyticus)是1種嗜鹽性革蘭氏陰性細(xì)菌,廣泛分布于海洋、河口及海洋生物中[1-3],可引起海水魚、蝦、貝類的疾病,給水產(chǎn)養(yǎng)殖業(yè)帶來較大的經(jīng)濟(jì)損失[4-6]。該菌也是沿海地區(qū)人類腹瀉和食物中毒的常見病原菌[7-9]。Kahla-Nakbi等從突尼斯地中海沿岸的患病烏頰海鯛(S parus aurata)和海鱸(Dicentrarchus labrax)體內(nèi)分離出溶藻膠弧菌,發(fā)現(xiàn)7株溶藻膠弧菌對(duì)這2種魚類有致病性,LD50的范圍從5.01×104～6.20×107CFU/尾[10]。溶藻膠弧菌致病相關(guān)的毒力因子包括載鐵體、蛋白酶類及外毒素。Yan等發(fā)現(xiàn)溶藻膠弧菌能夠強(qiáng)烈黏附到大黃魚的腸道黏液中,并且各種環(huán)境因素對(duì)其黏附性有很大的影響[11]。Lee等研究發(fā)現(xiàn),溶藻膠弧菌的胞外產(chǎn)物可引起日本對(duì)蝦血漿中抗脂多糖因子消失、血藍(lán)蛋白減少[12]。Chen等發(fā)現(xiàn)堿性絲氨酸蛋白酶是溶藻膠弧菌分泌的主要蛋白酶,也是對(duì)蝦的主要致病因子之一[13]。有關(guān)溶藻膠弧菌致病的機(jī)理仍有許多問題有待解決。

許多病原菌在不良環(huán)境中能形成活的非可培養(yǎng)狀態(tài)(Viable but nonculturable state,VBNC),用常規(guī)方法培養(yǎng)不能生長繁殖,但仍然具有代謝活性,VBNC狀態(tài)的細(xì)菌在一定條件下復(fù)蘇為可培養(yǎng)形式,部分病原菌在復(fù)蘇之后保持了致病性[14-15]。溶藻膠弧菌在低溫寡營養(yǎng)條件下能進(jìn)入活的非可培養(yǎng)狀態(tài),當(dāng)采用升溫和添加營養(yǎng)物質(zhì)時(shí)能復(fù)蘇為可培養(yǎng)狀態(tài)[16],本文的目的是進(jìn)一步研究VBNC狀態(tài)的溶藻膠弧菌復(fù)蘇后的生理特性和致病性,并研究其毒力相關(guān)基因tox R和tox S的表達(dá)情況,以揭示其疾病發(fā)生和傳播機(jī)制。

1 材料和方法

1.1 實(shí)驗(yàn)菌株

溶藻膠弧菌VIB 283(LMG 4408T,ATCC17749T)來自英國Heriot-Watt大學(xué)。

1.2 溶藻膠弧菌VBNC狀態(tài)的誘導(dǎo)及復(fù)蘇

實(shí)驗(yàn)用海水采自青島沿海水域,用孔徑0.22μm微孔濾膜過濾,以除去顆粒物質(zhì),121℃滅菌20 min。將培養(yǎng)至對(duì)數(shù)生長期的溶藻膠弧菌接種到滅菌的過濾海水中,使其終濃度為107CFU/mL,靜置于4℃冰箱里培養(yǎng),每隔一定時(shí)間取樣進(jìn)行檢測(cè)。VBNC狀態(tài)的溶藻膠弧菌采用添加營養(yǎng)物質(zhì)升溫的方法進(jìn)行復(fù)蘇。

1.3 復(fù)蘇的溶藻膠弧菌對(duì)環(huán)境應(yīng)激的敏感性

由VBNC狀態(tài)復(fù)蘇的溶藻膠弧菌細(xì)胞和正常生長的細(xì)胞分別在2216 E液體培養(yǎng)基中培養(yǎng)12 h,收集菌懸液,8 000 r/min,離心10 min收集沉淀細(xì)胞,用等體積PBS(p H=7.4)洗滌,稀釋至適當(dāng)濃度用于環(huán)境應(yīng)激試驗(yàn)。

紫外照射:用PBS將正常與復(fù)蘇的溶藻膠弧菌10倍梯度稀釋到102,103,104CFU/mL 3個(gè)濃度,充分混合均勻后取細(xì)胞懸液0.1 mL,分別涂布于2216 E平板,無菌條件下進(jìn)行紫外照射,紫外燈功率為15 W,照射距離為30 cm,照射時(shí)間分別為0,20,40,60和80 s,照射結(jié)束后置于暗處避免光修復(fù),于28℃培養(yǎng)24 h,進(jìn)行菌落計(jì)數(shù)并計(jì)算存活率。

熱激處理:用PBS將正常與復(fù)蘇的溶藻膠弧菌10倍梯度稀釋到102,103,104CFU/mL 3個(gè)濃度,再置于42℃水浴中分別處理0,30,60和90 min,定時(shí)取樣并迅速置于冰浴中冷卻至室溫,涂布2216 E平板,28℃培養(yǎng)24 h,進(jìn)行菌落計(jì)數(shù)并計(jì)算存活率[17]。

冷激處理:用PBS將正常與復(fù)蘇的溶藻膠弧菌10倍梯度稀釋到102,103,104CFU/mL 3個(gè)濃度,分別在4℃冰箱中處理0,30,60和90 min,定時(shí)取樣涂布2216 E平板,于28℃培養(yǎng)24 h,進(jìn)行菌落計(jì)數(shù)并計(jì)算存活率[18]。

1.4 RT-PCR檢測(cè)不同狀態(tài)溶藻膠弧菌毒力基因的表達(dá)

根據(jù)GenBank已有的溶藻膠弧菌的tox S和tox R基因序列,利用Primer 5.0分別設(shè)計(jì)2對(duì)簡(jiǎn)并引物。tox S基因引物:5’-CCACTGGCGGACAAAATAACC-3’;5’-AACAGTACCGTAGAACCGTGA-3’,擴(kuò)增目的片斷為640 bp。tox R基因引物:

5’-TTTGTTTGGCGTGAGCAAGGTTTT-3’;5’-GGTTATTT TGTCCGCCAGTGG-3’,擴(kuò)增目的片斷為595 bp。引物由上海生物工程公司合成。

正常和復(fù)蘇的溶藻膠弧菌細(xì)胞在28℃培養(yǎng)24 h,用無菌生理鹽水分別從2216 E斜面上洗下,4℃、10 000 r/min離心10 min收集菌體。VBNC狀態(tài)溶藻膠弧菌細(xì)胞于4℃、12 000 r/min離心30 min進(jìn)行收集。用Trizol(Invitrogen)試劑抽提正常狀態(tài)、復(fù)蘇狀態(tài)以及VBNC狀態(tài)菌體的總RNA,用無RNase的DNaseⅠ處理提取的RNA樣品。取4μL DNaseⅠ處理后的RNA,使用M-MLV逆轉(zhuǎn)錄酶合成cDNA第1條鏈。然后對(duì)cDNA第1條鏈進(jìn)行PCR擴(kuò)增。PCR體系為:10×PCR buffer 5μL;25 mmol/L MgCl23 μL;200μmol/L dNTP 1μL;cDNA模板0.5μL;1.25 UTaq聚合酶0.5μL和50μmol/L上游下游引物0.5 μL,用雙蒸水補(bǔ)至50μL。PCR反應(yīng)參數(shù)為:94℃預(yù)變性5 min;94℃變性1 min;52℃退火1 min;72℃延伸1 min;30個(gè)循環(huán)72℃延伸10 min,PCR產(chǎn)物于1%瓊脂糖凝膠電泳。

1.5 復(fù)蘇溶藻膠弧菌對(duì)斑馬魚的致病性

健康斑馬魚110尾隨機(jī)分為11組,每組10條,分別于2 L的容器中,20～25℃條件下飼養(yǎng),定期喂食。正常和復(fù)蘇的溶藻膠弧菌于2216 E液體培養(yǎng)基中過夜培養(yǎng),用滅菌的生理鹽水洗脫,稀釋成不同濃度(105,106,107,108和109CFU/mL)。每組分別腹腔注射20μL不同濃度的正常菌液和復(fù)蘇菌液,對(duì)照組腹腔注射生理鹽水20μL。注射的斑馬魚在相同的條件下飼養(yǎng)30 d,記錄死亡數(shù),計(jì)算半數(shù)致死量(LD50)[19]。

2 結(jié)果

2.1 復(fù)蘇溶藻膠弧菌對(duì)環(huán)境應(yīng)激的抵抗力

溶藻膠弧菌在自然海水中4℃保存90 d后進(jìn)入活的非可培養(yǎng)狀態(tài)。在活的非可培養(yǎng)狀態(tài)的溶藻膠弧菌懸液中加入終濃度為0.025%的酵母液,28℃培養(yǎng)16 h后,得到復(fù)蘇的細(xì)胞,經(jīng)16 S rRNA基因序列分析確定為溶藻膠弧菌。

在紫外照射下,正常狀態(tài)和復(fù)蘇的溶藻膠弧菌細(xì)胞的存活率隨時(shí)間延長而逐漸降低,照射80 s后2種狀態(tài)的細(xì)胞都失去培養(yǎng)能力,正常和復(fù)蘇細(xì)胞的存活率沒有明顯差別(見圖1A)。在42℃熱激條件下,正常和復(fù)蘇細(xì)胞的存活率隨著處理時(shí)間的延長迅速降低,90 min后細(xì)胞大部分失去培養(yǎng)能力,2種狀態(tài)細(xì)胞的存活率分別是12.38%和2.73%(見圖1B)。在4℃冷激條件下,正常和復(fù)蘇細(xì)胞存活率均沒有明顯降低, 90 min后正常狀態(tài)的細(xì)胞存活率保持在101.90%,復(fù)蘇的細(xì)胞存活率為94.55%(見圖1C)。

圖1 正常與復(fù)蘇狀態(tài)溶藻膠弧菌VIB 283在不同環(huán)境應(yīng)激的存活曲線:A,紫外輻射;B,熱激(42℃)處理;C,冷激(4℃)處理Fig.1 Survival curves of normal and resuscitative cells ofVibrio alginolyticusVIB 283 under different environmental stresses:A,ultraviolet radiation;B,heat shock;C,cold shock

2.2 RT-PCR檢測(cè)溶藻膠弧菌tox R和tox S基因的表達(dá)

溶藻膠弧菌VIB 283在正常和復(fù)蘇狀態(tài)tox R和tox S基因均有表達(dá),而在VBNC狀態(tài)下沒有檢測(cè)到表達(dá)(見圖2)。

2.3 溶藻膠弧菌的致病性

將正常狀態(tài)和復(fù)蘇狀態(tài)的溶藻膠弧菌分別腹腔注射斑馬魚,結(jié)果發(fā)現(xiàn)復(fù)蘇狀態(tài)的溶藻膠弧菌恢復(fù)了對(duì)斑馬魚的致病性,復(fù)蘇后菌株對(duì)斑馬魚的LD50為6.25 ×106CFU/尾,而正常野生菌株對(duì)斑馬魚的LD50為4.80×106CFU/尾,復(fù)蘇后的溶藻膠弧菌的毒力沒有明顯降低。

圖2 RT-PCR檢測(cè)不同狀態(tài)溶藻膠弧菌VIB 283tox R基因(A)和tox S基因表達(dá)(B)。Fig.2 RT-PCR detection oftox Rgene(A)andtox Sgene (B)ofVibrio alginolyticusVIB 283 in different status.

表1 溶藻膠弧菌VIB 283正常狀態(tài)和復(fù)蘇狀態(tài)細(xì)胞對(duì)斑馬魚的致病性Table 1 Virulence to zebra fish of normal and resuscitative cells ofVibrio alginolyticusVIB 283

3 討論

已報(bào)道形成VBNC狀態(tài)的細(xì)菌超過60種,其中大部分是人和動(dòng)物的致病菌。Baffone等報(bào)道溶藻膠弧菌在人工海水中保存28～30 d時(shí)進(jìn)入VBNC狀態(tài)[20]。研究發(fā)現(xiàn)溶藻膠弧菌VIB 283在自然海水中低溫保持90 d后進(jìn)入活的非可培養(yǎng)狀態(tài),進(jìn)入VBNC狀態(tài)的細(xì)胞形態(tài)發(fā)生變化,變?yōu)榍蛐?細(xì)胞體積縮小,當(dāng)復(fù)蘇為可培養(yǎng)形式后,細(xì)胞恢復(fù)為正常形態(tài)。復(fù)蘇的溶藻膠弧菌與正常細(xì)胞相比對(duì)紫外照射、冷激和熱激的抵抗力沒有顯著降低。表明溶藻膠弧菌從非可培養(yǎng)狀態(tài)復(fù)蘇為可培養(yǎng)狀態(tài)后對(duì)環(huán)境的適應(yīng)能力沒有顯著降低。

不同病原菌在VBNC狀態(tài)時(shí)的基因表達(dá)和毒性有一定差異。Rahman等發(fā)現(xiàn)痢疾志賀氏菌1型的VBNC細(xì)胞保留若干致病因素和潛在的毒力[21]。 Gonzalez-Escalona等發(fā)現(xiàn)VBNC狀態(tài)的霍亂弧菌tuf基因表達(dá)量下降,relA與rpoS基因表達(dá)量增加[22]。Colwell等發(fā)現(xiàn)注射VBNC狀態(tài)的霍亂弧菌能導(dǎo)致受測(cè)試志愿者的腹瀉癥狀,同時(shí)發(fā)現(xiàn)此菌在進(jìn)入VBNC狀態(tài)28 d后,仍能產(chǎn)生霍亂毒素,PCR檢測(cè)說明產(chǎn)生霍亂毒素的基因仍然存在[23]。Coutard報(bào)導(dǎo)處于VBNC狀態(tài)的副溶血弧菌可檢測(cè)到16S-23S rRNA和rpoS基因表達(dá),但沒有檢測(cè)到tdh1和tdh2基因表達(dá)[24]。Saux發(fā)現(xiàn)不同來源的創(chuàng)傷弧菌在人工海水形成VBNC時(shí)能檢測(cè)溶血素基因的表達(dá)[25]。Baffone報(bào)道從VBNC復(fù)蘇的副溶血弧菌暫時(shí)失去了對(duì)小鼠的致病性,而這些細(xì)胞在結(jié)扎的小鼠回腸模型(Ileal loop model)中傳代2次后又恢復(fù)了致病性[20]。作者研究發(fā)現(xiàn)復(fù)蘇的溶藻膠弧菌對(duì)斑馬魚的致病性與正常狀態(tài)的溶藻膠弧菌相比并沒有顯著降低。說明VBNC狀態(tài)的溶藻膠弧菌仍然保留著潛在的毒力,當(dāng)在一定的適宜環(huán)境下復(fù)蘇后即重新表達(dá)并產(chǎn)生毒力和感染性。

tox R基因在弧菌中廣泛分布,能對(duì)弧菌的多種毒力基因的表達(dá)進(jìn)行調(diào)控。tox R基因最初是在霍亂弧菌(Vibrio cholerae)中發(fā)現(xiàn),它對(duì)編碼霍亂毒素的ctx基因具有轉(zhuǎn)錄調(diào)控作用[26],隨后還發(fā)現(xiàn)tox R基因可以編碼1種跨膜蛋白,該蛋白對(duì)ctx、tcp及omp U、omp T基因的協(xié)同調(diào)控有重要作用[27-30]。tox S基因與tox R基因位于同一個(gè)操縱子,它處于tox R基因的下游。tox S基因能夠明顯增強(qiáng)由tox R基因調(diào)控的轉(zhuǎn)錄反應(yīng)[31]。Tox R和Tox S蛋白共同調(diào)控著tox T基因的表達(dá),而tox T蛋白作為毒力基因級(jí)聯(lián)調(diào)控的一部分,能夠激活至少8種不同的毒力基因啟動(dòng)子[28]。我們用RT-PCR沒有檢測(cè)到VBNC狀態(tài)的溶藻膠弧菌tox R和tox S基因的表達(dá),但當(dāng)其復(fù)蘇為可培養(yǎng)狀態(tài)時(shí),tox R和tox S基因的表達(dá)又恢復(fù)到可檢測(cè)水平。推測(cè)tox R和tox S基因?qū)Χ玖虻谋磉_(dá)調(diào)控作用可能與溶藻膠弧菌復(fù)蘇后恢復(fù)致病性相關(guān)。

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Abstract: Vibrio alginolyticusis widely distributed in marine environment.It is an opportunistic pathogen of human being and marine animals.The bacterium is able to adapt to environmental stresses and enter into the viable but nonculturable(VBNC)state.The VBNC cells could be recovered to culturable state under certain conditions.This study investigated the physiological characteristics and pathogenicity to zebra fish of the resuscitative cells.The sensitivity of the resuscitative cells to UV,high temperature (42℃)and cold stresses did not change obviously when compared with that of the normal cells.Reverse transcription-polymerase chain reaction(RT-PCR)was used to identify expression level of virulence relatedtoxSandtoxRgenes of the resuscitative cells.The expressions oftoxSandtoxRgenes were not detected in the VBNC cells but in the resuscitative cells.The resuscitative cells retained their virulence when they were intraperitoneally inoculated into zebra fish.The LD50of the wild-type cells and the resuscitative cells was 4.80×106CFU per fish and 6.25×106CFU per fish respectively,which showed no significant difference.

Key words: Vibrio alginolyticus;VBNC;physiological characteristics;gene expression;pathogenicity

責(zé)任編輯 于 衛(wèi)

Virulence Retention and Gene Expression of Vibrio alginolyticus VIB 283 Resuscitated from Viable but Nonculturable State

J IANG Ying-An1,CHEN Ji-Xiang1,J IA Jun-Tao2,GUO Qian-Ru3,ZHAO Ming-Jun1
(1.Ocean University of China,Qingdao 266003,China;2.Shandong Entry-Exit Inspection and Quarantine Bureau,Qingdao 266002,China;3.Guangdong Ocean University,Zhanjiang 524088,China)

O179.29

A

1672-5174(2010)09Ⅱ-142-05

國家自然科學(xué)基金項(xiàng)目(30972275);農(nóng)業(yè)部公益性行業(yè)專項(xiàng)(nyhyzx 07-046);國家質(zhì)檢總局科研項(xiàng)目(2010IK180)資助

2009-12-09;

2010-05-25

姜熒安(1986-),女,碩士生,研究方向:海洋微生物學(xué)。E-mail:trick203@163.com。

Tel:+86-532-82031561;Fax:+86-532-82031561;E-mail:betcen@ouc.edu.cn