Progress in research of viable but non-culturable state of Vibrio cholerae
CHEN Bao-li, KAN Biao
State Key Laboratory for Communicable Diseases Prevention and Control, Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Abstract
Vibrio cholerae, widely existing in marine and estuarine environments, is the causative agent of cholera. When V. cholerae cells are subjected to stringent conditions they may form viable but not culturable (VBNC) cells. These bacteria can recover from VBNC state and may pose risk to human. This paper summarizes the progress in the research of the VBNC state of V. cholerae for the better surveillance of V. cholerae.
霍乱弧菌活的非可培养状态研究进展
陈保立, 阚飙
中国疾病预防控制中心传染病预防控制所, 北京 昌平 102206
摘要
霍乱弧菌是霍乱的病原体,广泛存在于海洋及江河入海口环境中。在不良环境中霍乱弧菌会进入活的非可培养状态(viable but non-culturable state, VBNC),常规分离方法检测不到,在条件合适时VBNC状态的霍乱弧菌会复苏为可培养状态。本文对近年霍乱弧菌VBNC相关研究做了回顾,以期能够为霍乱弧菌越冬机制以及霍乱的监测提供参考。
内容大纲
1 VBNC状态的霍乱弧菌形态及生理生化上的改变
从患者中新分离的霍乱弧菌菌体呈短小弯曲的杆状，一般长1.5～2.0 μm，宽0.3～0.4 μm，运动极为活泼，在显微镜下可见“鱼群”样穿梭运动。霍乱弧菌进入VBNC状态的会发生一系列形态上的改变，常表现为菌体萎缩、体积变小、由杆状或弧状变为球杆状或球状。Chaiyanan等[5]用透射电镜和扫描电镜分别对在人造海水中培养的霍乱弧菌进行了为期2年的观察，他们发现在霍乱弧菌首先从杆状变成不规则的杆状(～1 μm)，然后变成球形(～0.8 μm)，最终会变成极小的球形(0.07～0.4 μm)，还发现以上3种形态的霍乱弧菌都可以彼此之间相互黏附在一起，形成团状。Alam等[6]也发现霍乱弧菌进入VBNC状态过程中会聚集在一起形成生物膜，而且只有处于生物膜内部的VBNC状态的霍乱弧菌能够复苏。虽然VBNC细菌的形态发生了显著的改变，但细菌的结构仍然完整，由细胞质、细胞膜、细胞壁及外膜构成，只是外膜呈波浪状并带有一些细微的纤维，细胞壁肽聚糖比正常细胞的稍薄[7]。
除形态上发生改变外，霍乱弧菌进入VBNC状态后在生理生化上也有着重要的改变。研究发现，随着时间的延长，低营养条件下的霍乱弧菌总脂及碳水化合物的含量急剧降低，DNA及蛋白的含量也持续降低，然而RNA的量却无明显变化[8]。
2 霍乱弧菌进入VBNC状态过程中基因表达的变化
霍乱弧菌进入VBNC状态以后，大多数基因的转录都会降低。Gonzalez-Escalona等[9]用定量
反转录-聚合酶链反应(RT-PCR)的方法研究了霍乱弧菌VBNC状态的压力反应基因rpoS和relA以及蛋白合成相关基因16S rRNA和tuf基因的转录水平，与对数期相比这些基因的转录水平都明显降低。Asakura等[10]同样发现相对于平台期，绝大部分基因的转录水平是降低，不过矛盾的是，Gonzalez-Escalona等[9]认为16S rRNA在VBNC状态下转录水平降低，而Asakura等[10]认为16S rRNA的转录水平并没有下降的趋势。霍乱弧菌进入VBNC状态过程仍有一些基因的转录会相对升高，Asakura等[10]发现处于VBNC状态的霍乱弧菌相对于生长平台期大约有100个基因存在上调5倍表达，其中大部分与细胞过程相关。
3 霍乱弧菌VBNC状态的诱导及复苏
环境中各种因素，如温度、pH值、营养条件、溶氧水平、甚至光照等发生改变不利于细菌生长繁殖时均可诱导细菌进入VBNC状态[11]。对于霍乱弧菌来讲，研究最多的是低温低营养条件下诱导其进入VBNC状态 。但是不同的菌株诱导进入VBNC状态的时间是不一样的，短的9天[9]，长的可达160天[12]。除菌株因素外，所用的培养液对诱导其进入VBNC状态也有着重要的影响。我们的实验表明在自然海水中培养的霍乱弧菌进入VBNC的时间要比在人造海水中时间长。
进入VBNC状态的霍乱弧菌可以在条件适宜的情况下复苏，Colwell等[13]首次证实兔肠断结扎实验可使VBNC状态下的霍乱弧菌复苏，而且复苏的霍乱弧菌具有毒力，能引起肠段内积液。Asakura等[10]证实，通过小鼠肠道注射，霍乱弧菌也能从VBNC状态复苏成可培养状态，并能引起小鼠死亡。志愿者的人体实验同样证实，处于VBNC状态的霍乱弧菌可以在人体肠道内复苏并致病，从志愿者的粪便中能够培养分离到霍乱弧菌[14]。处于VBNC状态的霍乱弧菌在体外同样可以复苏，Wai等[15]报道经过45 ℃ 60 s的水浴热激，VBNC状态的霍乱弧菌可以重新在普通琼脂平板上生长，但Ravel等[16]认为温度升高引起的霍乱弧菌菌数增加不是VBNC细胞的复苏而是残存的可培养细菌的再生长。最近Senoh等[17]将VBNC状态的霍乱弧菌与真核细胞混在一起培养，发现CHO、HT-29、Caco-2、T84、HeLa及Intestine 407五种细胞均可使霍乱弧菌从VBNC状态复苏到可培养状态。
此外对VBNC状态可维持时间存在着不同的看法，Asakura等[10]的实验表明在人造海水低温培养70天后霍乱弧菌完全进入VBNC状态，但这种状态只能维持约20天，88天时的霍乱弧菌在小鼠体内已经不能复苏。Alam等[6]则认为处于生物膜内的VBNC状态的霍乱弧菌可长期存在。
4 霍乱弧菌的非可培养状态的研究方法和技术
目前对于霍乱弧菌VBNC状态的研究主要是应用定量RT-PCR以及基因芯片技术检测VBNC状态基因表达情况 ，然而，无论是定量RT-PCR还是基于基因芯片的转录检测，都只能检测已知的基因，检测不到霍乱弧菌进入VBNC状态过程中的新的基因。RNA-seq即转录组测序是建立在二代测序基础上的转录组分析技术，可进行全基因组水平的基因表达差异研究，相对于芯片技术，定量更准确、重复性更高、检测范围更广，而且能发现新的转录本 。另外蛋白水平上的研究同样有助于理解这一特殊状态。传统的双向凝胶电泳(2-DE)技术有一定的局限性，如低丰度蛋白、极端等电点蛋白和疏水蛋白的鉴定效率低，多样本分析的重复性差等。同位素标记相对和绝对定量(iTRAQ)技术是近年来开发的一种新的蛋白质组学定量研究技术，具有较好的定量效果、较高的重复性，并可对多达8种不同样本同时进行定量分析 。应用RNA-seq以及iTRAQ技术对霍乱弧菌进入VBNC状态过程中不同时间点进行比较转录组以及比较蛋白组分析研究，将会有助于更深入的研究和理解VBNC这一细菌特殊的状态。
5 VBNC状态霍乱弧菌的检测
霍乱细菌进入VBNC状态以后，经典的培养分离方法无法检测到这些细菌的存在。为了检测到VBNC状态的细菌，目前建立多种检测技术，主要有以下几种。
5.1 基于DNA的检测方法
由于DNA分子比较稳定，即使细菌裂解后DNA仍然可以长期存在于环境中，常规的PCR法无法区分出环境中的致病菌的死活，对于混合样本也无法对活细菌进行定量，因此需要建立一种新的检测方法弥补这种不足。叠氮溴化丙锭(propidium monoazide，PMA)是一种DNA分子核酸染料，只能进入破损的细胞膜与DNA结合，而对于完整细胞膜内的DNA不影响[22]。PMA与DNA结合后在强光照射下会与水分子反应形成没有活性的羟胺，使其不能扩增，与PCR方法联合使用能够很好的弥补常规检测方法的不足。这种PMA-PCR方法已经有了很多的应用，不过目前还未在霍乱弧菌中得到应用[23]。另外PMA还可以与环介导等温扩增(loop-mediated isothermal amplification，LAMP)相结合，检测环境中活的致病菌[24]。由于LAMP全过程只需要一个恒定的温度，而且实验的结果可肉眼直接观察，不需要任何复杂昂贵的仪器，因此PMA-LAMP的联合应用对病原体的环境监测非常有意义。
5.2 基于mRNA的检测方法
相对于DNA和蛋白而言，mRNA半衰期短容易降解，因此检测细菌的mRNA作为区分死活细菌而言是一个很好的方法。Gonzalez-Escalona等[9]用定量RT-PCR的方法比较了饥饿状态以及VBNC状态的霍乱弧菌的基因表达情况，发现relA只在VBNC状态的霍乱弧菌中表达，因此可以将relA作为检测目标检测环境水中VBNC状态的细菌。Asakura等[10]还发现在VBNC状态下VC0230 ，VC1212(polB)，VC2132(fliG)以及VC2187(flaC)这些基因的表达量增加，并认为这些基因可以作为检测VBNC状态下霍乱弧菌的标记。最近Mishra等[25]用RT-qPCR的方法验证了这些基因在VBNC状态霍乱弧菌中表达。
5.3 基于荧光染料的检测方法
VBNC状态的细菌与死亡细菌一个重要的区别是细胞膜的完整性，处于VBNC状态的细菌细胞膜是完整的。根据这一特点，已筛选出来数种染料以区分死活细菌，其中LIVE/DEAD BacLight BacterialTM Viability Kits(Molecula Probes)是应用较为广泛的商业化的试剂盒 。该试剂盒由两种核酸染料组成，一种染料是SYTO9，能渗入完整的细胞膜，激发后呈绿色荧光；一种为PI，仅能渗到细胞膜破损的菌体内，激发后呈红色荧光，并且与SYTO9竞争核酸着染位点。在荧光显微镜下观察，发出绿色荧光的为活细胞，发出红色荧光的为死细胞。该试剂盒还可以与流式细胞仪联合使用对活细菌进行计数[28]。最近报道评价了一种快速检测分离活的霍乱弧菌的免疫荧光菌球检测(immunofluorescence-agglomerate assay，IFAG)方法[29]。该方法通过对样本中添加O1群或O139群特异的标记荧光的抗体，在荧光显微镜下观察和挑选特异凝集为荧光微球的细菌进行培养，并发现有一部分结果为阳性的样本无法培养，推测为进入VBNC状态的霍乱弧菌，可能是一个检测VBNC的反筛选提示方法。
除了以上几种应用较广方法外，还有其他的一些方法检测VBNC状态的细菌，比如酶活性分析[30]、放射自显影[31]等。另外Kuehl等[27]在MALDI-ToF/MS的基础上建立了区分死活细菌的方法，该方法不仅能区分死细菌和活细菌，还能区分复苏的细菌与正常的细菌。为了能更准确的检测样本中VBNC状态的细菌，实际应用中往往是两种或几种方法联合应用。发展更加准确快速廉价的活细菌检测技术对霍乱以及其他病原体的监测显得尤为迫切。
6 VBNC状态的霍乱弧菌对公共卫生的挑战
低温和低营养环境不利于霍乱弧菌的增殖，VBNC状态是霍乱弧菌抵抗这些不适宜环境的一种方式，使霍乱弧菌在环境中持续存在，并可在适宜条件下复苏和进入人群引起流行。目前对VBNC的研究还非常有限，对霍乱弧菌细菌进入VBNC以及从VBNC复苏的机制不清楚，现有的检测方法要么花费昂贵要么繁琐复杂，无法大批量的对样本进行检测。因此深入研究霍乱弧菌进入VBNC以及复苏的机制和条件，发展准确快速低廉的检测VBNC细菌的技术对霍乱的监测预警有着重要的意义。
除霍乱弧菌外，还有几十种致病菌可进入VBNC状态，传统的检测方法无法检测到这些潜在的危险，很容易造成监测上的遗漏，对于疾病防控是一个挑战。了解霍乱弧菌VBNC的研究进展对研究其他可进入VBNC的致病菌将有很大的参考意义。
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