对于检测到的敏感性和特异性显色琼脂培养基发展<em>副溶血性弧菌</em>及其他<em>弧菌</em>物种
Summary
Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.
Abstract
造成弧菌美国食源性感染呈上升趋势。在弧菌属，副溶血性弧菌是负责为广大弧菌机相关感染。因此， 弧菌之间的辨证准确。和检测五 。 副溶血性弧菌是非常重要的，以确保食品供应的安全。虽然分子技术也越来越普遍，这取决于培养的方法仍然经常做，他们被认为在某些情况下的标准方法。因此，一种新的生色琼脂培养基用提供用于隔离和临床相关弧菌的分化更好方法的目标测试。该协议相比，检测V的敏感性，特异性和检测限新的显色培养基和传统媒体之间溶血性弧菌 。 五，各副溶血性弧菌菌株（N = 22），重呈现多样化的血清型和起源的源头中使用。他们以前由食品和药物管理局（FDA）和美国疾病控制和预防中心（CDC）鉴定，并通过TLH -PCR在我们的实验室中进一步验证。在至少四个独立的试验中，这些菌株在35-37℃下接种在显色琼脂和硫代 – 柠檬酸盐 – 胆盐 – 蔗糖（TCBS）琼脂，它是用于培养该种类推荐的培养基，然后孵育24 -96小时。三V.溶血性弧菌菌株（13.6％）未对TCBS最优生长，然而表现出绿色菌落是否有增长。两种菌株（9.1％）没有取得对生色琼脂预期青色菌落。 五，非溶血性弧菌菌株（32）也被测试，以确定显色琼脂的特异性。在这些菌株，31没有增长或者表现出其他菌落形态。 五的平均回收率副溶血性弧菌在chromoge网卡琼脂为〜相对于补充有2％的NaCl的胰蛋白酶大豆琼脂96.4％。最后，新的生色琼脂是检测V.有效介质副溶血性弧菌和其他弧菌区别开来。
Introduction
作为弧菌属，V的构件副溶血是革兰氏阴性，非孢子形成，弯曲的，棒状细菌。它显示出在液体和半固体环境中的高运动性。 五大部分溶血性弧菌菌株是非致病对人类，但致病亚型在许多国家1,2-引起流行和大流行，因此这种被认为是一个重要的食源性病原体。 弧菌感染在美国的发病率自2000年3已经显示出上升的趋势，其中弧菌属，V.副溶血是报道最频繁的物种引起的疾病，在美国4,5。其他临床相关品种包括V.溶藻弧菌 ，V.创伤弧菌 ，V.霍乱 等疾病的一小部分是由多个物种同时引起的。
副溶血性弧菌是一种自然的我海洋水nhabitant，因此广泛分布于世界各地的海洋水域，包括河口。该品种于1950年发现了以下食物中毒在日本爆发。在美国，该物种首次在海水，沉积物和贝类在普吉特海湾地区6,7隔离。在海洋生物栖息地，如双壳贝类滤食动物，能够携带V.副溶血性弧菌作为他们的自然菌群8的一部分。正因为如此， 五在人类溶血性弧菌感染往往与受污染的海鲜，尤其是生的或未煮熟的贝类的消耗。当开放伤口暴露于海水，导致皮肤发生感染进入一个不太常见的途径。 五大部分副溶血性弧菌菌株不会引起人类疾病，但某些亚型窝藏致病因素，如耐热直接溶血素（TDH）是致病。食源性五，最普遍的症状副溶血性弧菌感染的腹泻和腹痛，接着恶心，呕吐，和发热。也报道头痛和发冷。平均潜伏期为15小时，但致病菌株9的足够量的食用后可高达96小时。这种疾病由两至三天持续。该肠胃炎引起的症状V.副溶血性在很大程度上是自限性的，因此，特殊处理是没有必要的。肠胃炎的轻症病例可口服补液得到有效治疗。更严重的疾病可以通过抗生素如四环素或环丙沙星10进行处理。死亡率为胃肠炎例约2％，但也可以是高达29％，为那些谁开发血流感染或败血症。任何人谁消耗的海鲜或有暴露于海水开放的伤口是V.的风险副溶血性弧菌感染。疾病，危及生命的败血症的更严重的形式，多见于有潜在的医疗合作亚群nditions 11，它包括酒精中毒，肝脏疾病，糖尿病，肾病，恶性肿瘤，和其它条件，导致削弱免疫应答。值得注意的是，这一群人也是在为收缩严重的疾病的风险较高所致V.创伤弧菌 ，它可以以类似于五自然栖息地找到副溶血性弧菌 。
副溶血性弧菌 ，使用硫代-柠檬酸盐-胆盐-蔗糖（TCBS）琼脂作为选择性和鉴别介质常规地分离。富集在碱性蛋白胨水可在TCBS琼脂先隔离。上TCBS推定菌落然后进一步在生化检查和/或分子检测靶向的种特异性基因的存在的阵列进行测试。基于PCR的方法经常被用来确认V的身份通过放大耐热溶血毒素基因，TLH 12 溶血性弧菌 。
不管通道的确认方法音色，它有隔离和分化V.的有效媒介是很重要的从副溶血性摆在首位的其他海洋弧菌。 TCBS已经经常被用来根据自己的能力发酵蔗糖12 弧菌属内分化的物种。阳性发酵反应伴随pH指示剂溴百里酚蓝的颜色变化。V.副溶血性弧菌菌落是相当独特的TCBS上，展出蓝绿色。然而，这种介质不能轻易区分V.溶藻弧菌和V.霍乱弧菌，蔗糖发酵变形杆菌可能会产生黄色菌落类似V.霍乱弧菌或V.溶藻弧菌 13。上TCBS， 五初始隔离副溶血性弧菌也可能误认为嗜水，邻单胞shigelloides和假单孢菌属14。迟发蔗糖FERM株entation可以与其它蔗糖非发酵弧菌 13，其包括五相混淆副溶血性弧菌 。 TCBS被发现是对大肠杆菌 ， 腐败假单胞菌等等不敏感。其他几个品种产量绿它们可能与五混淆灰色菌落副溶血性弧菌或V.创伤弧菌 15。其结果是，希望开发替代培养基具有更好的灵敏度和特异性朝向检测和分离V.副溶血性弧菌等密切相关的物种。
几个媒体替代最近已开发的。除了包含选择剂的，最掺入生色底物根据它们的差分酶活性来区分物种。例如，吲哚酚-β葡糖苷和吲哚酚β-D-半乳糖苷已被用作显色底物来区分V.第从这些五 ，溶血殖民地（这似乎蓝绿色） 霍乱 （紫色），由于其差的能力，以产生β葡糖苷酶和β半乳糖苷酶16。通过几组开发的显色培养基的不同配方进行了评估，报告履行同等或高于TCBS 17,18,19更好。使用显色介质的一个优点是，周围介质的着色最小从而促进特定菌落的分离。在这项研究中，我们评估了新制定的显色介质的检测和隔离五的能力霍乱弧菌 ，V.副溶血性弧菌和V.创伤弧菌 ;特别注重其分化能力V.副溶血来自其它物种。
Protocol
Representative Results
Discussion
本研究着重于文化传媒开发和评估。按照惯例，TCBS是选择性和鉴别培养基用于分离和检测V.副溶血性弧菌，霍乱弧菌和V.创伤弧菌 12。然而，限制已报道这个介质，例如无法区分V.从霍乱 弧菌等品种。蔗糖和pH指示剂是TCBS的分化剂。因此，产酸由蔗糖发酵导致介质的颜色变化。介质的着色是TCBS的一个缺点，因为它可能会掩盖菌落形态的观察。新开发的显?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢M. Channey，E.洲和K.托马斯为他们的项目援助。工程物资部分被加州州立理工大学资助。
Materials
| Reagent/Equipment | |||
| Agar | Fisher Scientific | DF0140-15-4 | may use other brands |
| Autoclave | Any | ||
| BHI powder | Fisher Scientific | DF0418-17-7 | may use other brands |
| Blender | Any | to blend oyster meat | |
| CampyGen gas generator | Hardy Diagnostics | CN035A | to provide a microaerophilic atmosphere; may use other brands |
| Chocolate agar plates | Hardy Diagnostics | E14 | may use other brands |
| Common PCR reagents (dNTPs, MgCl2, Taq Polymerase) | Any | or use PCR beads (Fisher Sci 46-001-014) | |
| Culture tubes | Fisher Scientific | S50712 | may use other brands |
| Eppendorf tubes | Fisher Scientific | S348903 | may use other brands |
| Gel doc | Any | ||
| HardyChrom Vibrio agar plates | Hardy Diagnostics | G319 | This study evaluates this medium |
| Incubator | Any | ||
| Inoculating loops | Fisher Scientific | 22-363-606 | 10 microliter-size was used in this study |
| NaCl | Fisher Scientific | BP358-212 | may use other brands |
| Oysters | Any | ||
| PBS | Fisher Scientific | R23701 | may use other brands |
| Petri dish | Fisher Scientific | FB0875713 | may use other brands |
| Pipette and tips | Any | Sterilized tips | |
| Primers for tlh | IDT DNA | ||
| Scale | Any | ||
| Spreader | Fisher Scientific | 08-100-11 | Beads may be used instead |
| Stomacher blender | Stomacher | 400 | Samples were homogenized at 200 rpm for 30 sec. Other homogenizer can be used. |
| Sterile filter bags for blenders | Fisher Scientific | 01-812-5 | |
| TCBS powder | Hardy Diagnostics | 265020 | This study evaluates this medium |
| Thermocycler | Any | ||
| TSB powder | Fisher Scientific | DF0370-07-5 | may use other brands |
| UV viewing cabinet | Any | Emit long-wave UV light | |
| Water bath | Any | ||
| Name | Sources | Catalog Number | Comments |
| Bacterial species and strains | |||
| Aeromonas hydrophila | ATCC | ||
| Candida albicans | ATCC | ||
| Campylobacter jejuni | ATCC | ||
| Escherichia coli | ATCC | ||
| Proteus mirabilis | ATCC | ||
| Pseudomonas aeruginosa | ATCC | ||
| Staphylococcus aureus | ATCC | ||
| Salmonella Choleraesuis | ATCC | ||
| Shigella boydii | ATCC | ||
| Shigella flexneri | ATCC | ||
| Shigella sonnei | ATCC | ||
| Vibrio alginolyticus | ATCC | ||
| V. cholerae (serotypes include O139, O1, non O1, El Tor biovars) | FDA, ATCC | ||
| V. damsela | FDA | ||
| V. fisherii | Environnement | ||
| V. fluvialis | CDC | ||
| V. furnissii | CDC | ||
| V. hollisae | FDA | ||
| V. metschnikovii | ATCC | ||
| V. mimicus | FDA | ||
| V. parahaemolyticus(serotypes include O3:K6, O1:K56, O4:K8, O5:K15, O8, etc) | ATCC, FDA, CDC, Environment | ||
| V. proteolyticus | FDA | ||
| V. vulnificus | FDA |
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