A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.
There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.
昆虫起到与食品有关的疾病的传播中起重要作用,因为它们可以传播病菌沾染到食物或食物接触的表面和器具1。其中昆虫,蝇,蟑螂,蚂蚁和表现有利于食源性病原体的传播行为。这些行为包括与腐烂的物质的关联,垃圾和粪便,endophily(进入建筑物),以及synanthropy(与人同居)2。 。食源性病原体如沙门氏菌 , 李斯特菌 , 弯曲杆菌 , 大肠杆菌 O157:H7和属Cronobacter(原崎肠杆菌 )的成员已经被报道由昆虫3-5被发送。住区(synanthropic)藏污纳垢苍蝇从他们的污染身体表面传递的病原体传播的机械食源性细菌。然而,食源性致病菌在苍蝇的消化道中存在可多达三倍比对自己的身体表面(身体,头部,腿和翅膀)5观察到更大。食源性致病菌也可以留在苍蝇的消化道的时间比在身体表面6,7,在某些情况下,更大的长度,所以能大量繁殖,定植蝇消化道4,8,9。这增加了苍蝇的矢势,因为他们可以通过排便和返流10,11进一步蔓延食源性致病菌。
如今,有改进的监视系统,其能够更迅速地检测食源性疾病的爆发。在执行食源性疫情调查,公共卫生官员寻找食物,可能是源(S)或感染的车辆(S)。调查人员还可以进行参与,找出食物是如何被污染的设施(或设施)的环境评估,并可能收集样本作为调查12的一部分。 DESP伊特有关昆虫为食源性病原携带者,链接昆虫病原体的载体造成特定的食源性疾病的爆发已经具有挑战性的科学文献的大量。这主要是因为昆虫没有被无菌收集作为环境采样方案中的食源性暴发调查的一部分。包括昆虫,特别是那些表现出有利于食源性病原体的传播行为,作为一种环境取样过程中,一个规范,快速,灵敏,可靠的协议来检测食源性致病菌从单一的昆虫需要到位的一部分。
传统的电镀技术,从昆虫食源性致病菌的检测都费力,取决于目标细菌在不同的文化传媒竞争力的增长,克服了昆虫的先天共生微生物的快速增长。大多数具有关联的昆虫与BA的研究cterial病原体已通过汇集在一起几种昆虫而不是识别针对每个个别的病原体的存在增加了该方法的灵敏度。因此,这些研究并没有区分在发现病原体13-18昆虫的身体部分。以确定是否食源性致病菌位于身体表面或在个体的昆虫消化道的能力是很重要的,因为这可能有流行病学影响,并可能导致不同的缓解策略。作为机械载体,蝇上的食物,土地的时间很短可以仅传送细菌水平低,从他们的身体表面,而那些苍蝇反刍和排便的食物增加在潜在的更高水平的感染的转印病原体的概率。因此,为了估算一个食源性病原体的流行每一个个体的昆虫,并区分昆虫,其中细菌p的主体部分是很重要的 athogen所在。
即使使用非培养的方法来检测食源性致病菌越来越多地被实施,它们还没有被商业上使用,以由一个单一的昆虫检测食源性致病菌。目前,有验证了在市场上可用于快速检测食源性致病菌的食物,正在使用的行业和监管机构的分子协议。这些方法包括对病原体在多种食物样本的检测DNA为基础的系统。虽然分子协议比传统电镀方法快,样品的富集仍需要获得的10 2个菌落形成单位所需的聚合酶链反应(PCR)的细菌病原体(CFU)系的方法19的灵敏度水平。此外,来自PCR阳性样品纯的细菌菌落隔离是必要的,以确认使用适当的方法的病原体。
内容“>该协议的目的是标准化用于检测食品和环境样品的病原体为从身体表面的检测食源性细菌和单飞散的消化道并进一步隔离这些市售的PCR为基础的系统从此处描述的协议的samples.The灵敏度病原体被第一校准用实验室饲养的成虫家蝇( 家蝇 ),这些实验与每个细菌病原体的连续稀释进料。该标准化协议随后用来调查100野外捕捉苍蝇为食源性致病菌从他们的身体表面和/或消化道运河的存在。这种标准化的协议将允许公共卫生实验室检测由昆虫造成的健康威胁,允许执行食源性疾病时,他们收集的环境取样计划的一部分的可能性疫情调查。已检测到的从野生昆虫食源性致病菌已经使用了各种各样的协议,可能不包括必要的信息,以准确地评估在食品或与食品有关的环境13,15的单个飞行的存在的与食物有关的风险以往的研究, 23,24。在这里,我们证明了使用该标准化的协议,它可以检测并隔离Cronobacter属。,S。肠炎,和L.单核细胞从身体表面和单苍蝇夹在野外的消化道。因为昆虫可以携带靶食源性病原体的数量少和高数量的其他土著菌群25,26的,该协议需要在特定培养基样品的初级(和有时辅助)富集以增加检测目标食源性病原体的敏感性。结果从PCR为基础的检测系统分别在大约30小时得到(对于DETEction Cronobacter属。和S.肠炎 )和48小时(用于后开始处理样品的检测单增李斯特菌 )的。因此,该协议是可靠以及快速和足够敏感以筛选的食源性病原体的存在的单个飞。
进行PCR阳性结果确认活菌和隔离是许多实验室的标准作业程序的一部分。此外,对于流行病学的目的,从PCR阳性样品纯的细菌培养都需要进一步确认和使用生化,免疫,或遗传方法血清型的食源性病原体。虽然没有误报,观察检测S.当肠道和L.从主体部件单野生捕获苍蝇的单核细胞增生 ,使用这个协议中,我们发现到的Cronobacter属误报50%的比率。这表明,基于PCR的检测系统,用于菌属Cronobact呃可以与其它肠杆菌科的高度复杂的小型生物群的苍蝇携带之间存在交叉反应。因此,隔离和从PCR阳性标本属Cronobacter纯菌落净化需要更多的选择性电镀比评估的其他病原体。
此协议已主要被标准化以筛选个体野外捕获苍蝇为Cronobacter属的存在。,S。肠炎,和L.使用商业的基于PCR的检测系统菌等 。但是,这个协议也容易适应屏体部分单苍蝇的其他食源性致病菌如肠出血性大肠杆菌 O157存在:H7(使用大肠杆菌 O157:H7 MP标准的检测试剂盒,或大肠杆菌 O157:H7实时检测试剂盒)和志贺毒素大肠杆菌大肠杆菌 (STEC)基团(使用实时STEC套件),获得的灵敏度> 80%(取消发布编辑数据)。另外,该协议可以潜在地适用于从已知的疾病(蟑螂和蚂蚁)载体等昆虫检测食源性病原体,但需要更多的这方面的研究。
食源性疾病暴发的调查是非常动态的,包括一个多步骤的过程,可根据具体情况和当地的环境正在调查12,27而有所不同。这些调查是重要的,因为它们提供了防止未来疾病的即时保护公众健康。此外,这些研究可以阐明由食源性微生物分布的新机制,提高导致新的研究领域28的重要问题。侦查技术以及标准化,快速,敏感的协议是必要的,从个人的昆虫检测食源性致病菌。这种标准化的协议打开了机会无菌采集昆虫,如苍蝇,WHICH可以矢量食源性细菌病原体,作为环境取样计划的一部分。可以从这个来获得的流行病学资料将利用在构建由昆虫传播的食源性致病菌的机制的一个准确的描述( 即照射时间长度:a。通过登陆与苍蝇落地,排粪和反刍飞)。
最后,尽管这里描述的商用基于PCR检测系统是切合实际的使用,并简化了PCR扩增和属级扩增子的可视化,它绝不是唯一的适当的系统。从富集的样品裂解物替代,可以使用通过使用公开可用的物种特异性引物对筛选的食源性病原体的存在。然而,检测灵敏度应之前,他们使用的证明。
The authors have nothing to disclose.
Thanks to Ben D. Tall, Yi Chen, and Thomas Hammak from the U.S. Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition (CFSAN) for critically reviewing the manuscript. The authors also thank Hannah Lee (research internship program, Joint Institute for Food Safety and Applied Nutrition (JIFSAN), University of Maryland) for laboratory assistance and David Weingaertner (FDA, CFSAN) for preparing the figure of the schematic overview shown in the video.
Bismuth sulfite (BS) agar | Fisher Scientific | R452402 | *Multiple suppliers. |
Brain heart infusion (BHI) broth | Becton, Dickson and Company | 299070 | *Pre-warmed to 37°C. Multiple suppliers. |
Brilliance Listeria agar (BLA) | Fisher Scientific | CM1080B | *Multiple suppliers. |
Buffered peptone water (BPW) | Becton, Dickson and Company | 212367 | *Pre-warmed to 37°C or 42°C. Multiple suppliers. |
Brilliance Cronobacter agar (Druggan-Forsythe-Iversen formulation/DFI) | Fisher Scientific | CM1055B | *Multiple suppliers. |
chromID Sakazakii Agar | bioMérieux | 43741 | *Call for information: 800.682.2666 |
R & F Enterobacter sakazakii (Cronobacter) Chromogenic Plating Medium | R & F Laboratories | Various | *Call for information: +1.630.969.530 |
R & F Enterobacter sakazakiii Enrichment Broth and supplement | R & F Laboratories | Various | *Call for information: +1.630.969.530 |
Hektoen enteric (HE) agar | Fisher Scientific | OXCM0419B | *Multiple suppliers. |
24 Listeria enrichment broth (24LEB) | Oxoid | CM1107 | *Freshly prepared at room-temperature. Multiple suppliers. |
Listeria selective enrichment supplement | Oxoid | SR0243 | *Multiple suppliers. |
Novobiocin | Fisher Scientific | OXSR0181E | *Multiple suppliers. Store at 2-8 °C |
Vancomycin hydrochloride hydrate | Sigma Aldrich | 861987 | Store at 2-8 °C |
Cefsulodin sodium salt hydrate | Sigma Aldrich | C8145 | Store at 2-8 °C |
Rappaport-Vassiliadis (RV) medium | Fisher Scientific | CM0669B | *Multiple suppliers. |
Tetrathionate (TT) Broth | Becton, Dickson and Company | 249120 | *Multiple suppliers. |
Trypticase soy agar (TSA) | Becton, Dickson and Company | 236930 | *Multiple suppliers. |
Xylose lysine desoxycholate (XLD) agar | Becton, Dickson and Company | 221284 | *Multiple suppliers. |
API Biochemical identification system | bioMérieux | Various | *Call for information: +1.800.682.2666 |
VITEK 2: Product Safety | bioMérieux | Various | *Call for information: +1.800.682.2667 |
BAX System Q7 | DuPont | N/A | |
BAX E. sakazakii Standard assay kit | DuPont | D11801836 | * |
BAX L. monocytogenes 24E assay kit | DuPont | D13608125 | * |
BAX Salmonella 2 Standard assay kit | DuPont | D14368501 | * |
Capping tool | DuPont | D11677028 | |
Decapping tool | DuPont | D11134095 | |
PCR tube rack/holder | DuPont | D12701663 | |
Featherweight forceps, wide tip | BioQuip | 4750 | Sterilize before use. Multiple suppliers. |
Fine point, straight tip forceps | BioQuip | 4731 | Sterilize before use. Multiple suppliers. |
Zirconia/silica beads, 0.5 mm | Bio Spec Products, Inc. | 11079105z | Multiple suppliers. |
Petri dishes – 60X15mm | Fisher Scientific | 08-772B | Multiple suppliers. |
Disposable inoculating loops, 10µL | Fisher Scientific | 22-363-606 | Multiple suppliers. |
L-shaped cell spreaders | Fisher Scientific | 14-665-230 | Multiple suppliers. |
Microcentrifuge tubes, 2 ml | Fisher Scientific | Various | Sterilize before use when needed. Secure lid is preferred. Multiple suppliers. |
Cluster tubes | Fisher Scientific | 05-500-13 | |
Cluster tubes caps | Fisher Scientific | 05-500-23 | |
Sodium hypochlorite (Liquid chlorine bleach) | N/A | N/A | *Dilute to 0.05% with water. Multiple suppliers. |
Sterile deionized water | N/A | N/A | Multiple suppliers. |
Sterile distilled water | N/A | N/A | Multiple suppliers. |
Ethyl alcohol 190 proof | N/A | N/A | *Dilute to 70% with water when needed. Multiple suppliers. |
Genie cell disruptor, 120V – for 1.5ml and 2.0ml microtubes | Scientific Industries, Inc. | SI-D238 | Multiple suppliers. |
Heating block | N/A | N/A | Multiple suppliers. |
Cooling block | N/A | N/A | Multiple suppliers. |
Recirculating water bath | N/A | N/A | Multiple suppliers. |
Stereo microscope | N/A | N/A | Multiple suppliers. |
Centrifuge | N/A | N/A | Multiple suppliers. |
Incubator | N/A | N/A | Multiple suppliers. |