Metagenomics-Guided Pathogen Discovery in Travelers' Diarrhea

Traveler's diarrhea (TD) is caused by enterotoxigenic Escherichia coli (ETEC), other pathogenic gram-negative pathogens, norovirus and some parasites. Nevertheless, standard diagnostic methods fail to identify pathogens in more than 30% of TD patients, so it is predicted that new pathogens or groups of pathogens may be causative agents of disease. A comprehensive metagenomic study of the fecal microbiomes from 23 TD patients and seven healthy travelers was performed, all of which tested negative for the known etiologic agents of TD in standard tests. Metagenomic reads were assembled and the resulting contigs were subjected to semi-manual binning to assemble independent genomes from metagenomic pools. Taxonomic and functional annotations were conducted to assist identification of putative pathogens. We extracted 560 draft genomes, 320 of which were complete enough to be enough characterized as cellular genomes and 160 of which were bacteriophage genomes. We made predictions of the etiology of disease in individual subjects based on the properties and features of the recovered cellular genomes. Three subtypes of samples were observed. First were four patients with low diversity metagenomes that were predominated by one or more pathogenic E. coli strains. Annotation allowed prediction of pathogenic type in most cases. Second, five patients were co-infected with E. coli and other members of the Enterobacteriaceae, including antibiotic resistant Enterobacter, Klebsiella, and Citrobacter. Finally, several samples contained genomes that represented dark matter. In one of these samples we identified a TM7 genome that phylogenetically clustered with a strain isolated from wastewater and carries genes encoding potential virulence factors. We also observed a very high proportion of bacteriophage reads in some samples. The relative abundance of phage was significantly higher in healthy travelers when compared to TD patients. Our results highlight that assembly-based analysis revealed that diarrhea is often polymicrobial and includes members of the Enterobacteriaceae not normally associated with TD and have implicated a new member of the TM7 phylum as a potential player in diarrheal disease. ]]>
Fever > 100.3°F Symptoms of acute gastroenteritis >/= 72 hours duration Known or suspected infection or co-infection with a non-bacterial pathogen, such as viral or parasitic gastroenteritis Bloody diarrhea History of inflammatory bowel disease Treatment with an antimicrobial agent within 30 days prior to screening Treatment with > 2 doses of an anti-diarrheal medicine within 24 hours prior to study entry, or the anticipated need for such therapy (including, but not limited to, bismuth subsalicylate, loperamide, and attapulgite) during study treatment Concomitant need or use of any other oral, intramuscular or intravenous antibacterial with expected activities against enteric bacterial pathogens including a fluoroquinolone, macrolide, trimethoprim-sulfamethoxazole, doxycycline, or other potentially effective drug Participation in other clinical research studies utilizing an investigational agent within one month prior to screening or within five half-lives of the investigational agent, whichever is longer Period of stay in the travel destination of < 72 hours Unstable or clinically significant medical conditions, such as uncontrolled diabetes, cancer, etc.) Kidney disease Seizure disorder Psychiatric condition requiring use of major tranquilizers, such as phenothiazines, butyrophenones, etc. Concurrent treatment with Class 1a (e.g.: disopyramide, procainamide, quinidine) or Class III (e.g.: amiodarone, bretylium, ibutilide, sotalol) antiarrhythmic agents (medications that affect heartbeat) Infection known or expected to be caused by a pathogen that is resistant to prulifloxacin History of allergy or serious adverse reaction to prulifloxacin or any other fluoroquinolone, such as ciprofloxacin Failed therapy with a fluoroquinolone for any reason within the past three months Unable to tolerate oral therapy Unable or unwilling to comply with the study protocol, including swallowing tablets, having blood drawn, and providing stool samples as scheduled ]]>

旅行者腹泻（Traveler's diarrhea, TD）多由肠产毒性大肠杆菌（enterotoxigenic Escherichia coli, ETEC）、其他致病性革兰阴性病原菌、诺如病毒（norovirus）及部分寄生虫引发。然而，标准诊断方法仍无法在超过30%的TD患者体内检出病原菌，因此推测可能存在尚未明确的病原菌或病原菌类群作为该病的致病原。本研究对23名TD患者与7名健康旅行者的粪便宏基因组进行了全面分析，所有受试者的标准检测结果均未检出已知的TD致病原。对宏基因组读长进行组装后，将得到的重叠群（contigs）进行半手动分箱（semi-manual binning），以从宏基因组混合序列中拼接出独立基因组；随后开展分类学与功能注释，以辅助推定病原菌的鉴定。本研究共提取得到560份草图基因组（draft genome），其中320份的完整性足以被鉴定为细胞基因组，剩余160份为噬菌体基因组（bacteriophage genome）。基于所获取的细胞基因组的属性与特征，我们对个体受试者的疾病病因进行了预测。研究共观察到三类样本亚型：第一类为4名患者，其宏基因组多样性较低，优势菌群为一株或多株致病性大肠杆菌；多数情况下可通过注释预测其致病类型。第二类为5名合并感染患者，同时感染大肠杆菌及肠杆菌科（Enterobacteriaceae）的其他成员，包括耐药性肠杆菌（Enterobacter）、克雷伯菌（Klebsiella）与柠檬酸杆菌（Citrobacter）。第三类为部分样本中存在所谓的宏基因组暗物质（dark matter）基因组。其中一例样本中，我们鉴定出一株TM7基因组，其系统发育聚类结果与一株分离自废水的菌株一致，且携带编码潜在毒力因子的基因。此外，部分样本中噬菌体读长占比极高；与TD患者相比，健康旅行者体内噬菌体的相对丰度显著更高。本研究结果表明，基于组装的分析揭示旅行者腹泻通常为多微生物感染，且包含通常与TD无关的肠杆菌科成员；同时我们将TM7菌门（TM7 phylum）的一个新成员认定为腹泻疾病的潜在致病原。以下为受试者排除标准：体温＞100.3°F；急性胃肠炎症状持续时长≥72小时；已知或疑似感染非细菌性病原菌（如病毒性或寄生虫性胃肠炎）；血性腹泻；炎症性肠病病史；筛查前30天内接受过抗菌药物治疗；研究入组前24小时内服用超过2剂止泻药物，或在研究治疗期间预期需要使用此类药物（包括但不限于碱式水杨酸铋（bismuth subsalicylate）、洛哌丁胺（loperamide）与凹凸棒石（attapulgite））；同时需要或正在使用任何其他预期可对抗肠道细菌性病原菌的口服、肌内或静脉抗菌药物，包括氟喹诺酮类（fluoroquinolone）、大环内酯类（macrolide）、甲氧苄啶-磺胺甲恶唑（trimethoprim-sulfamethoxazole）、多西环素（doxycycline）或其他潜在有效药物；筛查前1个月内或距研究用试验药物5个半衰期（以较长者为准）内参与过其他使用试验性药物的临床研究；在旅行目的地停留时长＜72小时；存在不稳定或具有临床意义的内科疾病，如未控制的糖尿病、恶性肿瘤等；肾脏疾病；癫痫发作障碍；需要使用大剂量镇静剂（如吩噻嗪类、丁酰苯类等）的精神疾病；同时使用Ⅰa类（如：丙吡胺（disopyramide）、普鲁卡因胺（procainamide）、奎尼丁（quinidine））或Ⅲ类（如：胺碘酮（amiodarone）、溴苄铵（bretylium）、伊布利特（ibutilide）、索他洛尔（sotalol））抗心律失常药物（影响心跳的药物）；已知或预期由对普卢利沙星（prulifloxacin）耐药的病原菌引发的感染；对普卢利沙星或其他氟喹诺酮类药物（如环丙沙星（ciprofloxacin））存在过敏或严重不良反应史；过去3个月内因任何原因使用氟喹诺酮类药物治疗失败；无法耐受口服治疗；无法或不愿遵守研究方案，包括按计划吞服药片、采血及提供粪便样本。