全基因定序於Schwarzengrund血清型沙門氏桿菌流行病研究的運用性探討

Application of Whole-Genome Sequencing in the Epidemiology of Salmonella enterica Serovar Schwarzengrund

李宜蓁(Jenny I-Chen Li)

指導教授 ： 周崇熙

國立臺灣大學/獸醫專業學院/獸醫學研究所/博士(2022年)

https://doi.org/10.6342/NTU202201182

摘要

二十年來，脈衝場凝膠電泳法（PFGE）對食源性疾病感染的檢測、調查和控制非常有效。然而，PFGE 數據庫是一個封閉系統，只有參與 PulseNet的實驗室才能使用。為了能對公共衛生之突如其來之威脅可有效檢測並能迅速反應，應要設計一個隨時可被不同實驗室存取與比較之資料庫。此時，全基因組測序（WGS）的發明提供一個嶄新的契機。WGS含有經數位化與標準化之資料，大眾可以隨時使用。迄今該技術尚未應用於細菌亞型分類、風險評估和重建質體。因此，據我們所知，本論文為首位以使用WGS 將 2000 年至 2018 年間 23 個流病無關（epidemiologically unrelated）來源菌株準確區分出 Salmonella enterica serovar Schwarzengrund 分離株，據以了解它們的致病因子以進行其風險評估，並重建質體以追踪的耐藥性（AMR）基因的轉移。
首先，加強沙門氏桿菌感控措施時，重要的是使用適當的分型工具以快速精準辨識污染源。本論文將WGS與其他基因分型方法進行比較，包括脈衝場凝膠電泳法 （PFGE）、多位點序列分型法 （MLST）和群聚且有規律間隔的短回文重複序列法（CRISPR），並評估它們的分型能力。在23 個流病無關的Schwarzengrund血清型沙門氏桿菌中，與 PFGE (DI = 0.938)、CRISPR (DI = 0.906) 和 MLST (DI = 0.463) 方法相比，本論文使用之WGS 具有最大的分辨率 (DI = 0.982)，可精準跟踪感染源。
再者，並非所有 Salmonella enterica serovar Schwarzengrund 分離株具有同等致病性， 我們使用WGS 描繪其潛在的致病因素以進行風險評估。結果顯示，所有分離株均表現出多重耐藥性，其中六株對ciprofloxacin具有耐藥性，大多是來自豬源的分離株。而在菌株中發現了很高的 IncFIB (86.9 %) 質體，此質體已知會增加其在雞盲腸定殖並引起腸外疾病的能力。此外，95.6% 的菌株含有第一型整合子並攜帶首次被鑑定出含有對trimethoprim、streptomycin、tetracycline、sulfonamide、chloramphenicol和gentamicin的耐藥性有關的五個不同基因卡匣。因此，WGS可以立即識別包括ciprofloxacin耐藥性、IncFIB(K) 質粒和第一型整合子在內的中潛在風險菌株，故可幫助公共衛生部門做出快速反應和有效決策。
最後，病原體溯源時不僅關注菌株，更應特別關注質體，因為即使在很短的時間內它們也可能在菌株間傳播。因此，本論文使用兩種不同的WGS方法重建質體，一為結合短序列片段和長序列片段來重建質體序列，另一種則利用Illumina 短序列片段回貼至長序列片段來重建序列。實驗還特別使用S1核酸酶脈衝場凝膠電泳 （S1-PFGE） 進一步驗證沙門氏桿菌菌株中檢測到質體的數量和大小。結果發現，Illumina 短序列片段回貼至長序列片段比短序列片段和長序列片段結合更為精準，可提供更高的解析度以說明菌株與耐藥性基因的傳播途徑。
總而言之，本論文透過WGS 的應用增強對沙門氏桿菌之監測效果，可更準確地識別病原體，評估中風險菌株並能更快速地做出決策，且能通過質體追踪進行更全面的分析。隨著 WGS 數據在全球基因組數據庫中逐步積累，WGS將成為全世界強大的武器，使人類能在各個領域與沙門氏桿菌鬥爭，推進健康一體（One Health）戰略以改善所有國家的公共衛生。

關鍵字

全基因定序 ； 沙門氏桿菌 ； 基因分型 ； 風險 ； 質體 ； 公共衛生

並列摘要

Over the past 20 years, the pulsed-field gel electrophoresis (PFGE) method has been extremely efficient in driving the detection, investigation, and control of food-borne infection outbreaks. However, the PFGE database is a closed system, as only PulseNet participating laboratories can have access to it. For effective detection and prompt response to sudden public health threats, a new strategy should be designed so these real-time data can be compared with other laboratories at all times. The advent of whole-genome sequencing (WGS) may provide such new opportunities. WGS data are inherently digital, standardized, and can be accessed at any time by the general public. However, this technique has not been applied to bacterial subtyping, hazard characterization, and plasmid reconstruction. Hence, to the best of our knowledge, this thesis is the first to use WGS to accurately differentiate Salmonella enterica serovar Schwarzengrund isolates from 23 epidemiologically unrelated sources from the year 2000 to 2018, to characterize their pathogenicity profiles for risk assessment, and to reconstruct plasmids for tracking antimicrobial resistance (AMR) genes transfer.
First, accurate identification of contamination sources with appropriate subtyping tools is crucial to reinforce Salmonella control measures. In this thesis, WGS was compared with alternative genotyping methods including PFGE, multilocus sequence typing (MLST), and clustered regular interspaced short palindromic repeats (CRISPR) to assess their discriminatory powers. Results showed that among 23 epidemiologically unrelated Salmonella enterica serovar Schwarzengrund isolates, WGS has the greatest resolution (DI = 0.982) compared to PFGE (DI = 0.938), CRISPR (DI = 0.906), and MLST (DI = 0.463) methods, supporting its advantage in the improvement of source tracking.
Considering that not all Salmonella enterica serovar Schwarzengrund isolates are equally pathogenic, we aim to delineate potential pathogenicity factors via WGS for risk assessment. Results showed that all studied isolates exhibited multidrug resistance, and six of those were resistant to ciprofloxacin phenotypically, mostly from pig sources. A high prevalence of IncFIB (86.9 %) plasmids was found among strains, which was known to increase the ability to colonize the chicken cecum and cause extra-intestinal disease. Moreover, 95.6% of the selected strains contained Class I integrons and harbor integrons with five different gene cassettes identified for the first time, which are associated with resistance to trimethoprim, streptomycin, tetracycline, sulfonamide, chloramphenicol, and gentamicin. Hence, potential moderate risk strains that include ciprofloxacin resistance, IncFIB(K) plasmids, and class I integrons, can be identified immediately to help public health sectors to give a rapid response and effective decisions.　
Lastly, for pathogen source tracking, studies should not only focus on strains but also their plasmids as they may transmit between strains frequently, even at short timescales. Hence, reconstruction of the plasmids using two different WGS approaches including a de novo assembly of both short and long reads and a reference-based assembly of Illumina short reads mapped onto the long reads approach were conducted. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) was further used to confirm the number and sizes of plasmids detected by in silico-based predictions in Salmonella strains. As result, reference-based surpassed de novo assembly in plasmid reconstruction, suggesting that it could provide a stepping-stone to higher resolution analyses and elucidate not only strain transmission but also antibiotic resistance transmission routes.
Taken together, this thesis has significantly improved Salmonella surveillance by using the applications of WGS, which can identify pathogens with higher accuracy, estimate moderate risk strains for faster decisions, and more comprehensive analysis with plasmid reconstruction. As WGS data accumulate in a global genomic database, it will provide the world with a strong weapon in the fight to combat Salmonella in all sectors and advance the One Health strategy for improving public health for all nations.

並列關鍵字

Whole-genome sequencing ； Salmonella ； Subtyping ； Risk ； Plasmid ； Public health

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