尋找黏附侵襲性大腸桿菌噬菌體之受體位置與克雷伯氏肺炎桿菌之磷黴素抗藥性機轉

Searching for adherent-invasive Escherichia coli phage receptor and mechanism(s) mediating low level fosfomycin resistance in Klebsiella pneumoniae

吳珈菱(Chia-Ling Wu)

指導教授 ： 王錦堂

國立臺灣大學/醫學院/微生物學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202001917

摘要

克隆氏症（Crohn's disease, CD）為發炎性大腸疾病（Inflammatory bowel disease, IBD）的一種，屬於慢性的腸道發炎疾病且容易復發。目前有研究指出，黏附侵襲性大腸桿菌（Adherent-invasive E. coli, AIEC）與克隆氏症有關，克隆氏症的治療方法主要針對疾病中的過度免疫反應，以抗發炎藥物為主，但近年來也有針對腸內菌的標靶療法被提出，噬菌體療法屬於其中之一。在本篇研究中，希望藉由建構AIEC的突變菌株庫，找出噬菌體所辨認的標的受體。先前本實驗室研究中，已經從污水中分離出可以感染LF82（AIEC之人類參考菌株）的噬菌體ФLF82，以及可感染LI60C3（小鼠腸胃道分離出的AIEC）的噬菌體ФLI60C3。我們一開始推測莢膜有可能作為噬菌體的受體而感染宿主，透過pKO3質體帶有四環黴素（Tetracycline）抗藥基因，去置換AIEC LF82莢膜形成的相關基因，成功建構了帶有抗藥性基因的AIEC LF82莢膜缺失菌株，但發現噬菌體對其感染效率並無減低。而後利用pUT-Km1質體帶有mini-Tn5跳躍基因建構AIEC LI60C3的跳躍子突變基因庫（Transposon mutant library），並收集了2688株突變菌株，實驗結果發現噬菌體對其感染能力並無下降，因此推測此突變基因庫中跳躍子插入的位置，並沒有剔除到影響AIEC噬菌體感染力的基因。
細菌抗藥性在全世界日趨嚴重，早期開發的磷黴素（Fosfomycin）也重新被檢視，磷黴素毒性低、殺菌力強，且對產生廣效性乙內醯胺酶（Extended spectrum beta-lactamase；ESBL）分解酶的腸內菌具有高度感受性，但也有抗藥性菌株的發現。本實驗室先前測試15株CRKP後發現，這些菌株對磷黴素具有抗性，且最小抑制濃度（Minimum inhibitory concentration；MIC）為256 µg/mL或512 µg/mL，稱為低抗性菌株。在本篇研究中，以定量逆轉錄聚合酶連鎖反應測定這些低抗性菌株之fosA基因，與對磷黴素具感受性之NTUH-K2044菌株相比並沒有過度表現，而後將其fosA基因序列進行定序，並同樣利用NTUH-K2044菌株進行比對，結果顯示低抗性菌株之FosA蛋白質的第91個胺基酸皆為纈胺酸（Valine），而NTUH-K2044則為異白胺酸（Isoleucine），我們推測此胺基酸的變異可能與低抗性菌株之抗藥機制相關，透過pKO3質體成功建構NTUH-K2044胺基酸置換突變株（Ile91Val）後，利用最小抑菌濃度試驗測得磷黴素對其最小抑制濃度由64 µg/ml上升至256 µg/ml，提升了4倍，證實這些低抗性菌株的抗藥機制與FosA蛋白質上第91個胺基酸的改變有關。

關鍵字

克隆氏症 ； 黏附侵襲性大腸桿菌 ； 噬菌體 ； 跳躍子突變基因庫 ； 克雷伯氏肺炎桿菌 ； 磷黴素 ； 抗藥性 ； FosA

並列摘要

Crohn's disease is a kind of inflammatory bowel disease, which belongs to chronic intestinal inflammatory disease and is prone to recurrence. Many studies indicate that adherent-invasive Escherichia coli（AIEC）are pathogenic bacteria associated with human Crohn's disease. Targeted therapies directed against intestinal bacteria were proposed, and phage therapy is one of them. In this study, we aim to use phage therapy to eliminate AIEC from the inflamed intestinal mucosa in mice and to identify the target receptor recognized by the phage through establishing AIEC transposon mutant library. In the previous laboratory study, ФLF82 and ФLI60C3, which can infect LF82（AIEC human reference strain）and LI60C3（AIEC isolated from mouse gastrointestinal tract), respectively, have been isolated from sewage. According to previous studies, whether the capsule was the receptor for AIEC phage to infect the host became our testing objective. The pKO3 plasmid carrying the Tetracycline resistance gene was utilized to replace the AIEC LF82 capsular-forming gene. However, the AIEC LF82 capsular-deficient strain was found to have no reduction in phage infectivity. We used the pUT-Km1 plasmid with the mini-Tn5 transposon gene to construct the AIEC LI60C3 transposon mutant library and collected 2688 mutant strains. The results showed that the infectivity of ФLI60C3 in these mutants is not declining. Thus, the insertion positions of the transposon in this mutant gene pool didn’t target the gene which might affect the infectivity of ФLI60C3.
With the issue of carbapenem-resistant Klebsiella pneumoniae（CRKP）as a rising concern, fosfomycin is one of the last-line antibiotic candidates due to its low toxicity and efficiency of clearing bacteria. Unfortunately, the fosfomycin resistant strains have been found. According to the previous study, we confirmed 15 CRKPs strains through the minimum inhibitory concentration（MIC）test of fosfomycin, and observed that these strains were low-resistant to fosfomycin（MIC = 256-512 µg/ml）. In this study, since the resistant mechanisms were unknown, we sequenced chromosomal fosA gene in these strains and used quantitative reverse transcription-polymerase chain reaction（qRT-PCR）to determine the expression level of fosA gene. The results showed that, compared with NTUH-K2044 strain which was susceptible to fosfomycin, fosA gene of low-resistant strains was not overexpressed. On the other hand, the results indicated that an amino acid substitution in the FosA protein（I91V）was detected. Based on this examination, we speculated that this missense mutation in FosA contributed to low level fosfomycin resistance. Subsequently, site-directed mutagenesis was used to generate fosA loci harboring individual missense mutation; introduction of the mutated genes into NTUH-K2044 strain, resulted in 64- to 256-fold increases in fosfomycin MICs. In conclusion, we hypothesized that novel individual FosA amino acid substitution（I91V）was responsible for low level fosfomycin resistance.

並列關鍵字

Crohn’s disease ； Adherent-invasive E. coli ； bacteriophage ； Klebsiella pneumoniae ； fosfomycin ； drug resistance ； FosA

參考文獻

1. Torres, J., et al., Crohn's disease. Lancet, 2017. 389(10080): p. 1741-55.

2. Wei, S.-C., et al., Management of Crohn's disease in Taiwan: consensus guideline of the Taiwan Society of Inflammatory Bowel Disease. Intestinal research, 2017. 15(3): p. 285-310.

3. Molodecky, N.A., et al., Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology, 2012. 142(1): p. 46-54.

4. Ng, S.C., et al., Incidence and phenotype of inflammatory bowel disease based on results from the Asia-pacific Crohn's and colitis epidemiology study. Gastroenterology, 2013. 145(1): p. 158-65.e2.

5. Kuo, C.J., et al., The Trend of Inflammatory Bowel Diseases in Taiwan: A Population-Based Study. Dig Dis Sci, 2015. 60(8): p. 2454-62.