Data_Sheet_1_Isolation and Characterization of a New Phage Infecting Elizabethkingia anophelis and Evaluation of Its Therapeutic Efficacy in vitro and in vivo.pdf

Elizabethkingia spp. are a group of non-fermentative, Gram-negative, catalase-positive, and non-motile bacilli. They can cause meningitis in neonates and immunosuppressed patients, and lead to high mortality. Considering the rising trend of drug resistance among bacteria pathogens, bacteriophage (phage) therapy is a potential alternative to antibiotics for treating multidrug-resistant bacterial infections. However, so far, no phages specific for Elizabethkingia spp. have been reported. Using a clinically isolated Elizabethkingia anophelis as the host, the phage TCUEAP1 was isolated from wastewater of Hualien Tzu Chi hospital. The phage particle of TCUEAP1 under electron microscopy was revealed to belong to the siphoviridae family, with a head size of 47 nm, and a tail dimension 12 nm in diameter and 172 nm in length. The one-step growth analysis showed that the latent period of TCUEAP1 was about 40 min with a rise period lasting about 20 min, yielding a burst size of approximately 10 PFU/cell. The adsorption rate of TCUEAP1 reached about 70% in 20 min. Using 20 isolates of Elizabethkingia spp. to test the host range of TCUEAP1, it displayed narrow spectrum infecting three strains of E. anophelis, but forming spot lysis on 16 strains. The sequence result showed that the genome of TCUEAP1 is a double-stranded DNA of 49,816 bp, containing 73 predicted open reading frames. Further genomic analysis showed TCUEAP1 to be a new phage with no resemblance to publicly available phage genomes. Finally, in a mouse intraperitoneal infection model, at 6 h after the bacterial injection, TCUEAP1 decreased the bacterial load by fivefold in blood. Also, TCUEAP1 rescued 80% of mice heavily infected with E. anophelis from lethal bacteremia. We hope that the isolation and characterization of TCUEAP1, the first phage infecting Elizabethkingia spp., can promote more studies of the phages targeting this newly emerging bacterial pathogen.

伊丽莎白菌属（Elizabethkingia spp.）是一类不发酵、革兰氏阴性、过氧化氢酶阳性且无动力的杆菌。该菌可引发新生儿及免疫功能低下人群的脑膜炎，且致死率较高。鉴于细菌病原体的耐药性问题日益严峻，噬菌体（bacteriophage，简称phage）疗法是治疗多重耐药细菌感染的潜在替代方案。然而截至目前，尚未有针对伊丽莎白菌属的特异性噬菌体被报道。本研究以临床分离的按蚊伊丽莎白菌（Elizabethkingia anophelis）为宿主，从花莲慈济医院的污水中分离得到噬菌体TCUEAP1。电子显微镜观察显示，TCUEAP1的噬菌体颗粒隶属于长尾噬菌体科（Siphoviridae），其头部直径为47 nm，尾部直径12 nm、长度172 nm。一步生长曲线分析结果表明，TCUEAP1的潜伏期约为40分钟，裂解上升期持续约20分钟，裂解量约为10 PFU/细胞。其吸附率在20分钟内可达约70%。利用20株伊丽莎白菌属菌株测试TCUEAP1的宿主谱，结果显示其宿主范围较窄：仅可感染3株按蚊伊丽莎白菌，但可在16株菌株上形成点状溶斑。序列分析结果显示，TCUEAP1的基因组为双链DNA，全长49816 bp，包含73个预测开放阅读框。进一步的基因组分析表明，TCUEAP1是一种新型噬菌体，与现有公开的噬菌体基因组均无显著同源性。在小鼠腹腔感染模型中，细菌接种6小时后，TCUEAP1可使血液中的细菌载量降低5倍。同时，TCUEAP1可将80%重度感染按蚊伊丽莎白菌的小鼠从致死性菌血症中挽救。本研究分离并鉴定了首株可感染伊丽莎白菌属的噬菌体TCUEAP1，期望其能推动更多针对该新兴细菌病原体的噬菌体相关研究。