Overcoming donor variability and risks associated with fecal microbiota transplants through bacteriophage-mediated treatments

Background: Fecal microbiota transplantation (FMT) and fecal virome transplantation (FVT, sterile filtrated donor feces) have been effective in treating recurrent Clostridioides difficile infections, possibly through bacteriophage-mediated modulation of the gut microbiome. However, challenges like donor variability, costly screening, coupled with concerns over pathogen transfer (incl. eukaryotic viruses) with FMT or FVT hinders their wider clinical application in treating less acute diseases. Methods: To overcome these challenges, we developed methods to broaden FVT's clinical application while maintaining efficacy and increasing safety. Specifically, we employed the following approaches: 1) Chemostat-fermentation to reproduce the bacteriophage FVT donor component and remove eukaryotic viruses (FVT-ChP), 2) solvent-detergent treatment to inactivate enveloped viruses (FVT-SDT), and 3) pyronin-Y treatment to inhibit RNA-virus replication (FVT-PyT). We assessed the efficacy of these processed FVTs in a C. difficile infection mouse model and compared them with untreated FVT (FVT-UnT), FMT, and saline. Results: FVT-SDT, FVT-UnT, and FVT-ChP reduced the incidence of mice reaching the humane endpoint (0/8, 2/7, and 3/8, respectively) compared to the FMT, FVT-PyT, and saline control (5/8, 7/8, and 5/7, respectively) and significantly reduced the load of colonizing C. difficile cells and toxin A/B levels. There was a potential elimination of C. difficile colonization, with 7 out of 8 mice treated with FVT-SDT testing negative with qPCR. In contrast, all other treatments exhibited the continued presence of C. difficile. Moreover, the results were supported by changes in the gut microbiome profiles, cecal cytokine levels and histopathological findings. Assessment of viral engraftment following FMT/FVT treatment and host-phage correlations analysis suggested that transfer of phages likely were an important contributing factor associated with treatment efficacy. Conclusions: This proof-of-concept study show that specific modifications to FVT hold promise in addressing challenges related to donor variability and infection risks. Two strategies lead to treatments significantly limiting C. difficile colonization in mice, with solvent/detergent treatment and chemostat-propagation emerging as promising approaches.

背景：粪便微生物群移植（Fecal microbiota transplantation, FMT）与粪便病毒组移植（Fecal virome transplantation, FVT，即供体粪便无菌滤液）已被证实可有效治疗复发性艰难梭菌（Clostridioides difficile）感染，其作用机制可能通过噬菌体介导调控肠道微生物组。然而，供体个体差异、筛查成本高昂，以及人们对粪便微生物群移植/粪便病毒组移植可能引发病原体传播（包括真核病毒）的担忧，阻碍了二者在轻症疾病治疗中的更广泛临床应用。

方法：为克服上述挑战，本研究开发了一系列方法，在保留治疗疗效的同时拓展粪便病毒组移植的临床应用范围并提升其安全性。具体而言，本研究采用了如下策略：1）恒化器发酵法复刻供体噬菌体组分并去除真核病毒（该处理方式命名为FVT-ChP）；2）溶剂-去污剂处理法灭活包膜病毒（该处理方式命名为FVT-SDT）；3）吡罗红-Y处理法抑制RNA病毒复制（该处理方式命名为FVT-PyT）。本研究在艰难梭菌感染小鼠模型中评估了上述经处理的粪便病毒组移植的疗效，并将其与未处理粪便病毒组移植（FVT-UnT）、粪便微生物群移植及生理盐水对照组进行比较。

结果：与粪便微生物群移植、FVT-PyT及生理盐水对照组（小鼠达到人道终点的比例分别为5/8、7/8、5/7）相比，FVT-SDT、FVT-UnT及FVT-ChP组小鼠达到人道终点的比例更低（分别为0/8、2/7、3/8），且显著降低了定植艰难梭菌的菌体负荷及毒素A/B水平。经FVT-SDT处理的小鼠中，8只里有7只的定量PCR（quantitative PCR, qPCR）检测结果为阴性，提示该处理方式或可彻底清除艰难梭菌定植；与之相比，其余所有处理组的小鼠仍可检测到艰难梭菌定植。此外，肠道微生物组谱变化、盲肠细胞因子水平及组织病理学结果均验证了上述结论。对粪便微生物群移植/粪便病毒组移植后病毒定植情况的评估，以及宿主-噬菌体相关性分析结果表明，噬菌体移植可能是影响治疗疗效的重要因素。

结论：本概念验证研究表明，对粪便病毒组移植进行特定改造，有望解决供体差异及感染风险相关的挑战。其中，溶剂-去污剂处理法与恒化器传代法两种策略可显著限制小鼠体内艰难梭菌定植，展现出良好的应用前景。