*3.5. Determination of Intestinal Metabolites with Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC)*

A new HPLC method was established to measure the different metabolites. The intestinal fluid was sterile-filtered directly into a vial using a 0.2 µm syringe filter. An Aminex HPX-87H 300 × 7.8 mm (Bio-Rad, Hercules, CA, USA) and a guard column were used for separation, in which 20 µL were injected and isocratically eluted with 0.005 M sulfuric acid at a flow rate of 0.450 mL/min and a temperature of 35 ◦C. The detection was done by measuring refractive index. With this method the metabolites glucose, galactose, succinic acid, lactic acid, acetic acid, propionic acid, ethanol, butyric acid and iso-valeric acid could be quantified simultaneously. Phosphate and citrate elute at the same time with this method and therefore could not be differentiated.

#### **4. Conclusions**

From the comparison of the groups among themselves and with the group treated with Vancomycin and integrating our previous data (8) published in Heidebrecht et al. (2019), several conclusions can be drawn. As described above, an active and antibody-induced mechanism must take place in the intestine which causes the survival of the animals. Our antibodies can inactivate foreign *C. difficile* antigens through various mechanisms of action. These include opsonization, activation of the complement system, agglutination, prevention of adhesion and direct neutralization. It is unlikely, or at least not the primary mechanism of action, that cell adhesion is prevented, and direct neutralization of the *C. difficile* cells occurs. Had this been the case, there would not have been initial spore germination and cell growth [5], but the cells would have been directly eliminated. The later decline in cell growth between days six and eight indicates that the host's own immune system was activated (which is a delayed process), either by activation of the complement system or by direct labelling of the cells for the host's own defense cells. However, since cell decline was also observed in some control groups (e.g., control WPI), the deduced

conclusion is that both indirect regeneration of intestinal microbial diversity (as observed in all groups except the antibiotics group) and direct antibody-induced inactivation were crucial for cell and spore decline of *C. difficile.* In other words, prevention of recurrences (only observed in the groups treated with active antibodies) can only be achieved if both mechanisms are given the chance to work, which is the case for Ig-based CDI treatment instead of antibiotics.

**Author Contributions:** Conceptualization: H.-J.H., U.K., M.W.P.; methodology, formal, analysis: H.-J.H., I.L., S.R., C.H., interpretation of results: H.-J.H., U.K., M.W.P., I.L., S.R., C.H.; writing original draft preparation: H.-J.H., writing—review and editing: all authors; supervision, U.K., M.W.P.; funding acquisition U.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was partly funded by our industry partner, Biosys Health, who provided input into study design, but had no role in data collection and analysis, interpretation or decision to pub-lish or preparation of the manuscript.

**Institutional Review Board Statement:** The animals were acclimatized for five days prior to the study in the biosafety level laboratories at University of North Texas Health Science Center in accordance with NIH guidelines. All procedures were carried out in accordance with the approved protocol of the Institutional Animal Care and Use Committee (IACUC-2016-0015).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data is available in this manuscript.

**Acknowledgments:** We thank everybody who supported this study for their helpful contributions, especially the Microbiome core facility members Angela Sachsenhauser, Caroline Ziegler and Klaus Neuhaus.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Table A1.** Concentration of Different Metabolites in Intestinal Fluid Measured by RP-HPLC.



**Table A1.** *Cont.*

**Table A2.** Individual pairwise correlation coefficients and *p*-values.


#### **References**

