Beneficial modulation of the gut microbiome by leachates of Penicillium purpurogenum in the presence of clays: A model for the preparation and efficacy of historical Lemnian Earth

Abstract

The experiments presented here are based on the reconfiguration of an ancient medicine, Lemnian Earth (LE) (terra sigillata, stamped earth, sphragis), an acclaimed therapeutic clay with a 2500-year history of use. Based on our hypothesis that LE was not a natural material but an artificially modified one involving a clay-fungus interaction, we present results from experiments involving the co-culture of a common fungus, Penicillium purpurogenum (Pp), with two separate clay slurries, smectite and kaolin, which are the principal constituents of LE. Our results show: (a) the leachate of the Pp+smectite co-culture is antibacterial in vitro, inhibiting the growth of both Gram-positive and Gram-negative bacteria; (b) in vivo, supplementation of regular mouse diet with leachates of Pp+smectite increases intestinal microbial diversity; (c) Pp+kaolin does not produce similar results; (d) untargeted metabolomics and analysis of bacterial functional pathways indicates that the Pp+smectite-induced microbiome amplifies production of short-chain fatty acids (SCFAs) and amino acid biosynthesis, known to modulate intestinal and systemic inflammation. Our results suggest that the combination of increased microbial diversity and SCFA production indicates beneficial effects on the host microbiome, thus lending support to the argument that the therapeutic properties of LE may have been based on the potential for modulating the gut microbiome. Our experiments involving reconfigured LE open the door to future research into small molecule-based sources for promoting gut health.

Fig 1. Illustration of the workflow of experiments and analyses of experimental results, as described in this paper.

Penicillium purpurogenum was co-cultured with smectite or kaolin clays (2); co-cultures were filtered (3) and their leachates were tested for antibacterial activity in vitro (4) to establish reduction in Gram-positive and Gram-negative bacterial numbers (4a); this step was followed by targeted analysis of fungal metabolites present in each sample (5). The leachates of both co-cultures were then fed to mice, as a supplement to normal chow (6) during in vivo experiments; mouse stool was removed at day 0 and day 14. The effect of the leachates on the mouse microbiome was investigated via bacterial DNA sequencing (6a) and metabolomic analysis (6b).

Fig 2. Reduction of E.Coli and S.Aureus populations upon incubation with the leachates of Pp-control, Pp+kaolin and Pp+smectite.

The dashed red line indicates 60% reduction of each bacterial population.

Fig 3. Diversity estimates for faecal microbiota from leachate-fed mice.

Rarefied richness of bacterial ASVs shown in (A) with lines connecting different categories where values were significantly different (according to ANOVA; * p<0.05 ** p<0.01 *** p<0.001)., (B-D) show principal coordinate analysis (PcoA) plots with each axis showing the percentage variability explained by that axis, and where ellipses represent 95% CI of standard error for a given time point. Three distance matrices are used, Bray-Curtis (B) to reflect changes in composition, UniFrac (C) (to reflect changes in phylogeny), and HMS (D) (Hierarchical Meta-storms to reflect changes in function). (E) shows the top 25 most abundant genera recovered for each sample types.

Fig 4. Firmicutes to Bacteroidetes ratio plotted using R’s ggstatsplot package [49] with ASVs collated at Firmicutes/Bacteroidetes level using the taxonomy identified through SILVA SSU Ref NR database release v.138.

Fig 5. MS ion counts of SCFA conjugates with carnitine identified in murine stool samples when fed with Pp+smectite, Pp+kaolin and PBS.