Fourteen healthy volunteers from the Laboratories of Centre for Infectious Diseases, Faculty of Veterinary Medicine, University of Leipzig and Laboratory for Polymicrobial Infections and Biofilms, Charité Universitäts Medizin Berlin (24-64 years of age, mean 39 years, 5 males and 9 females) have taken 3 × 2 capsules (3 × 800 mg) Activomin^® orally for 10 d followed by 3 × 1 capsule (3 × 400 mg) for 35 d. Two stool samples were collected a week prior to therapy and one stool sample on days 10, 31 and 45.

The study was approved by the ethics commission of University of Leipzig. The collection of fecal samples for fluorescence in situ hybridization (FISH) diagnosis of dysbiosis was approved by the ethics commission of the Charité University Hospital.

Colonic microbiota were investigated using FISH analysis of Carnoy fixated and paraffin embedded stool cylinders[4]. Multicolor FISH simultaneously using 3 differently stained FISH probes (C3 - orange, FITC-dobe - green, C5 - dark red) and counterstained with DAPI for DNA structures was performed on 4 μm longitudinally cut sections of punched-out stool cylinders. Sections were placed on SuperFrost plus slides.

A Nikon e600 fluorescence microscope was used. The images were photo-documented with a Nikon DXM 1200F color camera and software (Nikon, Tokyo, Japan).

Bacteria were quantified using group specific C3 probes. The FITC marked universal probe was used in each hybridization to evaluate the number of all bacteria, C5 marked probes with a different to C3 probes specificity were used to exclude unspecific binding. Only signals that hybridized with a specific FISH probe and the universal FISH probe, but did not hybridize with specific FISH probes from unrelated bacterial groups, were evaluated.

Bacterial concentrations of homogeneous populations were enumerated visually in one of the 10 × 10 fields of the ocular raster corresponding to 10 μm × 10 μm of the section surface at magnification of 1000. This number was assigned to concentration of 1 × 10⁹ bacteria/mL, which was most equivalent to the calculation formula, which we had used previously[4].

In case of uneven distribution of bacteria over the microscopic field, the positive signals were enumerated in ten fields of the ocular raster along the gradient of distribution and divided by ten.

Forty-one bacterial FISH probes were applied, Table 1. The exact specification of the FISH probes and hybridization conditions are available in public resources[5]. The names of the FISH probes are listed according to abbreviations of the probeBase online resource (http://www.microbial-ecology.net/probebase/credits.asp). The Fprau probe is described in 6[6].

The FISH probes were arranged in Table 1 to four functional groups described previously: essential bacteria, individual pioneer bacteria, individual substantial and individual marginal or accidental bacteria[7].

Bacteria detected with EREC (mainly Roseburia), Bac303 (Bacteroides), Fprau (Faecalibacterium prausnitzii) probes are always present in healthy human subjects and together contribute about half of the colonic microbiome. They are obviously essential for colonic bio-fermentation.

All other bacterial groups are individual, present only in some of the subjects in substantial concentrations (mean ≥ 0.1 × 10⁹/mL) or marginal concentrations (mean < 0.1 × 10⁹/mL).

Four FISH probes including Bif153 (Bifidobacteriaceae), Cdif198 (Clostridium difficile), Ebac1790 (Enterobacteriaceae) and Clit135 (Clostridium lituseburense) represent individual bacterial groups with pioneer function, which are found prevalent in newborns, after antibiotic treatment and convalescence patients, but are seldom found in low concentration in healthy persons.

Differences between groups were evaluated using the twosided t-Student U test. Data are presented as mean ± SD, P <0.05 was considered statistically significant.

Table 1 summarizes changes in the mean concentrations of single bacterial groups prior to and during supplementation with Activomin^®. Bacteria in the Table 1 are arranged to sets of essential, individual pioneer, individual substantial and individual marginal bio-fermenting groups.

The mean microbial concentration after 45 d of supplementation of humic acids increased 14% in the essential groups (P < 0.01), 28% (NS) in the individual pioneer groups and 41% (P < 0.002) in the individual substantial groups. The accidental bacterial groups with initially marginal concentrations demonstrated no response to humic acids.

The response to humic acids of single bacterial groups was principally the same as in all functional sets of substantial bacteria. The concentrations of most bacterial groups within essential (2 of 3) pioneer (3 of 4) and individual substantial groups (19 of 28) increased in rates of 20% to 60%. In most cases, the increase was observed already at day 10 and continued to day 45. In groups with comparatively low initial mean concentrations (Ebac1790, Cdif198, Chis150, Eram997, Lab158, Veil223) an increase could be higher than 70% and up to 96%, but the contribution of these groups to the overall bacterial numbers was relatively low. Only the concentrations of bacteria detected with Bac303 (Bacteroides) and Myc657 (mycolic acid-containing Actinomycetes) FISH probes decreased under humic acids supplementation, but was statistically not significant, because of the high variance and low number of probands.

The increase in concentrations of microbiota was caused by preexisting groups, and not due to emerging new microorganisms. The individual microbial profile remained constant. In none of the test persons did the ratio of positive/negative individual groups change more than 5%.

Humic acid supplementation did not affect microbial diversity. Mean percent of substantial individual bacterial groups positive for bacteria for each person was nearly the same over time with 72%; 74%; 76%; 72% at the control days accordingly.

The patterns in distribution of single bacterial groups over the stool cylinder differed depending on the species but remained the same in the mucus close transient zone and in the center of the fecal cylinder regardless of humic acids supplementation.

Patients demonstrate increasing interest in medical treatments that are not part of mainstream medicine. Critical argumentation is important, but difficult to do when not evaluated with scientific methods. Humins are a product of microbial metabolism and an important mediator of microbial interactions and activity. As natural fertilizers humins are used for medical indications since ancient times. It is believed that the human microbiome is the main target of humic activity. However no data exist on the impact of humins on the human microbiome.

An evergrowing number of studies demonstrate the involvement of the colonic microbiome in obesity, digestive, endocrine, inflammatory and auto-immune and neurologic disorders. Different approaches are proposed to consolidate and improve the colonic microbiome. The research hotspot is to move beyond description and to introduce substances and therapies with proven controlled graduate effects on the microbiome.

The presented results show, that orally applied humic acids have a profound effect on the healthy colonic microbiome. Although the effects on single microbial groups were multidirectional, the sum concentrations of all colonic microbiota increased 20% to 30%. The increase occurred in the preexisting microbial groups without changes in the bacterial diversity of the microbiome.

The main message of our study is, that humic acids may be an interesting substrate for the development of defined drugs, which deliberately control colonic fermentation in conditions where it is suppressed (post-antibiotics, convalescence) or altered (metabolic disorders, inflammation, obesity etc.), and are a serious amendment/alternative to fecal transplantation or probiotics.

FISH - fluorescence in situ hybridization Cy3, FITC, Cy5, DAPI - different fluorescent dyes corresponding to orange, green, dark red and blue colours. Fluorescence in situ hybridization (FISH) combines the specific identification of microorganisms and the morphological aspect and is as a consequence especially helpful for these purposes. Each single bacterium possesses 10³-10⁵ ribosomes of which each ribosome owns the same copy of ribosomal RNA. Some of the regions of the rRNA are strain-specific, others are universal for species, families or even kingdoms. Oligonucleotides synthesized complimentary to rRNA sequences and labelled with fluorescent dye are called FISH probes. When added to samples containing bacteria, FISH probes hybridize with the rRNA of the bacterial ribosomes. No additional enhancement of fluorescence is necessary and bacteria can be visualized directly due to the large number of ribosomes in each bacterium.

Although the scientific literature reveals a large number of medical trials with dietary supplements of humic acids conducted all over the world. None of the previous studies investigated effects of humic acids on the colonic microbiome.