Shifts in the microbiome during anti tuberculosis therapy in an animal model

Abstract

Tuberculosis (TB) is a worldwide pandemic caused by the bacterial agent Mycobacterium tuberculosis (Mtb). The first-line treatment of the disease is invasive and consists of a combination of four antimicrobials to be administered for a period of at least six months. Antimicrobial administration is known to affect the diversity and structure of the gastro-intestinal microbiome by rapidly decreasing bacterial taxa present. Recently, the effect of TB and TB treatment on the gastro-intestinal microbiome has gained attention, since it is suggested that the microbiome plays a role in host immunity. In this study, the impact of different TB treatments on the gastro-intestinal microbiome of Kramnik mice was evaluated. To achieve this, an effective method for DNA extraction of the gastro-intestinal microbiome is necessary to successfully identify all bacteria present. To effectively extract DNA from the representative gastro-intestinal microbiome, commercially available kits namely QIAamp DNA microbiome kit (Qiagen, Germany) and the GenElute™ Stool DNA isolation kit (Sigma, USA) were compared to the NWU lysis method with and without purification steps. Based on these results, DNA from the gastro-intestinal environment was extracted using a similar commercial kit to determine microbiome composition from samples treated with TB therapies. TB therapies included standard TB therapy, Pheroid®-based TB therapy, and the Pheroid® formulation excluding active pharmaceutical ingredients (APIs). All DNA from the microbiome were analyzed with 16S amplicon sequencing using the Illumina Miseq platform. DNA data was analyzed using bioinformatics and community profiling was achieved by using MicrobiomeAnalyst. The various extraction methods did not indicate any significant differences in microbiome composition or structure of the gastro-intestinal environment. Therefore, extraction methods with similar lysis steps are suggested to be an effective approach for successful DNA isolation from the gastro-intestinal microbiome. However, inter-individual differences were observed between mice under the same environmental conditions. Exposure to all selected therapies had a significant impact on the microbiome diversity compared to Mtb infected mice, by decreasing the overall diversity of the microbiome. Members of the phylum Bacteroidota were significantly decreased and Firmicutes significantly increased. The effect of Pheroid®-based TB therapy on the microbiome did not differ significantly from standard TB therapy. However, the presence of members of the family Saccharimonadaceae was significantly more abundant in the microbiome of mice recovering from Pheroid®-based TB therapy, which is positively correlated with the immune response. Mice treated with the Pheroid® carriers (formulation excluding APIs) had a significantly higher abundance of Lactobacillus in their microbiome, which is associated with lipid metabolism. Mice treated with the latter did not indicate a significant reduction of Mtb in the lungs, but survived longer than the infected animals. It is suggested that the Pheroid® formulation has a possible immune support function. The proliferation v of commensal bacteria Parasutterella was observed in the microbiome of mice recovering from the Pheroid® carriers, indicating normalization of the microbiome after exposure. Therefore, the use of Pheroid® as an alternative for TB drug delivery does not necessarily improve the state of the microbiome during treatment, but might have the potential to stabilize the gastro-intestinal environment after treatment has ceased