Effect of Probiotics on the Fecal Resistome During Helicobacter Pylori Eradication Therapy

Antibiotic use is one of the most common therapeutic strategies of modern medicine. Together with the significant beneficial effects of antibiotic use, side effects accompany the use of these drugs, including the increasing appearance of antibiotic-resistant microbes, a global public health problem. Other significant side effects include gastrointestinal-related symptoms such as diarrhea, pain, intestinal discomfort, and flatulence, which can be the consequence of dysbiosis. A common strategy to limit antibiotic-associated side effects is the addition of probiotics, as S. boulardii CNCM I-745 (Sb). The use of bacterial probiotics to reduce antibiotic-associated side effects has several potential limitations, including the destruction of probiotics, development of probiotics strains resistant to antibiotics, and passage of antibiotic-resistant genes to pathogenic bacteria through horizontal gene transfer. Sb is a probiotic yeast whose benefits on intestinal dysbiosis have been associated with establishing a favorable growth environment for the normal intestinal microbiota. Due to the yeast nature of this probiotic, the limitations of bacterial-probiotics cannot be accounted for during Sb use. Consequently, it is crucial to study the molecular mechanism elicited by Sb on intestinal microbiota, including changes in resistome.

In our previous study regarding the supplementation with Sb on the treatment against H. pylori (Hp) infection, patients that received the probiotic had a significantly lower frequency of gastrointestinal symptoms. Additionally, those patients have a higher number of bacterial diversity evenness (P=0.0156), higher abundance of Enterobactereacea, and lower abundance of Bacteroides and Clostridia upon treatment completion and one month later. Bacteroides and Clostridia have been previously implicated as antibiotic multi-resistant pro-inflammatory strains. The objective of the current project is to characterize the resistome of individuals treated with anti-H. pylori therapy in the presence or absence of Sb in fecal samples.

Specific aims To determine the resistome changes before and after Hp eradication treatment in patients that have received or not Sb.

To compare the resistome patterns between patients treated for Hp infection that have received or not SB, at the time of completion of treatment and one month after finishing them.

Based on the shotgun metagenomics, the researchers seek to characterize the microbiome and resistome in fecal samples and understand the microbiome's functional characteristics, including virulence genes, metabolic pathways, and mobile genetic elements.

Methodology DNA obtained from the previous microbiome characterization will be used in this project. In case DNA is not enough, the researchers will extract DNA from the frozen stored samples using the Power Soil kit.

DNA will be shipped to UNC Chapel Hill or the University of Minnesota, where genomic libraries will be constructed. Hi-Seq Illumina runs will be used to produce a sequencing deepness of 10 million per sample.

FASTQC will be used for reads quality control and multiQC as reports generator from the raw reads. Trimmomatic will be used to trim Illumina adapters and to cut reads on the average quality. As human DNA is present on the fecal samples, the investigators have to remove the host DNA using the mapping software BWA and SAMtools. Using the Resfinder database, the investigators must align the filtered reads and generate a count table using ResistomeAnalyzer. Finally, the researchers can create rarefaction curves based on the table made using AMRPlusPlus.

Expected results The researchers expect to find a higher diversity and abundance of antibiotic resistance genes (genome intrinsic and carried by mobile genetic elements) on the group that did not receive Sb at the end of treatment and one month after its completion. This would be correlated with the lower abundance of Clostridia and Bacteroides encountered in the 16S rRNA gene microbiome characterization.

The investigators will also be able to sub-characterize the resistome diversity and abundance of antibiotic resistance genes encountered typically on Enterobacteriaceae. A previous analysis found a higher abundance of Enterobacteriaceae in the group that received Sb (previously reported in a mouse model by Sovran et al. 2018). Additionally, since the researchers will obtain the raw reads on the shotgun sequencing, they could further characterize the bacteria modulated by the use of Sb at strain level (Bacteroides, Clostridia, and Enterobacteriaceae). Together, the researchers expect to obtain valuable data on the use of Sb regarding antibiotic resistance and further microbiome characteristics, including genes of bacterial pathogenicity. These results will contribute to clarify the beneficial effects of Sb addition in patients treated for Hp infection.