Detection of Helicobacter pylori Microevolution and Multiple Infection from Gastric Biopsies by Housekeeping Gene Amplicon Sequencing

Abstract

Despite the great efforts devoted to research on <i>Helicobacter pylori</i>, the prevalence of single-strain infection or <i>H. pylori</i> mixed infection and its implications in the mode of transmission of this bacterium are still controversial. In this study, we explored the usefulness of housekeeping gene amplicon sequencing in the detection of <i>H. pylori</i> microevolution and multiple infections. DNA was extracted from five gastric biopsies from four patients infected with distinct histopathological diagnoses. PCR amplification of six <i>H. pylori</i>-specific housekeeping genes was then assessed on each sample. Optimal results were obtained for the <i>cgt</i> and <i>luxS</i> genes, which were selected for amplicon sequencing. A total of 11,833 <i>cgt</i> and 403 <i>luxS</i> amplicon sequences were obtained, 2042 and 112 of which were unique sequences, respectively. All <i>cgt</i> and <i>luxS</i> sequences were clustered at 97% to 9 and 13 operational taxonomic units (OTUs), respectively. For each sample from a different patient, a single OTU comprised the majority of sequences in both genes, but more than one OTU was detected in all samples. These results suggest that multiple infections with a predominant strain together with other minority strains are the main way by which <i>H. pylori</i> colonizes the human stomach.