Use of Alignment-Free Phylogenetics for Rapid Genome Sequence-Based Typing of Helicobacter pylori Virulence Markers and Antibiotic Susceptibility

Whole-genome sequencing is becoming a leading technology in the typing and epidemiology of microbial pathogens, but the increase in genomic information necessitates significant investment in bioinformatic resources and expertise, and currently used methodologies struggle with genetically heterogeneous bacteria such as the human gastric pathogen Helicobacter pylori. Here we demonstrate that the alignment-free analysis method feature frequency profiling (FFP) can be used to rapidly construct phylogenetic trees of draft bacterial genome sequences on a standard desktop computer and that coupling with in silico genotyping methods gives useful information for comparative and clinical genomic and molecular epidemiology applications. FFP-based phylogenetic trees of seven gastric Helicobacter species matched those obtained by analysis of 16S rRNA genes and ribosomal proteins, and FFP- and core genome single nucleotide polymorphism-based analysis of 63 H. pylori genomes again showed comparable phylogenetic clustering, consistent with genomotypes assigned by using multilocus sequence typing (MLST). Analysis of 377 H. pylori genomes highlighted the conservation of genomotypes and linkage with phylogeographic characteristics and predicted the presence of an incomplete or nonfunctional cag pathogenicity island in 18/276 genomes. In silico analysis of antibiotic susceptibility markers suggests that most H. pylori hspAmerind and hspEAsia isolates are predicted to carry the T(2812)C mutation potentially conferring low-level clarithromycin resistance, while levels of metronidazole resistance were similar in all multilocus sequence types. In conclusion, the use of FFP phylogenetic clustering and in silico genotyping allows determination of genome evolution and phylogeographic clustering and can contribute to clinical microbiology by genomotyping for outbreak management and the prediction of pathogenic potential and antibiotic susceptibility.