A validated sequence difference ratio framework distinguishes intrahost evolution from superinfection in Helicobacter pylori mixed infections

Background The inability to quantitatively distinguish endogenous within-host evolution (mutation, homologous recombination, and heterologous recombination) from superinfection as the origin of intrahost Helicobacter pylori genetic diversity remains a critical bottleneck constraining precision therapeutics. Methods We developed a novel gene-based sequence difference ratio (SDR) framework utilizing single-nucleotide polymorphism counts and percentile-derived regional reference ranges to quantitatively classify the origins of mixed infections, including homologous and heterologous recombination (interspecies horizontal gene transfer). A dual-hospital cohort (34 patients, 114 flagellar isolates; 28 patients, 152 housekeeping isolates) was enrolled after excluding those with recent antibiotic or acid-suppressant exposure. Isolates were recovered using a patented single-colony culture system (US 18/278,425; EP 21927415.6) to eliminate conventional streaking bias. The model was validated using phylogenetic analysis, hierarchical clustering, and geographical divergence assessment. Results The SDR framework achieved the first dedicated quantitative classification of five distinct H. pylori mixed infection mechanisms: mutation, homologous recombination, heterologous recombination, combined mutation–homologous recombination, and superinfection. Flagellar gene profiling identified a 51.5% mixed infection rate (17/33), with mutations as the dominant pattern (82.4%, 14/17); recombination involved the conserved flagellar genes flgE and flaA . Housekeeping gene analysis yielded a 14.8% superinfection rate (4/27), including the first documented triple-strain case; recombination involved the conserved housekeeping genes atpA and yphC . Strain H42 exhibited heterologous recombination in trpC (SDR 23.89–25.66%), providing the first nucleotide-resolution evidence of cross-species genetic exchange. Convergent mutations in fliD and mutY indicated niche-specific selective pressure. SDR thresholds were strongly correlated with evolutionary distance. Conclusion We report the first validated quantitative framework for distinguishing intrahost microbial evolution (mutation, homologous recombination, and heterologous recombination) from superinfection in H. pylori . These findings challenge the long-standing single-strain therapeutic paradigm, and this scalable approach lays a universal foundation for the precise management of infectious diseases.