A validated sequence difference ratio framework distinguishes intrahost evolution from superinfection in Helicobacter pylori mixed infections
- 1Health Science Center, Ningbo University
- 2Laboratory of Helicobacter Pylori Section, Professional Committee of Clinical Microbiology, Chinese Society for Microbiology, Ningbo University
- 3Department of Gastrology, Ninghai First Hospital
Abstract
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.
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BibTeX
@article{gu2026validated,
title = {{A validated sequence difference ratio framework distinguishes intrahost evolution from superinfection in Helicobacter pylori mixed infections}},
author = {Haiying Gu and Zhouhong Shi and Weiqin Chen and Weiwei Ye and Cheng Zhang and Airu Li},
journal = {Frontiers in Microbiology},
year = {2026},
month = oct,
doi = {10.3389/fmicb.2026.1948969},
url = {https://www.frontiersin.org/articles/10.3389/fmicb.2026.1948969/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - A validated sequence difference ratio framework distinguishes intrahost evolution from superinfection in Helicobacter pylori mixed infections
AU - Haiying Gu
AU - Zhouhong Shi
AU - Weiqin Chen
AU - Weiwei Ye
AU - Cheng Zhang
AU - Airu Li
PY - 2026
DA - 2026/10/06
JO - Frontiers in Microbiology
DO - 10.3389/fmicb.2026.1948969
UR - https://www.frontiersin.org/articles/10.3389/fmicb.2026.1948969/full
AB - 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.
SN - 1664-302X
ER -