Low-Abundance and Fragmentary Helicobacter pylori DNA Detected in Phenotypically Negative Gastric Biopsies Using...

Accurate detection and monitoring of antimicrobial resistance (AMR) in Helicobacter pylori mainly rely on phenotypic methods and culture, which can sometimes fail when bacterial load is low or after recent treatment. We investigated whether gastric biopsies classified as H. pylori-negative by standard diagnostic techniques still contain detectable bacterial DNA, including regions linked to AMR, and assessed whether selected DNA fragments can mediate allelic exchange in vitro. Gastric biopsies from 46 dyspeptic patients in the Democratic Republic of the Congo (including 23 phenotypically positive and 23 phenotypically negative individuals) were analyzed using long-read amplicon sequencing of seven resistance-associated loci, selective whole-genome amplification (sWGA) followed by long-read sequencing of H. pylori-enriched reads, and a proof-of-concept natural transformation assay. Phenotypically negative biopsies exhibited significantly lower sequencing depth across multiple loci (including 23S rRNA, gyrA, gyrB, and pbp1A; p = 0.003-0.014), indicating a reduced H. pylori DNA burden. However, AMR-associated mutations linked to various antibiotic classes were found in both groups. sWGA enabled recovery of fragmentary H. pylori sequence data from phenotypically negative samples, including reads that map to resistance- and virulence-associated genes. In vitro, 23S rRNA A2143G amplicons from both phenotypically positive and negative biopsies produced clarithromycin-resistant transformants in strain 26695. These findings indicate that phenotypically negative gastric biopsies might contain low-abundance and fragmentary H. pylori DNA. Although certain DNA fragments can mediate allelic exchange under controlled in vitro conditions, these results do not confirm bacterial viability, active infection, or clinically relevant in vivo resistance transfer. Therefore, they should be interpreted with caution in molecular AMR surveillance and detection contexts.