Abstract: Helicobacter pylori infection which affects more than half of the world population, is considered the most important risk factor in the pathogenesis of gastric cancer [1, 2], and its eradication might confer long-term protection against gastric adenocarcinoma in high-risk populations [3]. The treatment usually combines antibiotics and an acid-reducing proton pump inhibitor [4]. Nevertheless, increasing antibiotic resistance and ineffective treatments led the World Health Organization to designate clarithromycin-resistant H. pylori as a high-priority bacterium for antibiotic research and development [5]. These findings call for alternative strategies to control H. pylori. Phages, viruses that infect bacteria, show effectiveness in the treatment of pathogenic bacteria [6], however very little is known about H. pylori phages [7].

Due to the difficulties in isolating H. pylori strictly lytic phages and envisaging the possibility of using temperate phages to control H. pylori infections, we develop a method to identify prophages in H. pylori genomes and to induce their lytic cycle.  We used a collection of 74 H. pylori-clinical strains to screen for prophage genes based on PCR profiling. Strains encoding putative prophages were further sequenced. Subsequently, after genome analysis, prophage induction was attempted in strains with intact prophages, using UV radiation and mitomycin C. The isolated phages were further characterized.

Based on the results of the PCR-detection method, 14 strains, with a high probability of having prophages in their genome were sequenced, resulting in a total of 12 prophages identified in silico. The developed methodology increased the efficiency of identifying strains with complete prophages to 83.3 %. Induction strategies against strains with positive-prophage sequences resulted in the release of three new phages with a podovirus-like structure. They present a genome length range between 29 705 and 31 162 bp with a G+C content of around 37 %. No tRNAs or antibiotic-resistance genes were identified. Interestingly, the three phages were relatively stable from pH 3 to 11 and at 37 ◦C, suggesting that they are adapted to the human stomach environment. Furthermore, two phages had the capability to suppress H. pylori population levels for up to 24 h post-infection at multiplicities of infection of 0.01, 0.1, and 1. The phylogenomic tree conclusively demonstrated that these new phages are phylogenetically related to all other reported H. pylori phages because, with one exception, they all belong to the same family and genus, even though all of them have different geographical origins.

Overall, our findings reveal a novel area for future investigations representing a significant contribution to the knowledge of H. pylori phages and providing valuable insights into their potential use in phage therapy.

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