Helicobacter pylori inactivation and virulence gene damage using a supported sensitiser for photodynamic therapy

Abstract

About half of the world's population is currently infected with Helicobacter pylori, which is involved in the development of several gastro-duodenal pathologies. The increasing number of antibiotic resistance reduces the effectiveness of the first-line therapy, so new strategies to improve the H. pylori eradication rates are needed. Antimicrobial Photodynamic Therapy (APDT) benefits from photogenerated reactive oxygen species, such as singlet oxygen, which inactivate microorganisms by means of photosensitising dyes and visible light. Therefore, it could be a suitable alternative for H. pylori eradication in the gastro-duodenal tract, particularly in patients infected with antibiotic resistant strains. We evaluated APDT against H. pylori, in vitro, using a new photosensitising material (PSM) based on a ruthenium(II) complex covalently bound to micrometric glass beads. Five H. pylori isolates (classified according to cagA genotype, and metronidazole-clarithromycin resistance) were used. Bacteria were mixed with the PSM and incubated in the dark or illuminated by blue light. Aliquots (min 1', 2', 5', 15' and 30') were cultured and colonies were counted after 2-3 days. A 99.99999% decrease was detected in the number of colonies in the irradiated wells where the bacterium was mixed with the PSM, compared to non-illuminated wells or with irradiated wells without PSM. It was also confirmed that DNA is a molecular target for oxidant species released during APDT (evaluated by alkaline gel electrophoresis after endonuclease III incubation, ureC and cagA RT-PCR, and bacterial fingerprint). Results were independent of cagA gene and antibiotic resistances.

Keywords: APDT; AXMFYAOBYBXMLC-UHFFFAOYSA-H; Antibiotic resistance; CFU; Ct; H. pylori; Helicobacter pylori; LED; Oxidative damage; PSM; Photodynamic therapy; Photosensitiser; RAPD; ROS; RT-PCR; Singlet oxygen; TSA; TSB; antimicrobial photodynamic therapy; cagA; colony-forming units; cycle threshold; cytotoxin associated gene A; light-emitting diode; photosensitising material; random amplified polymorphic DNA; reactive oxygen species; real-time polymerase chain reaction; trypticase soy broth; tryptone soy agar; ureC; urease encoding gene.