Recovery of Helicobacter pylori ATCC43504 from a viable but not culturable state: regrowth or resuscitation?

Studies were conducted following the formation and characterization of the coccoid morphology of Helicobacter pylori. H. pylori ATCC43504 was incubated in brucella broth plus 2% fetal calf serum at three different temperatures: 37 degrees C, room temperature and 4 degrees C in a microaerophilic environment, and readings were taken at 2, 7, 15, 30 and 45 days. At control times, the total and the viable count, viability tests with tetrazolium salts, and ultrastructural studies were carried out. On solid media, H. pylori became nonculturable after 7 days of incubation at room temperature and 4 degrees C, and after 15 days of incubation at 37 degrees C. At these times of incubation, after subculturing in liquid medium under the same conditions, the growth of H. pylori was detected until the 15th day from cultures incubated at 4 degrees C and until the 30th day from cultures stored at 37 degrees C, and at room temperature. Ultrastructural studies showed a gradual reduction of integrity of bacterial cells that remained stable at 30 and 45 days of incubation: 30% of whole cells of bacteria incubated at 37 degrees C and room temperature and 50% in bacteria incubated at 4 degrees C. The viability of the VNC (viable nonculturable) state was assessed by studying the reduction of tetrazolium salts INT (p-iodonitrophenyl tetrazolium violet) and CTC (cyanoditolyl tetrazolium chloride) to their respective formazans and this was linked to the cellular respiration. At 45 days of incubation, when bacterial regrowth was not observed in solid or in liquid medium, different resuscitation methods were applied to evaluate a possible resuscitation of VNC H. pylori. No significant growth on solid medium was observed.