L. Cellini et al., RECOVERY OF HELICOBACTER-PYLORI ATCC43504 FROM A VIABLE BUT NOT CULTURABLE STATE - REGROWTH OR RESUSCITATION, APMIS. Acta pathologica, microbiologica et immunologica Scandinavica, 106(5), 1998, pp. 571-579
Studies were conducted following the formation and characterization of
the coccoid morphology of Helicobacter pylori. H. pylori ATCC43504 wa
s incubated in brucella broth plus 2% fetal calf serum at three differ
ent temperatures: 37 degrees C, room temperature and 4 degrees C in a
microaerophilic environment, and readings were taken at 2, 7, 15, 30 a
nd 45 days. At control times, the total and the viable count, viabilit
y tests with tetrazolium salts, and ultrastructural studies were carri
ed 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 subcul
turing in liquid medium under the same conditions, the growth of H. py
lori was detected until the 15th day from cultures incubated at 4 degr
ees C and until the 30th day from cultures stored at 37 degrees C, and
at room temperature. Ultrastructural studies showed a gradual reducti
on of integrity of bacterial cells that remained stable at 30 and 45 d
ays of incubation: 30% of whole cells of bacteria incubated at 37 degr
ees 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 the
ir respective formazans and this was linked to the cellular respiratio
n. At 45 days of incubation, when bacterial regrowth was not observed
in solid or in liquid medium, different resuscitation methods were app
lied to evaluate a possible resuscitation of VNC H. pylori. No signifi
cant growth on solid medium was observed.