Sequential statistical improvement of the liquid cultivation of Helicobacter pylori.
BACKGROUND
Colonization of the gastric mucosa by Helicobacter pylori is one of the most important causes of acute and chronic gastric pathologies in humans. Achieving the growth of H. pylori in liquid media is of great importance in the development of clinical studies. In this study, we developed a sequential optimization strategy based on statistical models to improve the conditions of liquid culture of H. pylori.


METHODS
Four statistical models were sequentially used. First, a Box-Behnken design was used to select the best process conditions (shaking speed, inoculum concentration, and final volume of culture). Secondly, a general factorial design was used to evaluate the influence of adding gel blocks or gel beads (shape and composition). Then a D-optimal reduce design was carried out to allow the selection of the most influential factors in increasing the cell concentration (culture media components). Finally, another Box-Behnken design was used to optimize the concentration of the culture media components previously selected.


RESULTS
After 12 hours of liquid culture a concentration of 25 x 10(8) cells per mL (9.4 log(10) cells per mL) of H. pylori was obtained, compared with a predicted 32 x 10(8) (9.5 log(10) cells per mL), which means between 1 and 5 log(10) units higher than some previous reports.


CONCLUSIONS
The sequential statistical approach increased the planktonic H. pylori cell culture. The final culture media and conditions were: Brain Heart Infusion, blood agarose (1.5% w/v), lamb's blood (3.18% v/v), DENT (0.11% v/v), and Vitox (0.52% v/v) at 60 rpm and 37 degrees C with filtered CO2 (5% v/v) bubbled directly into the culture media in a final volume of 76.22 mL.
Duque-Jamaica
Rocio
R
Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Carrera de Microbiología Industrial, Cra. 7 N degrees 40-62. Bogotá, DC, Colombia.
Arévalo-Galvis
Azucena
A
Poutou-Piñales
Raúl A
RA
Trespalacios
Alba A
AA
D001431:Bacteriological Techniques; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D046508:Culture Techniques / Q000379:methods; D016480:Helicobacter pylori / Q000737:chemistry / Q000254:growth & development* / Q000378:metabolism; D015233:Models, Statistical
D003470:Culture Media