Helicobacter pylori free-living and biofilm modes of growth: behavior in response to different culture media LUCINDA J. BESSA, 1 ROSSELLA GRANDE, 2 DONATO DI IORIO, 3 MARA DI GIULIO, 2 EMANUELA DI CAMPLI 2 and LUIGINA CELLINI 2 Departments of 1 Biomedical Sciences, 2 Pharmacy, and 3 Medical Oral Science and Biotechnology, University ‘G. d’Annunzio’ – Chieti-Pescara, Italy Bessa LJ, Grande R, Di Iorio D, Di Giulio M, Di Campli E, Cellini L. Helicobacter pylori free-living and biofilm modes of growth: behavior in response to different culture media. APMIS 2012. The physiological behavior of Helicobacter pylori in different growth conditions was investigated to approach its growth standardization. H. pylori free-living and biofilm modes of growth were assessed in four different liquid culture media (Brucella broth, brain heart infusion broth and Ham’s F-12 supple- mented with 2% fetal calf serum and Ham’s F-12 without serum). Free-living growth was monitored dur- ing 72 h in each medium and characterized for bacterial density, culturability, viability and morphology. The biofilm formation in the same media was evaluated for biomass production, colony forming unit (CFU) counts and microscopic visualization. Afterward, using Ham’s F-12, the effect of amoxicillin and clarithromycin at sub- minimum inhibitory concentrations (sub-MICs) was evaluated on H. pylori biofilm formation and luxS gene expression. Differences in the free-living growth were observed between the media supplemented with serum and Ham’s F-12 without serum. Biofilm formation was significantly dependent on the growth media used. Ham’s F-12 seems to be a good medium to support both growth phenotypes of H. pylori. Moreover, sub-MICs of antibiotics increased the biofilm formation and affected the luxS gene expression. Optimizing the growth conditions of H. pylori, especially in the biofilm mode, will be helpful to perform more accurate in-depth studies that will allow increasing the knowledge about H. pylori biofilm, which should be a target to eradicate resistant infection. Key words: Helicobacter pylori; culture media; free-living growth; biofilm formation; sub-MICs of antibiotics. Luigina Cellini, Department of Pharmacy, University ‘G. d’Annunzio’ – Chieti-Pescara, Via dei Vestini 31, Chieti 66100, Italy. e-mail: l.cellini@unich.it The pathogen Helicobacter pylori is the etiologic agent of gastritis and peptic ulcer disease and is also associated with the develop- ment of gastric adenocarcinoma and MALT- lymphoma (1, 2). The fastidious nature of this Gram-negative microorganism has been widely recognized and numerous solid and liquid media have been developed for its isolation and culture (3). Despite all the difficulties to grow this microorganism in vitro, growth in liquid media is particularly more challenging than in solid media (4). H. pylori growth has already been described in several complex and non-spe- cific liquid media such as Brucella broth (BB), Mueller-Hinton broth (MHB), brain heart infu- sion broth (BHIB), tryptic soy broth (TSB), but none of these media seemed to be particularly advantageous over the others (4–6). Moreover, good growth in those complex liquid media requires the addition of supplements, such as fetal bovine serum, b-cyclodextrins (7), horse serum (8), bovine serum albumin (9) and yeast extract (10). Ham’s F-12, a chemically defined liquid medium, has been so far the only Received 24 August 2012. Accepted 3 October 2012 1 APMIS © 2012 The Authors APMIS © 2012 APMIS DOI 10.1111/apm.12020