The treatment of Helicobacter pylori with antimicrobial agents has largely been ineffective, and susceptibility results are in disagreement with those obtained by standard in vitro testing. The bactericidal effect of amoxicillin was tested in an in vitro model by using sessile bacteria attached to HEp-2 cells; this bactericidal effect was compared with that against planktonic bacteria. Viable cell counts were performed by standard procedures after 1, 6, and 24 h of contact with the antibiotic at different concentrations. A bactericidal effect (99.9% kirling) was observed against sessile bacteria after 24 h with concentrations of only 10, 1, and 0.1 mg/liter, while against planktonic bacteria it was also noted at concentrations of 0.01 and 0.001 mg/liter, and the effect was observed after 6 h with the three highest concentrations. When the results for five strains were studied by analysis of variance at 6 and 24 h, the main variable was the antibiotic concentration, followed by the culture conditions, e.g., planktonic or sessile bacteria, the strain tested, and the time of contact. A decreased pH of 5.4 did not affect the action of amoxicillin. The bactericidal effect of the combination of amoxicillin and metronidazole was additive against sessile H. pylori.Helicobacter pylori is recognized as an important factor in the development of peptic ulcers (11). While peptic ulcer relapse occurs in up to 80% of cases during the year following the first diagnosis, eradication of H. pylori has been shown to improve greatly the natural history of the disease. In some studies in which eradication is obtained, the relapse rate is almost nil (25). However, eradication is difficult to obtain. There is no single agent which can achieve eradication in a significant percentage of cases (16). Amoxicillin treatment has given the best results, with approximately 10 to 20% eradication (3). These results contrast with the apparently high susceptibility of H. pylori in vitro to most antimicrobial agents, including amoxicillin (8,10,13,15,20), which indicate that the traditional MIC determination is not adequate for testing this mucosa-associated bacterium.The concept of biofilm has been proposed by Costerton (6). By studying industrial ecosystems, Costerton (6) showed that attached organisms (sessile bacteria) differ markedly from floating organisms (planktonic bacteria) that are present in the same ecosystem and differ even more markedly from cells that are grown in pure culture in laboratory media. Cell line. An HEp-2 epithelial cell line was used. The growth medium was Eagle minimum essential medium supplemented with 10% fetal bovine serum and 1% nonessential amino acids.General conditions of the test. Cells were seeded in 25-cm2 flasks (Nunc) to obtain a subconfluent monolayer after 24 h of growth (105 cells).A suspension of 24-h-old H. pylori was prepared and inoculated onto the HEp-2 cells to obtain a concentration of approximately 5 x 106 CFU per flask (2 x 105 CFU/cm2).The cells and the bacteria were incubated for 2 h in a C02-e...