Characterization of monospecies biofilm formation by Helicobacter pylori

Abstract

As all bacteria studied to date, the gastric pathogen Helicobacter pylori has an alternate lifestyle as a biofilm. H. pylori forms biofilms on glass surfaces at the air-liquid interface in stationary or shaking batch cultures. By light microscopy, we have observed attachment of individual, spiral H. pylori to glass surfaces, followed by division to form microcolonies, merging of individual microcolonies, and growth in the third dimension. Scanning electron micrographs showed H. pylori arranged in a matrix on the glass with channels for nutrient flow, typical of other bacterial biofilms. To understand the importance of biofilms to the H. pylori life cycle, we tested the effect of mucin on biofilm formation. Our results showed that 10% mucin greatly increased the number of planktonic H. pylori while not affecting biofilm bacteria, resulting in a decline in percent adherence to the glass. This suggests that in the mucus-rich stomach, H. pylori planktonic growth is favored over biofilm formation. We also investigated the effect of specific mutations in several genes, including the quorum-sensing gene, luxS, and the cagE type IV secretion gene. Both of these mutants were found to form biofilms approximately twofold more efficiently than the wild type in both assays. These results indicate the relative importance of these genes to the production of biofilms by H. pylori and the selective enhancement of planktonic growth in the presence of gastric mucin.

FIG. 1.

Progression of biofilm formation by H. pylori SD14, as shown in light micrographs of fixed and carbol fuchsin-stained H. pylori SD14 cells at the air-liquid interface on borosilicate glass coverslips. (A) Single bacteria attached to glass at 3 days of stationary growth in broth. Attached bacteria began dividing in two dimensions to form a microcolony at 3 to 4 days. Bar, 10 μm. (B) Individual microcolonies merged into a dense biofilm at the air-liquid interface at >4 days of growth. Bar, 25 μm.

FIG. 2.

SEM micrograph of a 4-day-old biofilm of H. pylori SD14 grown as a stationary batch culture. Bar, 1 μm.

FIG. 3.

Effect of mucin on biofilm production. Biofilm formation of H. pylori strains in the presence of 0 to 10% mucin was compared using the MTT respiratory assay (strain N6) (A and B) and direct counts (strain SD4) (C and D). Percent adherence (A and C) was calculated as described in the text for the two assays. In panels B and D, dark bars represent quantitation of biofilm bacteria and light bars indicate absolute values for planktonic bacteria. In both assays, note that while the percent adherence decreased with 1 and 10% mucin, the planktonic bacteria greatly outnumbered the biofilm bacteria (which remain relatively unchanged in number) in both assays. Error bars represent means ± standard deviations.

FIG. 4.

Effect of the luxS mutation on biofilm formation by two different strains of H. pylori. (A) Three-day-old H. pylori SD3 and SD3luxS biofilms were quantitated using the respiratory assay, as described in the text. (B) SD14 and SD14luxS were grown in biofilms for 3 days and quantitated using the direct count assay, as described in Materials and Methods. Error bars represent means ± standard deviations.

FIG. 5.

Effect of the cagE mutation on biofilm formation. (A) H. pylori SD4 and four different cagE isolates were grown as biofilms for 3 days and quantitated using the respiratory assay, as described in the text. (B) Strains SD4 and SD4cagE were grown as biofilms and assayed by direct counts, as described in Materials and Methods. Light bars represent results from biofilms grown for 3 days, while dark bars indicate values for 5-day-old biofilms. Error bars represent means of four experiments ± standard deviations.