Impact of environmental and genetic factors on biofilm formation by the probiotic strain Lactobacillus rhamnosus GG

Abstract

Lactobacillus rhamnosus GG (ATCC 53103) is one of the clinically best-studied probiotic organisms. Moreover, L. rhamnosus GG displays very good in vitro adherence to epithelial cells and mucus. Here, we report that L. rhamnosus GG is able to form biofilms on abiotic surfaces, in contrast to other strains of the Lactobacillus casei group tested under the same conditions. Microtiter plate biofilm assays indicated that in vitro biofilm formation by L. rhamnosus GG is strongly modulated by culture medium factors and conditions related to the gastrointestinal environment, including low pH; high osmolarity; and the presence of bile, mucins, and nondigestible polysaccharides. Additionally, phenotypic analysis of mutants affected in exopolysaccharides (wzb), lipoteichoic acid (dltD), and central metabolism (luxS) showed their relative importance in biofilm formation by L. rhamnosus GG.

FIG. 1.

Comparison of capacities of biofilm formation by different lactobacilli. Capacities of biofilm formation by eight Lactobacillus strains were compared under different culture conditions: i.e., mTSB medium, MRS medium (with and without [w/o] glucose), and AOAC medium. The results are expressed compared to biofilm formation of L. rhamnosus GG in mTSB medium (positive control), which was taken as 100% (dotted line). The error bars represent standard deviations of eight biological repeats. The data shown are representative of at least three independent experiments.

FIG. 2.

Influence of MRS medium factors on biofilm formation by L. rhamnosus GG. (A) Different components were omitted (−) from MRS medium to investigate their effect on biofilm formation. Additionally, the influence of these components was investigated after addition (+) to mTSB medium in the same concentration as that present in MRS medium. The results are expressed compared to biofilm formation of L. rhamnosus GG in mTSB medium (positive control), which was taken as 100% (dotted line). (B) The effect of the same components on 72-h growth in suspension (OD₆₀₀) was investigated after omission from MRS medium (−) and addition to mTSB medium (+).

FIG. 3.

Influence of gastrointestinal tract-mimicking conditions on biofilm formation by L. rhamnosus GG. The effect of addition of mucus (2.5 g/liter), inulin-type prebiotics (20 g/liter), bile (0.05 to 2.0%), and lactoferrin (100 μg/ml) to mTSB (black) and AOAC medium (gray) was investigated. Additionally, the influence of changing the pH and osmolarity to 0.3 M NaCl of the biofilm medium was assessed. Biofilm formations in the supplemented mTSB and AOAC media were compared to those of L. rhamnosus GG in unsupplemented mTSB and AOAC media (positive controls, taken as 100% [dotted line]), respectively.

FIG. 4.

Influence of genetic factors on biofilm formation by L. rhamnosus GG. (A) The influence of EPS was investigated after overexpression of antisense wzb RNA in strain CMPG5344 of L. rhamnosus GG in different growth media: mTSB, AOAC, and MRS medium without glucose (MRS − glc). Biofilm formation was then compared to that of L. rhamnosus GG transformed with the empty cloning vector pLAB1301 (positive control). Erythromycin was added for stable maintenance of the plasmids. (B) The influence of d-alanylation of LTA on biofilm formation was investigated by analysis of the phenotype of a dltD mutant, CMPG5540, under the same conditions. Biofilm formation was then compared to that of wild-type L. rhamnosus GG grown under the same conditions (positive control). (C) The influence of central metabolism was investigated by analysis of the phenotype of the luxS mutant CMPG5412 under different conditions. Biofilm formation of the mutants was compared to that of wild-type L. rhamnosus GG (positive control) under the same conditions, which was taken as 100% (dotted line).

FIG. 5.

Comparison of levels of EPS production by L. rhamnosus GG in different media. Two EPS fractions were differentiated: EPS-b and EPS-r. EPS fractions were isolated from stationary-phase cultures of L. rhamnosus GG grown in MRS medium (OD₆₀₀, ∼2.0), AOAC medium (OD₆₀₀, ∼1.5), and mTSB medium (OD₆₀₀, ∼0.5). Since the final OD differs considerably in the different media, results are expressed as μg of glucose equivalents produced per 10⁹ CFU.