Biofilm formation by Campylobacter jejuni is increased under aerobic conditions

Abstract

The microaerophilic human pathogen Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in the developed world. During transmission through the food chain and the environment, the organism must survive stressful environmental conditions, particularly high oxygen levels. Biofilm formation has been suggested to play a role in the environmental survival of this organism. In this work we show that C. jejuni NCTC 11168 biofilms developed more rapidly under environmental and food-chain-relevant aerobic conditions (20% O(2)) than under microaerobic conditions (5% O(2), 10% CO(2)), although final levels of biofilms were comparable after 3 days. Staining of biofilms with Congo red gave results similar to those obtained with the commonly used crystal violet staining. The level of biofilm formation by nonmotile aflagellate strains was lower than that observed for the motile flagellated strain but nonetheless increased under aerobic conditions, suggesting the presence of flagellum-dependent and flagellum-independent mechanisms of biofilm formation in C. jejuni. Moreover, preformed biofilms shed high numbers of viable C. jejuni cells into the culture supernatant independently of the oxygen concentration, suggesting a continuous passive release of cells into the medium rather than a condition-specific active mechanism of dispersal. We conclude that under aerobic or stressful conditions, C. jejuni adapts to a biofilm lifestyle, allowing survival under detrimental conditions, and that such a biofilm can function as a reservoir of viable planktonic cells. The increased level of biofilm formation under aerobic conditions is likely to be an adaptation contributing to the zoonotic lifestyle of C. jejuni.

FIG. 1.

Biofilm formation by the C. jejuni NCTC 11168 motile variant (11168Mot), the nonmotile variant (11168Non-mot), and the flaAB mutant (11168Mot::flaAB) after incubation for 2 days at 37°C under static microaerobic (filled bars) or aerobic (open bars) conditions or under 10% CO₂ in air (shaded bars). Results shown are averages from three independent experiments, each performed in triplicate. (A) Crystal violet assay for biofilm formation. (B) Congo red assay for biofilm formation. (C) Viable planktonic cells from biofilm supernatants from the crystal violet assay. The error bars represent standard deviations calculated from the three independent experiments performed.

FIG. 2.

Biofilm formation by the C. jejuni NCTC 11168 motile variant over 3 days under static microaerobic (filled bars) and aerobic (open bars) incubation conditions, as determined using crystal violet staining. Results are averages from three independent experiments, each containing triplicate samples. The error bars represent standard deviations calculated from the three independent experiments performed.

FIG. 3.

Representative light microscopy photographs of C. jejuni biofilms after 2 days of incubation at 37°C. Slides were stained with 1% crystal violet and were photographed at ×400 magnification. Images show 11168Mot (A and B) and 11168Non-mot (C and D) grown under microaerobic (A and C) and aerobic (B and D) conditions.

FIG. 4.

(A) Viable cells in the supernatant after 2 days of aerobic biofilm formation (prewash) (dark shaded bars), after a wash with sterile PBS (open bars), and after 24 h of static incubation in fresh brucella broth under either aerobic (filled bars) or microaerobic (light shaded bars) conditions at 37°C. Values are means from least three independent experiments, and error bars represent standard deviations calculated from the three independent experiments. (B) Model of a C. jejuni biofilm showing the fate of cells following release from a preformed biofilm. After release from a biofilm, planktonic cells can proliferate under favorable conditions or may reattach to an existing biofilm. Cells may also die under conditions that preclude biofilm formation (i.e., in a fast flowing system).