High-Throughput Microfluidic Method To Study Biofilm Formation and Host-Pathogen Interactions in Pathogenic Escherichia coli

Abstract

Biofilm formation and host-pathogen interactions are frequently studied using multiwell plates; however, these closed systems lack shear force, which is present at several sites in the host, such as the intestinal and urinary tracts. Recently, microfluidic systems that incorporate shear force and very small volumes have been developed to provide cell biology models that resemble in vivo conditions. Therefore, the objective of this study was to determine if the BioFlux 200 microfluidic system could be used to study host-pathogen interactions and biofilm formation by pathogenic Escherichia coli. Strains of various pathotypes were selected to establish the growth conditions for the formation of biofilms in the BioFlux 200 system on abiotic (glass) or biotic (eukaryotic-cell) surfaces. Biofilm formation on glass was observed for the majority of strains when they were grown in M9 medium at 30°C but not in RPMI medium at 37°C. In contrast, HRT-18 cell monolayers enhanced binding and, in most cases, biofilm formation by pathogenic E. coli in RPMI medium at 37°C. As a proof of principle, the biofilm-forming ability of a diffusely adherent E. coli mutant strain lacking AIDA-I, a known mediator of attachment, was assessed in our models. In contrast to the parental strain, which formed a strong biofilm, the mutant formed a thin biofilm on glass or isolated clusters on HRT-18 monolayers. In conclusion, we describe a microfluidic method for high-throughput screening that could be used to identify novel factors involved in E. coli biofilm formation and host-pathogen interactions under shear force. Biofilms are defined as bacterial communities encased in a selfproduced polymeric matrix that is attached to a surface (1). The ability to form a biofilm is virtually a universal trait of bacteria and other microorganisms. Growth as a biofilm offers protection against hostile environments, the immune response, and bactericidal concentrations of antibiotics or disinfectants (1). Biofilms have frequently been studied using the 96-microtiter plate model because it is a platform that requires small volumes and is suited for high-throughput screening (1). The major flaws of the microtiter model are that it is a closed system and does not incorporate shear force. It is generally accepted that biofilms will develop in the presence of shear force in the environment (1). The MBEC assay, formerly known as the Calgary Biofilm Device, was developed to incorporate shear force into high-throughput screens (2); however, this model is a closed system. In a closed system, dispersing signals and metabolic waste accumulate, and nutrients become depleted. These events are not always favorable for biofilm studies and may lead to the rapid dispersal of the biofilm before it is quantified.Open systems, which have both a continuous flow of fresh medium and shear force, have been developed and are frequently used in the laboratory (1). These include the drip-flow reactor, flow cells, perfused biofilm fermenters, the CDC biofilm reactor, the rotating-disc re...

Biofilm-Forming Abilities of Shiga Toxin-Producing Escherichia coli Isolates Associated with Human Infections

“…As previously observed, static or dynamic conditions used to form biofilms can influence the biofilm-forming capacity of isolates (26). This is especially observed for O145 isolates that formed stronger biofilms under dynamic conditions than under static conditions.…”

“…STEC biofilm production was characterized using the previously determined static and dynamic conditions (26). STEC organisms were grown in M9 supplemented with 0.4% glucose media in polystyrene microplates incubated at 30°C for 24 h, and biofilms were evaluated by crystal violet staining.…”

“…The biofilm formation assay was performed as previously described (26). Briefly, overnight cultures at 37°C in LB media were diluted (1:100) in 5 ml of M9 medium with glucose (0.4%, wt/vol) and minerals (1.16 mM MgSO 4 , 2 M FeCl 3 , 8 M CaCl 2 , and 16 M MnCl 2 ) and incubated for 24 h at 37°C.…”

“…Growth of biofilms in the BioFlux 200 device (Fluxion Biosciences, South San Francisco, CA) was performed as described before (26). Briefly, E. coli cultures were prepared as described above, and 1 ml from the 24-h culture was pelleted and resuspended in fresh prewarmed (30°C) M9 medium with glucose (0.4%, wt/vol) to an optical density at 600 nm (OD 600 ) of ϳ1.…”

“…These cultures were diluted (1:100) in M9 medium supplemented with glucose and minerals and were inoculated in triplicates into the microtiter plates (Costar 3370; Corning, NY). For all biofilm assays, the biofilm-positive STEC strain EDL 933 (26) and the biofilm-negative STEC isolate BJH-21 were used as positive and negative biofilm controls, respectively. Moreover, wells filled with sterile media were used as the blank value.…”