An in vitro model for the cultivation of polymicrobial biofilms under continuous-flow conditions

Abstract

The airways of people with cystic fibrosis (CF) are often chronically colonised with a diverse array of bacterial and fungal species. However, little is known about the relative partitioning of species between the planktonic and biofilm modes of growth in the airways. Existing in vivo and in vitro models of CF airway infection are ill-suited for the long-term recapitulation of mixed microbial communities. Here we describe a simple, in vitro continuous-flow model for the cultivation of polymicrobial biofilms and planktonic cultures on different substrata. Our data provide evidence for inter-species antagonism and synergism in biofilm ecology. We further show that the type of substratum on which the biofilms grow has a profound influence on their species composition. This happens without any major alteration in the composition of the surrounding steady-state planktonic community. Our experimentally-tractable model enables the systematic study of planktonic and biofilm communities under conditions that are nutritionally reminiscent of the CF airway microenvironment, something not possible using any existing in vivo models of CF airway infection.

Decreased efficacy of antimicrobial agents in a polymicrobial environment

“…Antimicrobial resistance is known to be increased (compared with the planktonic mode of growth) [ 40 ] in biofilms. However, we have recently demonstrated that the continuous-flow setup can be used to cultivate polymicrobial biofilms [ 19 ] and current efforts are aimed at examining how antibiotic challenge affects the population dynamics in this growth mode too. We are also expanding the species repertoire in the system.…”

“…The bacterial/fungal strains used in this study were: P. aeruginosa PAO1 (PA; strain MPAO1 from Colin Manoil), S. aureus ATCC 25923 (SA), and C. albicans SC314 (CA). Artificial sputum medium (ASM) was freshly prepared for every experiment as described in [ 19 ].…”

“…The continuous-flow culture vessel was assembled, inoculated and incubated as described in [ 13 ]. We also refer the reader to [ 19 ] for an illustrated, step-by-step guide on the assembly and use of the continuous-flow model. In brief, the continuous-flow culture vessel comprises a 100 mL Duran bottle fitted with a 4-port HPLC GL80 screwcap lid.…”

Challenges and opportunities in the development of novel antimicrobial therapeutics for cystic fibrosis

“…O'Brien et al outlines a model derived from the porcine lung model that uses a continuous flow of media, allowing establishment of steady-state polymicrobial biofilms and planktonic cultures (Fig. 3) [55]. Cubes of agar or porcine bronchiole inoculated with bacteria are suspended in a beaker with continuous ASM inflow and waste outflow.…”

“…A continuous flow model of polymicrobial infection [55]. Artificial sputum medium (ASM) is pumped into a culture vessel containing 5cm 3 cubes of agar or ex vivo porcine lung, which serve as a solid substratum for bacteria to grow.…”