Spatially configurable bacterial co-culture using a microfluidic device.
Co-culture in nanoliter volumes enhances competition for nutrients, metabolite cross-talk between complementary species, and cross-suppression via antimicrobial and quorum-sensing molecules. To perform nanoliter co-cultures, we utilized microfluidic devices that consist of an array of 1.57 nL-volume cylindrical chambers (200 µm diameter x 50 µm height) connected to a single 50 µm high outer channel by a 125 µm long channel with a 10 x 10 µm cross-section (Fig. 1A, adapted from[17]). The co-culture chambers are primed with a bacterial suspension by applying vacuum to de-gas the PMDS and then flowing the suspension though the channels and into the chambers. Once the chambers are loaded, the outer channel is washed with fresh media. For co-culture experiments, species can be cultured together directly in the inner chamber (Fig. 1B). PDMS devices are optically transparent and are bonded directly to glass coverslips, allowing detailed imaging of interactions between species on the glass surface using an inverted microscope.
We cultured a GFP-expressing strain of Staphylococcus aureusinside the nanoliter chambers, and observed that it achieved confluence after approximately 6 hours at 37°C when loaded at a concentration of 1x106 cells/mL[17]. To test whether the volume of the inner chamber effected bacterial growth, we fabricated a series of devices with altered chamber heights. The height of the outer chamber was adjusted to 200 µm to improved loading and washing steps, and the height of the inner chamber was tested at 10, 30, 50, and 100 µm, corresponding to 1.6, 9.42, 15.7, and 31.4 pL respectively. In these devices, S. aureus exhibited increasingly restricted, clustered growth patterns as the volume was reduced, suggesting rapid consumption of available nutrients or accelerated sensing of quorum signaling molecules in reduced volumes (Fig. S1A-E). The device with 50 µm high chambers and outer channels was utilized for all subsequent experiments unless otherwise stated.
Exclusion zones form around Actinomyces graevenitzii microcolonies in co-culture with Streptococcus.
Species from the family Actinomycetaceae and genusStreptococcus are amongst the most common isolated from oral biofilms, particularly dental plaque. Both Actinomycetaceae andStreptococcal species grew well as mono-cultures within our microfluidic devices. To study the co-culture characteristics ofActinomycetaceae and Streptococcal species in our microfluidic chambers, we co-loaded 3 species of Actinomycetaceae (3 strains of S. odontolyticus , 3 strains of A. naeslundii , and 2 strains of A. graevenitzii ) in combination with 4 species ofStreptococcus (1 strain of S. salivarius , 3 strains ofS. mitis , 2 strains of S. oralis , and 1 strain of S. cristatus ), 56 combinations in total (Fig. 2, Fig S2). Detailed microscopy of the microfluidic chambers was performed at 8 hours.
Observations in nanoliter chambers with co-cultures of either S. cristatus or S. salivarius A64PA33 with A. graevenitzii (either FO530 or FO582 strains) revealed a striking absence of physical association between the species. Otherwise confluent streptococcal cells appeared to be unable to grow in proximity to A. graevenitzii microcolonies, resulting in formation of an ”exclusion zone” bordering the A. graevenitzii (Fig 2C).Exclusion zones did not form around A. graevenitzi i microcolonies in co-culture with S. mitis (strains ATCC 903 orATCC 49456 ) or S. oralis (strains FFB47 orFCB39 ), or around either of the other actinomyces or schaalia species tested (Fig 2B). These observations rule out any physical exclusion of streptococcus cells from space inhabited by actinomyces cells. Instead, the exclusion appears more likely due to local production of a toxic metabolite or inhibitory compound with considerable species specificity. Importantly, we did not observe any separation between macroscopic co-cultures performed using traditional co-culture protocols (Fig. S3).