Discussion
We utilized PDMS microfluidic devices to observe microbial mono- and co-cultures of oral isolates in nanoliter volumes. The gas permeability of PDMS facilitates loading of the dead-end chamber through a single channel by applying vacuum, while the coverslip provides optical clarity for imaging approaches and a physical surface on which the microbes can grow. In Actinomyces and Streptococcus co-cultures, we observed formation of a physical “exclusion zones” bordering Actinomyces graevenitzii microcolonies with certain species ofStreptococcus . Interestingly, exclusion zones were also observed with a GFP-expressing strain of Staphylococcus aureus , allowing us to perform detailed analysis of exclusion zone formation using automated imaging approaches. These studies supported a model in which exclusion zone formation is triggered by interaction of specific species.
While it appears that the exclusion zone represents a physical space containing no living bacteria, it is unclear whether this is due to physical exclusion for matrix deposition, suppression of proliferation in this area by quorum signaling, or active killing of invading cells by a toxic metabolite. While the formation of exclusion zones only occurred between Actinomyces graevenitzii with S. cristatus, S. salivarius and S. aureus , it is unlikely that the formation of a simple physical barrier explains the interactions. Additionally, the kinetics of S. aureus coverage in co-culture with A. graevenitzii , which appear to show an initial increase followed by a decrease, suggest that formation of the exclusion zone involves death of existing S. aureus cells in that area. Macroscopic co-culture on solid media did not result in any visible cross-inhibition betweenA. graevenitizii and S. aureus colonies, highlighting the importance of small volume culture and high-resolution analysis for identification of such interactions. One limitation of the microfluidic chamber design used is the inability to rapidly fix and stain cells to gain a more detailed understanding of their structure, due to the excessive time taken for fixatives and labelling compounds to diffuse through the connecting channel. Either way, this phenomenon provides clear visualization of an antagonistic relationship between competing bacterial species and may provide some insight regarding community structures in the oral cavity.
These interactions are particularly interesting in the context of well-known opportunistic pathogens such as S. aureus . Modulation of inflammatory responses in these experiments may be a direct response to compounds released by the bacteria during co-culture, or may simply follow from suppression of S. aureus proliferation, which we previously demonstrated to be important for effective neutrophil recruitment[17].
In cases where S. aureus is identified in oral lesions, it is often isolated in the company of other opportunistic pathogens such asCandida albicans[25] , infections with which are generally associated with loss of microbiome stability. Thus, exclusion ofS. aureus by A. graevenitzii builds on the concept that established and stable commensal communities are important to prevent colonization of a niche by pathogens.
Dissecting the complexity of microbial community structure and its role in the health and disease in the oral cavity remains a focus of ongoing research. Physical and metabolic characteristics facilitate successful colonization of diverse oral surfaces and ongoing survival in this complex and dynamic environment[6]. Many oral microbes have proven challenging to culture, often because they require the presence of one or more speicies in consortia to process specific metabolites[28]. In addition to such relationships, microbial community structure is also dictated by antagonism driven by competition for space and nutrients[29].
Overall, low-volume techniques such as microchambers and droplet-based microfluidics may enhance quorum sensing and competition for nutrients[9], better mimicking in vivo conditions. The microfluidic technique overcome the limiations of traditional bulk suspension co-culture approaches, which provide limited spatiotemporal information regarding interspecies interactions and often simply result in domination by the faster-growing species.
Data availablility: All data presented will be made available by the authors upon request.
Author Contributions: Fatemeh Jalali: Data curation (lead), Investigation (lead), Methodology (equal), Visualization (equal), Writing – Original Draft Preparation (supporting).Felix Ellett: Formal Analysis (lead), Investigation (supporting), Methodology (equal), Visualization (equal), Supervision (supporting), Writing – Original Draft Preparation (lead), Writing – Review and Editing (equal). Pooja Balani: Resources (supporting). Margaret Duncan: Conceptualization (equal), Resources (equal), Writing – Review and Editing (equal). Floyd Dewhirst: Conceptualization (equal) , Resources (equal), Writing – Review and Editing (equal). Gary Borisy: Conceptualization (equal), Resources (equal), Writing – Review and Editing (equal).Daniel Irimia: Conceptualization (equal), Project Administration (lead), Supervision (lead), Writing – Review and Editing (equal).
Acknowledgements: This work was supported by a grant from the National Institute of Dental and Craniofacial Research (DE024468). Microfabrication was conducted at the BioMEMS Resource Center at Massachusetts General Hospital, supported by a grant from the National Institute of Biomedical Imaging and Bioengineering (EB002503). Dr. Felix Ellett was supported by a fellowship from the Executive Committee for Research at Massachusetts General Hospital.
Conflict of Intererest: None declared
Ethics Statement: None required